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Sample records for amiloride

  1. Amiloride

    Science.gov (United States)

    ... usually used in combination with other diuretics ('water pills') to treat high blood pressure and heart failure ... skip the missed dose and continue your regular dosing schedule. Do not take a double dose to ...

  2. Amiloride interacts with renal α- and β-adrenergic receptors

    International Nuclear Information System (INIS)

    Howard, M.J.; Mullen, M.D.; Insel, P.A.

    1987-01-01

    The authors have used radioligand binding techniques to assess whether amiloride and certain analogues of amiloride (ethylisopropyl amiloride and benzamil) can bind to adrenergic receptors in the kidney. They found that amiloride could compete for [ 3 H]rauwolscine (α 2 -adrenergic receptors), [ 3 H]prazosin (α 1 -adrenergic receptors), and [ 125 I]iodocyanopindolol (β-adrenergic receptors) binding in rat renal cortical membranes with inhibitor constants of 13.6 /plus minus/ 5.7, 24.4 /plus minus/ 7.4, and 8.36 /plus minus/ 13.5 μM, respectively. Ethylisopropyl amiloride and benzamil were from 2- to 25-fold more potent than amiloride in competing for radioligand binding sites in studies with these membranes. In addition, amiloride and the two analogues competed for [ 3 H]prazosin sites on intact Madin-Darby canine kidney cells and amiloride blocked epinephrine-stimulated prostaglandin E 2 production in these cells. They conclude that amiloride competes for binding to several classes of renal adrenergic receptors with a rank order of potency of α 2 > α 1 > β. Binding to, and antagonism of, adrenergic receptors occurs at concentrations of amiloride that are lower than previously observed nonspecific interactions of this agent

  3. Amiloride inhibits rat mucosal ornithine decarboxylase activity and DNA synthesis

    International Nuclear Information System (INIS)

    Ulrich-Baker, M.G.; Wang, P.; Fitzpatrick, L.; Johnson, L.R.

    1988-01-01

    Refeeding fasted rats induces a dramatic trophic response in gastrointestinal mucosa and is associated with elevations in both rate of DNA synthesis and ornithine decarboxylase (ODC) activity. The signal for these increases is unknown. Amiloride prevents cell alkalinization by blocking Na + -H + exchange at apical epithelial cell membranes. In study 1, rats were fasted 48 h, treated with amiloride (0.5 to 500 mg/kg), and refed for 4 h. Refeeding increased ODC activities in the jejunal mucosa (X8) and liver (X19) but not in the oxyntic gland mucosa. In the jejunum, but not the liver, the activation of ODC was completely abolished by 100 mg/kg amiloride. In study 2, the rate of DNA synthesis was determine by measuring the rate of [ 3 H]thymidine incorporation 16 h after refeeding. Refeeding resulted in significantly increased rates of DNA synthesis over fasted levels, and amiloride at 100 mg/kg significantly reduced the elevations in the jejenum and liver. In conclusion, amiloride inhibits the postprandial increases in jejunal ODC activity and DNA synthesis in the jejunum and liver. The results indicate that (1) the Na + -H + antiport is essential to the increased ODC activity in the jejunum and liver after a meal and (2) increases in DNA synthesis and their suppression by amiloride are not necessary linked to ODC activity

  4. Amiloride inhibits rat mucosal ornithine decarboxylase activity and DNA synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Ulrich-Baker, M.G.; Wang, P.; Fitzpatrick, L.; Johnson, L.R. (Univ. of Texas Health Science Center, Houston (USA))

    1988-03-01

    Refeeding fasted rats induces a dramatic trophic response in gastrointestinal mucosa and is associated with elevations in both rate of DNA synthesis and ornithine decarboxylase (ODC) activity. The signal for these increases is unknown. Amiloride prevents cell alkalinization by blocking Na{sup +}-H{sup +} exchange at apical epithelial cell membranes. In study 1, rats were fasted 48 h, treated with amiloride (0.5 to 500 mg/kg), and refed for 4 h. Refeeding increased ODC activities in the jejunal mucosa (X8) and liver (X19) but not in the oxyntic gland mucosa. In the jejunum, but not the liver, the activation of ODC was completely abolished by 100 mg/kg amiloride. In study 2, the rate of DNA synthesis was determine by measuring the rate of ({sup 3}H)thymidine incorporation 16 h after refeeding. Refeeding resulted in significantly increased rates of DNA synthesis over fasted levels, and amiloride at 100 mg/kg significantly reduced the elevations in the jejenum and liver. In conclusion, amiloride inhibits the postprandial increases in jejunal ODC activity and DNA synthesis in the jejunum and liver. The results indicate that (1) the Na{sup +}-H{sup +} antiport is essential to the increased ODC activity in the jejunum and liver after a meal and (2) increases in DNA synthesis and their suppression by amiloride are not necessary linked to ODC activity.

  5. Streptococcus faecalis susceptibility to amiloride depends on medium pH.

    Science.gov (United States)

    Giunta, S; Galeazzi, L; Turchetti, G; Grilli, G; Groppa, G

    1988-10-01

    Amiloride is one of the major molecular probes in basic and applied investigations on the physiology of cation transport in animal cells. In these cells the drug also exerts growth inhibitory activity. Recently, we discovered that amiloride causes growth inhibition also on bacterial cells. In this paper we report that medium pH influences amiloride activity on Streptococcus faecalis. The lowering of external pH causes a drop in the susceptibility of this bacterium to amiloride up to an almost complete resistance. This finding, constitutes a novel aspect of the in vitro experimental pharmacology of this diuretic potentially useful also in clinical pharmacology and in animal cell investigations.

  6. Effect of amiloride, or amiloride plus hydrochlorothiazide, versus hydrochlorothiazide on glucose tolerance and blood pressure (PATHWAY-3): a parallel-group, double-blind randomised phase 4 trial.

    Science.gov (United States)

    Brown, Morris J; Williams, Bryan; Morant, Steve V; Webb, David J; Caulfield, Mark J; Cruickshank, J Kennedy; Ford, Ian; McInnes, Gordon; Sever, Peter; Salsbury, Jackie; Mackenzie, Isla S; Padmanabhan, Sandosh; MacDonald, Thomas M

    2016-02-01

    Potassium depletion by thiazide diuretics is associated with a rise in blood glucose. We assessed whether addition or substitution of a potassium-sparing diuretic, amiloride, to treatment with a thiazide can prevent glucose intolerance and improve blood pressure control. We did a parallel-group, randomised, double-blind trial in 11 secondary and two primary care sites in the UK. Eligible patients were aged 18-80 years; had clinic systolic blood pressure of 140 mm Hg or higher and home systolic blood pressure of 130 mmHg or higher on permitted background drugs of angiotensin-converting enzyme inhibitors, angiotensin-receptor blockers, β blockers, calcium-channel blockers, or direct renin inhibitors (previously untreated patients were also eligible in specific circumstances); and had at least one component of the metabolic syndrome in addition to hypertension. Patients with known diabetes were excluded. Patients were randomly assigned (1:1:1) to 24 weeks of daily oral treatment with starting doses of 10 mg amiloride, 25 mg hydrochlorothiazide, or 5 mg amiloride plus 12·5 mg hydrochlorothiazide; all doses were doubled after 12 weeks. Random assignment was done via a central computer system. Both participants and investigators were masked to assignment. Our hierarchical primary endpoints, assessed on a modified intention-to-treat basis at 12 and 24 weeks, were the differences from baseline in blood glucose measured 2 h after a 75 g oral glucose tolerance test (OGTT), compared first between the hydrochlorothiazide and amiloride groups, and then between the hydrochlorothiazide and combination groups. A key secondary endpoint was change in home systolic blood pressure at 12 and 24 weeks. This trial is registered with ClinicalTrials.gov, number NCT00797862, and the MHRA, Eudract number 2009-010068-41, and is now complete. Between Nov 18, 2009, and Dec 15, 2014, 145 patients were randomly assigned to amiloride, 146 to hydrochlorothiazide, and 150 to the combination group

  7. Amiloride and SN-6 suppress audiogenic seizure susceptibility in genetically epilepsy-prone rats.

    Science.gov (United States)

    Quansah, Hillary; N'Gouemo, Prosper

    2014-09-01

    We have recently reported that amiloride, a potent and nonselective blocker of acid-sensing ion channels, prevents the development of pilocarpine-induced seizures and status epilepticus. Amiloride is also known to suppress the activity of Na(+) /Ca(2+) and Na(+) /H(+) exchangers that have been implicated in the pathophysiology of seizures. Here, we evaluated the effects of amiloride, SN-6 (a potent blocker of Na(+) /Ca(2+) exchangers) and zoniporide (a potent blocker of Na(+) /H(+) exchangers) on acoustically evoked seizures (audiogenic seizures, AGS) in genetically epilepsy-prone rats (GEPR-3s), a model of inherited generalized epilepsy. Male, six-week-old GEPR-3s were used. The GEPR-3s were tested for AGS susceptibility before and after treatment with various doses of amiloride, SN-6, and zoniporide (1, 3, 10, and 30 mg/kg; per os). We found that pretreatment with amiloride and SN-6 markedly reduced the incidence and severity of AGS in the GEPR-3s. In contrast, administration of zoniporide only minimally reduced the incidence and severity of AGS in the GEPR-3s. A combination of noneffective doses of SN-6 and zoniporide also suppressed AGS susceptibility in the GEPR-3s. These findings suggest acid-sensing ion channels and the Na(+) /Ca(2+) exchanger may play an important role in the pathophysiology of inherited AGS susceptibility in the GEPR-3s. © 2014 John Wiley & Sons Ltd.

  8. Additive hypocalciuric effects of amiloride and hydrochlorothiazide in patients treated with calcitriol.

    Science.gov (United States)

    Alon, U; Costanzo, L S; Chan, J C

    1984-01-01

    To compare the effects of hydrochlorothiazide (HCTZ) alone and in combination with amiloride on urinary calcium excretion we performed 14 acute studies on 7 patients with vitamin-D-induced calciuria. Each patient was studied first with HCTZ alone, and 1-32 weeks later with the same or lower dose of HCTZ combined with amiloride. Administration of HCTZ alone did not change UCaV during the 1st day of therapy, and caused a significant reduction from 44.4 +/- 28.8 to 26.0 +/- 14.4 mg/m2/24 h (p less than 0.02) on the 4th day. In contrast, combined diuretic regimen caused a significant reduction in UCaV from 36.0 +/- 16.7 to 23.6 +/- 14.4 mg/m2/24 h (p less than 0.02) on the 1st day and further reduction to 13.3 +/- 6.9 mg/m2/24 h (p less than 0.01) on the 4th day. UCaV on the 4th day was significantly lower with the HCTZ-amiloride combination (p less than 0.05). The combined therapy caused a greater reduction in FECa/FENa than HCTZ alone on the 1st day (p less than 0.02) and on the 4th day (p less than 0.01). HCTZ-induced hyperkaluria, hypokalemia and alkalosis were prevented by the addition of amiloride. In another patient, low-dose HCTZ-amiloride had a maximal dissociative effect on FECa/FENa and was more effective than HCTZ given alone in a double dose. 3 patients were treated with the low-dose HCTZ-amiloride regimen for a total of 23 months. UCaV was kept persistently low.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Amiloride lowers blood pressure and attenuates urine plasminogen activation in patients with treatment-resistant hypertension

    DEFF Research Database (Denmark)

    Stolzenburg Oxlund, Christina; Buhl, Kristian Bergholt; Jacobsen, Ib A

    2014-01-01

    In conditions with albuminuria, plasminogen is aberrantly filtered across the glomerular barrier and activated along the tubular system to plasmin. In the collecting duct, plasmin activates epithelial sodium channels (ENaC) proteolytically. Hyperactivity of ENaC could link microalbuminuria....../proteinuria to resistant hypertension. Amiloride, an ENaC inhibitor, inhibits urokinase-type plasminogen activator. We hypothesized that amiloride (1) reduces blood pressure (BP); (2) attenuates plasminogen-to-plasmin activation; and (3) inhibits urine urokinase-type plasminogen activator in patients with resistant...

  10. Amiloride causes changes in the mechanical properties of hair cell bundles in the fish lateral line similar to those induced by dihydrostreptomycin

    NARCIS (Netherlands)

    Wiersinga-Post, JEC; van Netten, SM

    1998-01-01

    Amiloride is a known blocker of the mechano-electrical transduction current in sensory hair cells. Measurements of cupular motion in the lateral line organ of fish now show that amiloride concurrently changes the micromechanical properties of the hair cell bundles. The effects of amiloride on the

  11. Effect of the bases flanking an abasic site on the recognition of nucleobase by amiloride.

    Science.gov (United States)

    Rajendran, Arivazhagan; Zhao, Chunxia; Rajendar, Burki; Thiagarajan, Viruthachalam; Sato, Yusuke; Nishizawa, Seiichi; Teramae, Norio

    2010-06-01

    We explain here the various non-covalent interactions which are responsible for the different binding modes of a small ligand with DNA. The combination of experimental and theoretical methods was used. The interaction of amiloride with thymine was found to depend on the bases flanking the AP site and different binding modes were observed for different flanking bases. Molecular modeling, absorption studies and binding constant measurements support for the different binding patterns. The flanking base dependent recognition of AP site phosphates was investigated by (31)P NMR experiments. The thermodynamics of the ligand-nucleotide interaction was demonstrated by isothermal titration calorimetry. The emission behavior of amiloride was found to depend on the bases flanking the AP site. Amiloride photophysics in the context of AP-site containing DNA is investigated by time-dependent density functional theory. Flanking bases affect the ground and excited electronic states of amiloride when binding to AP site, which causes flanking base-dependent fluorescence signaling. The various noncovalent interactions have been well characterized for the determination of nucleic acid structure and dynamics, and protein-DNA interactions. However, these are not clear for the DNA-small molecule interactions and we believe that our studies will bring a new insight into such phenomena. Copyright 2010 Elsevier B.V. All rights reserved.

  12. Effect of amiloride on arachidonic acid and histamine release from rat mast cells

    DEFF Research Database (Denmark)

    Linnebjerg, H.; Hansen, Harald S.; Jensen, B.

    1989-01-01

    The effect of a putative Na/H exchange inhibition on histamine and [C]arachidonic acid ([C]AA) release has been examined in rat peritoneal mast cells, using either addition of amiloride or removal of extracellular Na. The cells were stimulated by non-immunological agents, i.e. calcium ionophore A...

  13. Interaction of antihypertensive drug amiloride with metal ions in micellar medium using fluorescence spectroscopy

    International Nuclear Information System (INIS)

    Gujar, Varsha; Pundge, Vijaykumar; Ottoor, Divya

    2015-01-01

    Steady state and life time fluorescence spectroscopy have been employed to study the interaction of antihypertensive drug amiloride with biologically important metal ions i.e. Cu 2+ , Fe 2+ , Ni 2+ and Zn 2+ in various micellar media (anionic SDS (sodium dodecyl sulfate), nonionic TX-100 (triton X-100) and cationic CTAB (cetyl trimethyl ammonium bromide)). It was observed that fluorescence properties of drug remain unaltered in the absence of micellar media with increasing concentration of metal ions. However, addition of Cu 2+ , Fe 2+ and Ni 2+ caused fluorescence quenching of amiloride in the presence of anionic micelle, SDS. Binding of drug with metal ions at the charged micellar interface could be the possible reason for this pH-dependent metal-mediated fluorescence quenching. There were no remarkable changes observed due to metal ions addition when drug was present in cationic and nonionic micellar medium. The binding constant and bimolecular quenching constant were evaluated and compared for the drug–metal complexes using Stern–Volmer equation and fluorescence lifetime values. - Highlights: • Interaction of amiloride with biologically important metal ions, Fe 2+ , Cu 2+ , Ni 2+ and Zn 2+ . • Monitoring the interaction in various micelle at different pH by fluorescence spectroscopy. • Micelles acts as receptor, amiloride as transducer and metal ions as analyte in the present system. • Interaction study provides pH dependent quenching and binding mechanism of drug with metal ions

  14. Spectrophotometric method for estimation of amiloride in bulk and tablet dosage form

    Directory of Open Access Journals (Sweden)

    Aitha Vijaya Lakshmi

    2015-01-01

    Full Text Available Introduction: Amiloride chemically, 3,5-diamino-6-chloro-N-(diaminomethylene pyrazine-2-carboxamide. It is used in the management of congestive heart failure, available as Amifru tab, Amimide. It causes adverse effects like Nausea, diarrhea and dizziness. Materials: 0.1 N Hydrochloric acid, 0.1 N Sodium hydroxide and 1 mg/ml amiloride drug solution were required. Spectral and absorbance measurements were made using ELICO UV-160 double beam Spectrophotometer. Method: Amiloride drug solution concentration range of 25 to 125ug/ml in 0.1N HCl medium was scanned over the wave length range of 235-320 against blank prepared in 0.1N NaOH solution. Two wavelengths are selected one at positive peak 245 nm and another at negative peak 290 nm, the amplitude is calculated from these values. Results and Discussion: The sum of the absolute values at these wavelengths is called amplitude. The amplitude is proportional to the amount of drug. High accuracy, reproducibility and low t-values were reported from the calibration curve plotted with the amplitude verses amount of drug. So the proposed method is simple, less time consuming and it can be successfully adopted for the estimation of amiloride.

  15. Amiloride Improves Endothelial Function and Reduces Vascular Stiffness in Female Mice Fed a Western Diet

    Directory of Open Access Journals (Sweden)

    Luis A. Martinez-Lemus

    2017-06-01

    Full Text Available Obese premenopausal women lose their sex related cardiovascular disease protection and develop greater arterial stiffening than age matched men. In female mice, we have shown that consumption of a Western diet (WD, high in fat and refined sugars, is associated with endothelial dysfunction and vascular stiffening, which occur via activation of mineralocorticoid receptors and associated increases in epithelial Na+ channel (ENaC activity on endothelial cells (EnNaC. Herein our aim was to determine the effect that reducing EnNaC activity with a very-low-dose of amiloride would have on decreasing endothelial and arterial stiffness in young female mice consuming a WD. To this end, we fed female mice either a WD or control diet and treated them with or without a very-low-dose of the ENaC-inhibitor amiloride (1 mg/kg/day in the drinking water for 20 weeks beginning at 4 weeks of age. Mice consuming a WD were heavier and had greater percent body fat, proteinuria, and aortic stiffness as assessed by pulse-wave velocity than those fed control diet. Treatment with amiloride did not affect body weight, body composition, blood pressure, urinary sodium excretion, or insulin sensitivity, but significantly reduced the development of endothelial and aortic stiffness, aortic fibrosis, aortic oxidative stress, and mesenteric resistance artery EnNaC abundance and proteinuria in WD-fed mice. Amiloride also improved endothelial-dependent vasodilatory responses in the resistance arteries of WD-fed mice. These results indicate that a very-low-dose of amiloride, not affecting blood pressure, is sufficient to improve endothelial function and reduce aortic stiffness in female mice fed a WD, and suggest that EnNaC-inhibition may be sufficient to ameliorate the pathological vascular stiffening effects of WD-induced obesity in females.

  16. Aberrant glomerular filtration of urokinase-plasminogen activator in nephrotic syndrome leads to amiloride-sensitive plasminogen activation in urine

    DEFF Research Database (Denmark)

    Stæhr, Mette; Buhl, Kristian Bergholt; Andersen, René F

    2015-01-01

    In nephrotic syndrome, aberrant glomerular filtration of plasminogen and conversion to active plasmin in pre-urine is thought to activate proteolytically ENaC and contribute to sodium retention and edema. The ENaC blocker amiloride is an off-target inhibitor of urokinase-type plasminogen activator...... with aberrant filtration of uPA across the injured glomerular barrier. Amiloride inhibits urine uPA activity which attenuates plasminogen activation and urine protease activity in vivo. Urine uPA is a relevant target for amiloride in vivo....

  17. Interaction of antihypertensive drug amiloride with metal ions in micellar medium using fluorescence spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Gujar, Varsha; Pundge, Vijaykumar; Ottoor, Divya, E-mail: divya@chem.unipune.ac.in

    2015-05-15

    Steady state and life time fluorescence spectroscopy have been employed to study the interaction of antihypertensive drug amiloride with biologically important metal ions i.e. Cu{sup 2+}, Fe{sup 2+}, Ni{sup 2+} and Zn{sup 2+} in various micellar media (anionic SDS (sodium dodecyl sulfate), nonionic TX-100 (triton X-100) and cationic CTAB (cetyl trimethyl ammonium bromide)). It was observed that fluorescence properties of drug remain unaltered in the absence of micellar media with increasing concentration of metal ions. However, addition of Cu{sup 2+}, Fe{sup 2+} and Ni{sup 2+} caused fluorescence quenching of amiloride in the presence of anionic micelle, SDS. Binding of drug with metal ions at the charged micellar interface could be the possible reason for this pH-dependent metal-mediated fluorescence quenching. There were no remarkable changes observed due to metal ions addition when drug was present in cationic and nonionic micellar medium. The binding constant and bimolecular quenching constant were evaluated and compared for the drug–metal complexes using Stern–Volmer equation and fluorescence lifetime values. - Highlights: • Interaction of amiloride with biologically important metal ions, Fe{sup 2+}, Cu{sup 2+}, Ni{sup 2+} and Zn{sup 2+}. • Monitoring the interaction in various micelle at different pH by fluorescence spectroscopy. • Micelles acts as receptor, amiloride as transducer and metal ions as analyte in the present system. • Interaction study provides pH dependent quenching and binding mechanism of drug with metal ions.

  18. Active urea transport by the skin of Bufo viridis: Amiloride- and phloretin-sensitive transport sites

    Energy Technology Data Exchange (ETDEWEB)

    Rapoport, J.; Abuful, A.; Chaimovitz, C.; Noeh, Z.; Hays, R.M. (Soroka Medical Center, Beersheva (Israel) Albert Einstein College of Medicine, New York, NY (USA))

    1988-09-01

    Urea is actively transported inwardly (J{sub i}) across the skin of the green toad Bufo viridis. J{sub i} is markedly enhanced in toads adapted to hypertonic saline. The authors studied urea transport across the skin of Bufo viridis under a variety of experimental conditions, including treatment with amiloride and phloretin, agents that inhibit urea permeability in the bladder of Bufo marinus. Amiloride (10{sup {minus}4} M) significantly inhibited J{sub i} in both adapted and unadapted animals and was unaffected by removal of sodium from the external medium. Phloretin (10{sup {minus}4} M) significantly inhibited J{sub i} in adapted animals by 23-46%; there was also a reduction in J{sub i} in unadapted toads at 10{sup {minus}4} and 5 {times} 10{sup {minus}4} M phloretin. A dose-response study revealed that the concentration of phloretin causing half-maximal inhibition (K{sub {1/2}}) was 5 {times} 10{sup {minus}4} M for adapted animals. J{sub i} was unaffected by the substitution of sucrose for Ringer solution or by ouabain. They conclude (1) the process of adaptation appears to involve an increase in the number of amiloride- and phloretin-inhibitable urea transport sites in the skin, with a possible increase in the affinity of the sites for phloretin; (2) the adapted skin resembles the Bufo marinus urinary bladder with respect to amiloride and phloretin-inhibitable sites; (3) they confirm earlier observations that J{sub i} is independent of sodium transport.

  19. Amiloride-insensitive sodium channels are directly regulated by actin cytoskeleton dynamics in human lymphoma cells.

    Science.gov (United States)

    Sudarikova, Anastasia V; Tsaplina, Olga A; Chubinskiy-Nadezhdin, Vladislav I; Morachevskaya, Elena A; Negulyaev, Yuri A

    2015-05-22

    Sodium influx mediated by ion channels of plasma membrane underlies fundamental physiological processes in cells of blood origin. However, little is known about the single channel activity and regulatory mechanisms of sodium-specific channels in native cells. In the present work, we used different modes of patch clamp technique to examine ion channels involved in Na-transporting pathway in U937 human lymphoma cells. The activity of native non-voltage-gated sodium (NVGS) channels with unitary conductance of 10 pS was revealed in cell-attached, inside-out and whole-cell configurations. NVGS channel activity is directly controlled by submembranous actin cytoskeleton. Specifically, an activation of sodium channels in U937 cells in response to microfilament disassembly was demonstrated on single-channel and integral current level. Inside-out experiments showed that filament assembly on cytoplasmic membrane surface caused fast inactivation of the channels. Biophysical characteristics of NVGS channels in U937 cells were similar to that of epithelial sodium channels (ENaCs). However, we found that amiloride, a known inhibitor of DEG/ENaC, did not block NVGS channels in U937 cells. Whole-cell current measurements revealed no amiloride-sensitive component of membrane current. Our data show that cortical actin structures represent the main factor that controls the activity of amiloride-insensitive ENaC-like channels in human lymphoma cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Amiloride-sensitive channels in type I fungiform taste cells in mouse

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    Clapp Tod R

    2008-01-01

    Full Text Available Abstract Background Taste buds are the sensory organs of taste perception. Three types of taste cells have been described. Type I cells have voltage-gated outward currents, but lack voltage-gated inward currents. These cells have been presumed to play only a support role in the taste bud. Type II cells have voltage-gated Na+ and K+ current, and the receptors and transduction machinery for bitter, sweet, and umami taste stimuli. Type III cells have voltage-gated Na+, K+, and Ca2+ currents, and make prominent synapses with afferent nerve fibers. Na+ salt transduction in part involves amiloride-sensitive epithelial sodium channels (ENaCs. In rodents, these channels are located in taste cells of fungiform papillae on the anterior part of the tongue innervated by the chorda tympani nerve. However, the taste cell type that expresses ENaCs is not known. This study used whole cell recordings of single fungiform taste cells of transgenic mice expressing GFP in Type II taste cells to identify the taste cells responding to amiloride. We also used immunocytochemistry to further define and compare cell types in fungiform and circumvallate taste buds of these mice. Results Taste cell types were identified by their response to depolarizing voltage steps and their presence or absence of GFP fluorescence. TRPM5-GFP taste cells expressed large voltage-gated Na+ and K+ currents, but lacked voltage-gated Ca2+ currents, as expected from previous studies. Approximately half of the unlabeled cells had similar membrane properties, suggesting they comprise a separate population of Type II cells. The other half expressed voltage-gated outward currents only, typical of Type I cells. A single taste cell had voltage-gated Ca2+ current characteristic of Type III cells. Responses to amiloride occurred only in cells that lacked voltage-gated inward currents. Immunocytochemistry showed that fungiform taste buds have significantly fewer Type II cells expressing PLC signalling

  1. Insulin activates single amiloride-blockable Na channels in a distal nephron cell line (A6).

    Science.gov (United States)

    Marunaka, Y; Hagiwara, N; Tohda, H

    1992-09-01

    Using the patch-clamp technique, we studied the effect of insulin on an amiloride-blockable Na channel in the apical membrane of a distal nephron cell line (A6) cultured on permeable collagen films for 10-14 days. NPo (N, number of channels per patch membrane; Po, average value of open probability of individual channels in the patch) under baseline conditions was 0.88 +/- 0.12 (SE)(n = 17). After making cell-attached patches on the apical membrane which contained Na channels, insulin (1 mU/ml) was applied to the serosal bath. While maintaining the cell-attached patch, NPo significantly increased to 1.48 +/- 0.19 (n = 17; P less than 0.001) after 5-10 min of insulin application. The open probability of Na channels was 0.39 +/- 0.01 (n = 38) under baseline condition, and increased to 0.66 +/- 0.03 (n = 38, P less than 0.001) after addition of insulin. The baseline single-channel conductance was 4pS, and neither the single-channel conductance nor the current-voltage relationship was significantly changed by insulin. These results indicate that insulin increases Na absorption in the distal nephron by increasing the open probability of the amiloride-blockable Na channel.

  2. Changes in urinary excretion of water and sodium transporters during amiloride and bendroflumethiazide treatment

    DEFF Research Database (Denmark)

    Jensen, Janni M; Mose, Frank H; Kulik, Anna-Ewa O

    2015-01-01

    AIM: To quantify changes in urinary excretion of aquaporin2 water channels (u-AQP2), the sodium-potassium-chloride co-transporter (u-NKCC2) and the epithelial sodium channels (u-ENaC) during treatment with bendroflumethiazide (BFTZ), amiloride and placebo. METHODS: In a randomized, double......-blinded, placebo-controlled, 3-way crossover study we examined 23 healthy subjects on a standardized diet and fluid intake. The subjects were treated with amiloride 5 mg, BFTZ 1.25 mg or placebo twice a day for 4.5 d before each examination day. On the examination day, glomerular filtration rate was measured...... by the constant infusion clearance technique with (51)Cr-EDTA as reference substance. To estimate the changes in water transport via AQP2 and sodium transport via NKCC2 and ENaC, u-NKCC2, the gamma fraction of ENaC (u-ENaCγ), and u-AQP2 were measured at baseline and after infusion with 3% hypertonic saline. U...

  3. Simultaneous Determination of Amiloride and Hydrochlorothiazide in a Compound Tablet by Diffuse Reflectance Spectroscopy and Chemometrics

    Science.gov (United States)

    Tang, J.; Li, X.; Feng, Y.; Liang, B.

    2016-09-01

    This paper studies the simultaneous determination of amiloride hydrochloride (AMH) and hydrochlorothiazide (HCTZ) in amiloride hydrochloride tablets by ultraviolet-visible-shortwave near-infrared diffuse reflectance spectroscopy (UV-Vis-swNIR DRS) and chemometrics. Quantitative models for the two components were established by partial least squares (PLS) and support vector regression (SVR), respectively. For the PLS models of AMH and HCTZ, the determination coefficient R2 of the calibration set was 0.9503 and 0.9538, and the coefficient R2 of the prediction set was 0.8983 and 0.9260, respectively. The root mean square error of the calibration set (RMSEC) was 0.8 mg and 8.1 mg, while the root mean square error of the prediction set (RMSEP) was 1.0 mg and 8.7 mg, respectively. For the SVR models of AMH and HCTZ, the R2 of the calibration set was 0.9668 and 0.9609; the R2 of the prediction set was 0.9145 and 0.9446, respectively. The RMSEC was 0.7 and 7.5 mg, and the RMSEP was 0.9 and 8.9 mg, respectively. The results show that SVR modeling has a satisfactory prediction effect. The proposed method based on UV-vis-swNIR and chemometrics is efficient, nondestructive, and expected to be used for online quality monitoring in the production of drugs.

  4. Potential Roles of Amiloride-Sensitive Sodium Channels in Cancer Development

    Directory of Open Access Journals (Sweden)

    Siguang Xu

    2016-01-01

    Full Text Available The ENaC/degenerin ion channel superfamily includes the amiloride-sensitive epithelial sodium channel (ENaC and acid sensitive ionic channel (ASIC. ENaC is a multimeric ion channel formed by heteromultimeric membrane glycoproteins, which participate in a multitude of biological processes by mediating the transport of sodium (Na+ across epithelial tissues such as the kidney, lungs, bladder, and gut. Aberrant ENaC functions contribute to several human disease states including pseudohypoaldosteronism, Liddle syndrome, cystic fibrosis, and salt-sensitive hypertension. Increasing evidence suggests that ion channels not only regulate ion homeostasis and electric signaling in excitable cells but also play important roles in cancer cell behaviors such as proliferation, apoptosis, invasion, and migration. Indeed, ENaCs/ASICs had been reported to be associated with cancer characteristics. Given their cell surface localization and pharmacology, pharmacological strategies to target ENaC/ASIC family members may be promising cancer therapeutics.

  5. Successful Management of Refractory Type 1 Renal Tubular Acidosis with Amiloride

    Directory of Open Access Journals (Sweden)

    Patrick Oguejiofor

    2017-01-01

    Full Text Available A 28-year-old female with history of hypothyroidism, Sjögren’s Syndrome, and Systemic Lupus Erythematosus (SLE presented with complaints of severe generalized weakness, muscle pain, nausea, vomiting, and anorexia. Physical examination was unremarkable. Laboratory test showed hypokalemia at 1.6 mmol/l, nonanion metabolic acidosis with HCO3 of 11 mmol/l, random urine pH of 7.0, and urine anion gap of 8 mmol/l. CT scan of the abdomen revealed bilateral nephrocalcinosis. A diagnosis of type 1 RTA likely secondary to Sjögren’s Syndrome was made. She was started on citric acid potassium citrate with escalating dosages to a maximum dose of 60 mEq daily and potassium chloride over 5 years without significant improvement in serum K+ and HCO3 levels. She had multiple emergency room visits for persistent muscle pain, generalized weakness, and cardiac arrhythmias. Citric acid potassium citrate was then replaced with sodium bicarbonate at 15.5 mEq every 6 hours which was continued for 2 years without significant improvement in her symptoms and electrolytes. Amiloride 5 mg daily was added to her regimen as a potassium sparing treatment with dramatic improvement in her symptoms and electrolyte levels (as shown in the figures. Amiloride was increased to 10 mg daily and potassium supplementation was discontinued without affecting her electrolytes. Her sodium bicarbonate was weaned to 7.7 mEq daily.

  6. Regulación por proteasas del canal de sodio sensible al amiloride (ENaC Amiloride sensitive sodium channels (ENaC and their regulation by proteases

    Directory of Open Access Journals (Sweden)

    Luciano Galizia

    2011-04-01

    Full Text Available El ENaC es un canal que permite el movimiento de Na+ desde el líquido luminal hacia las células en numerosos epitelios reabsortivos y también en otros tejidos como la placenta. ENaC juega un papel crucial en la homeostasis de los electrolitos y volumen de líquido extracelular. Es regulado por numerosas hormonas, incluyendo la aldosterona y bloqueado por el diurético amiloride. El ENaC está formado por tres subunidades homólogas α, β y γ que forman el poro por el cual se mueven los iones Na+. Dos factores regulan la actividad del ENaC. 1 el número de canales insertos en la membrana celular y 2 la probabilidad de apertura o tiempo en que se encuentra abierto el canal. El número de canales es el resultado de un balance entre su síntesis y degradación. La probabilidad de apertura depende de la proteólisis de zonas específicas de las subunidades α y γ por múltiples proteasas dentro de la célula y en el espacio extracelular. Entre las proteasas más estudiadas se encuentran la furina, prostasina, elastasa, plasmina y tripsina. Existen sustancias endógenas que bloquean la actividad de estas proteasas como la aprotinina, la bikunina y la nexina-1 y la expresión de las proteasas y sus inhibidores es regulada a su vez por la aldosterona, la tasa de movimiento de Na y el TFGβ. En este trabajo presentamos algunos ejemplos de esta regulación y su potencial papel en condiciones normales y en ciertas enfermedades como la fibrosis quística, renales e hipertensión.ENaC is a channel that mediates entry of Na+ from the luminal fluid into the cells in many reabsorbing epithelia and it is also expressed in human placenta. ENaC is crucial in the control of electrolyte and extracellular volume homeostasis. ENaC is regulated by several hormones, including aldosterone and blocked by amiloride and its analogs. ENaC channels are composed by three homologous subunits, α, β and γ that form the pore where Na ions are transported. Two factors

  7. NHE1 inhibition by amiloride- and benzoylguanidine-type compounds. Inhibitor binding loci deduced from chimeras of NHE1 homologues with endogenous differences in inhibitor sensitivity

    DEFF Research Database (Denmark)

    Pedersen, Stine F; King, Scott A; Nygaard, Eva B

    2007-01-01

    NHE1). Although highly homologous to the amiloride- and HOE-sensitive human NHE1 (hNHE1), AtNHE1 is insensitive to HOE-type and PaNHE1 to both amiloride- and HOE-type compounds. Here we generated chimeras to "knock in" amiloride and HOE sensitivity to PaNHE1, and we thereby identified several NHE1...... regions involved in inhibitor interaction. The markedly different inhibitor sensitivities of hNHE1, AtNHE1, and PaNHE1 could not be accounted for by differences in transmembrane (TM) region 9. Replacing TM10 through the C-terminal tail of PaNHE1 with the corresponding region of AtNHE1 partially restored...... sensitivity to amiloride and the related compound 5'-(N-ethyl-N-isopropyl)amiloride (EIPA) but not to HOE694. This effect was not due to the tail region, but it was dependent on TM10-11, because replacing only this region with that of AtNHE1 also partially restored amiloride and EIPA but not HOE sensitivity...

  8. Amiloride-enhanced gene transfection of octa-arginine functionalized calcium phosphate nanoparticles

    Science.gov (United States)

    Tenkumo, Taichi; Kamano, Yuya; Egusa, Hiroshi; Sasaki, Keiichi

    2017-01-01

    Nanoparticles represent promising gene delivery systems in biomedicine to facilitate prolonged gene expression with low toxicity compared to viral vectors. Specifically, nanoparticles of calcium phosphate (nCaP), the main inorganic component of human bone, exhibit high biocompatibility and good biodegradability and have been reported to have high affinity for protein or DNA, having thus been used as gene transfer vectors. On the other hand, Octa-arginine (R8), which has a high permeability to cell membrane, has been reported to improve intracellular delivery systems. Here, we present an optimized method for nCaP-mediated gene delivery using an octa-arginine (R8)-functionalized nCaP vector containing a marker or functional gene construct. nCaP particle size was between 220–580 nm in diameter and all R8-functionalized nCaPs carried a positive charge. R8 concentration significantly improved nCaP transfection efficiency with high cell compatibility in human mesenchymal stem cells (hMSC) and human osteoblasts (hOB) in particular, suggesting nCaPs as a good option for non-viral vector gene delivery. Furthermore, pre-treatment with different endocytosis inhibitors identified that the endocytic pathway differed among cell lines and functionalized nanoparticles, with amiloride increasing transfection efficiency of R8-functionalized nCaPs in hMSC and hOB. PMID:29145481

  9. Membrane events and ionic processes involved in dopamine release from tuberoinfundibular neurons. II. Effect of the inhibition of the Na+-Ca++ exchange by amiloride

    International Nuclear Information System (INIS)

    Taglialatela, M.; Amoroso, S.; Canzoniero, L.M.; Di Renzo, G.F.; Annunziato, L.

    1988-01-01

    In the present study we investigated the effect of amiloride, a rather specific inhibitor of the membrane Na+-Ca++ exchange system, on the release of endogenous dopamine (DA) and previously taken-up [3H]DA from tuberoinfundibular dopaminergic neurons. Amiloride (300 microM) stimulated either endogenous DA or [3H]DA release. Amiloride-induced stimulation of [3H]DA release was prevented in a Ca++-free plus ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid medium. Amiloride, at the same concentration, reinforced both high K+- and electrically-induced stimulation of [3H]DA release. These results are explained on the basis of the ability of amiloride in blocking the Na+-Ca++ exchange system, therefore causing an elevation of intracellular Ca++ levels in resting conditions, and a further accumulation of Ca++ ions after high K+- or electrically elicited opening of voltage-operated channels specific for Ca++ ions. The enhanced intracellular Ca++ availability may trigger the stimulation of neurotransmitter release. In addition, amiloride was able to block in a dose-dependent manner (70-300 microM) the ouabain-induced [3H]DA release, suggesting that, when intracellular concentrations of Na+ are increased by the blockade of Na+,K+-adenosine triphosphatase the Na+-Ca+;+ exchange carrier reverses its resting mode of operation, mediating the influx of extracellular Ca++ ions. Amiloride, by blocking the Na+-Ca++ exchange mechanism, prevents the ouabain-elicited entrance of extracellular Ca++ ions, therefore inhibiting [3H]DA release stimulated by the cardioactive glycoside. Collectively, the results of the present study seem to be compatible with the idea that the Na+-Ca++ exchange mechanism is involved in the regulation of [3H]DA release from tuberoinfundibular dopaminergic neurons, through the regulation of Ca++ movements across the plasma membrane

  10. Regulación por proteasas del canal de sodio sensible al amiloride (ENaC

    Directory of Open Access Journals (Sweden)

    Luciano Galizia

    2011-04-01

    Full Text Available El ENaC es un canal que permite el movimiento de Na+ desde el líquido luminal hacia las células en numerosos epitelios reabsortivos y también en otros tejidos como la placenta. ENaC juega un papel crucial en la homeostasis de los electrolitos y volumen de líquido extracelular. Es regulado por numerosas hormonas, incluyendo la aldosterona y bloqueado por el diurético amiloride. El ENaC está formado por tres subunidades homólogas α, β y γ que forman el poro por el cual se mueven los iones Na+. Dos factores regulan la actividad del ENaC. 1 el número de canales insertos en la membrana celular y 2 la probabilidad de apertura o tiempo en que se encuentra abierto el canal. El número de canales es el resultado de un balance entre su síntesis y degradación. La probabilidad de apertura depende de la proteólisis de zonas específicas de las subunidades α y γ por múltiples proteasas dentro de la célula y en el espacio extracelular. Entre las proteasas más estudiadas se encuentran la furina, prostasina, elastasa, plasmina y tripsina. Existen sustancias endógenas que bloquean la actividad de estas proteasas como la aprotinina, la bikunina y la nexina-1 y la expresión de las proteasas y sus inhibidores es regulada a su vez por la aldosterona, la tasa de movimiento de Na y el TFGβ. En este trabajo presentamos algunos ejemplos de esta regulación y su potencial papel en condiciones normales y en ciertas enfermedades como la fibrosis quística, renales e hipertensión.

  11. Amiloride interferes with platelet- activating factor-induced respiratory burst and MMP-9 release in bovine neutrophils independent of Na+/H+ exchanger 1.

    Science.gov (United States)

    Larrazabal, C S; Carretta, M D; Hidalgo, M A; Burgos, R A

    2017-09-01

    Cytoplasmic pH homeostasis is required for an appropriate response in polymorphonuclear neutrophils (PMNs). In these cells, chemotaxis and reactive oxygen species (ROS) production are reduced by the use of Na + /H + exchanger (NHE-1) inhibitors, but these results are mainly obtained using amiloride, a non-selective NHE-1 inhibitor. In bovine PMNs, the role of NHE-1 in functional responses has not been confirmed yet. The aim of this study was to determine the role of NHE-1 using amiloride and zoniporide in pH regulation, ROS production, matrix metalloproteinase 9 (MMP-9) release and calcium flux in bovine PMNs induced by the platelet activation factor (PAF), additionally we evaluated the presence of NHE-1 and NHE-2 mRNA Our data show the presence only of NHE-1 but not NHE-2 in bovine PMNs. Amiloride or zoniporide inhibited the intracellular alkalization induced by PAF without affecting calcium flux. Amiloride diminished ROS production and MMP-9 release, while zoniporide enhanced ROS production without change the MMP-9 release induced by PAF. Our work led us to conclude that changes in intracellular pH induced by PAF are regulated by NHE-1 in bovine neutrophils, but the effects of amiloride on ROS production and MMP-9 release induced by PAF are not NHE-1 dependent. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. TRPM5-dependent amiloride- and benzamil-insensitive NaCl chorda tympani taste nerve response.

    Science.gov (United States)

    Ren, ZuoJun; Rhyu, Mee-Ra; Phan, Tam-Hao T; Mummalaneni, Shobha; Murthy, Karnam S; Grider, John R; DeSimone, John A; Lyall, Vijay

    2013-07-01

    Transient receptor potential (TRP) subfamily M member 5 (TRPM5) cation channel is involved in sensing sweet, bitter, umami, and fat taste stimuli, complex-tasting divalent salts, and temperature-induced changes in sweet taste. To investigate if the amiloride- and benzamil (Bz)-insensitive NaCl chorda tympani (CT) taste nerve response is also regulated in part by TRPM5, CT responses to 100 mM NaCl + 5 μM Bz (NaCl + Bz) were monitored in Sprague-Dawley rats, wild-type (WT) mice, and TRP vanilloid subfamily member 1 (TRPV1) and TRPM5 knockout (KO) mice in the presence of resiniferatoxin (RTX), a TRPV1 agonist. In rats, NaCl + Bz + RTX CT responses were also monitored in the presence of triphenylphosphine oxide, a specific TRPM5 blocker, and capsazepine and N-(3-methoxyphenyl)-4-chlorocinnamid (SB-366791), specific TRPV1 blockers. In rats and WT mice, RTX produced biphasic effects on the NaCl + Bz CT response, enhancing the response at 0.5-1 μM and inhibiting it at >1 μM. The NaCl + Bz + SB-366791 CT response in rats and WT mice and the NaCl + Bz CT response in TRPV1 KO mice were inhibited to baseline level and were RTX-insensitive. In rats, blocking TRPV1 by capsazepine or TRPM5 by triphenylphosphine oxide inhibited the tonic NaCl + Bz CT response and shifted the relationship between RTX concentration and the magnitude of the tonic CT response to higher RTX concentrations. TRPM5 KO mice elicited no constitutive NaCl + Bz tonic CT response. The relationship between RTX concentration and the magnitude of the tonic NaCl + Bz CT response was significantly attenuated and shifted to higher RTX concentrations. The results suggest that pharmacological or genetic alteration of TRPM5 activity modulates the Bz-insensitive NaCl CT response and its modulation by TRPV1 agonists.

  13. Frog ANP increases the amiloride-sensitive Na(+) channel activity in urinary bladder cells of Japanese tree frog, Hyla japonica.

    Science.gov (United States)

    Yamada, Toshiki; Matsuda, Kouhei; Uchiyama, Minoru

    2007-01-01

    We examined the mechanism of amiloride-sensitive Na(+) channels (ENaC) activated by fANP in epithelial cells of frog urinary bladder by using a cell-attached patch-clamp technique. ENaC activities in the epithelial cells were significantly increased following administration of both 10(-9)M fANP and 10(-5)M 8-Br-cGMP. Both fANP and 8-Br-cGMP, however, failed to activate the ENaC in the presence of 10(-6)M amiloride. In addition, 8-Br-cGMP failed to activate the ENaC in the presence of a PKA inhibitor, KT-5720. In the next experiment, we measured both cGMP and cAMP production levels after treatment of fANP on the frog urinary bladder cells. Frog ANP significantly increased cGMP production, but not the cAMP production. Taken together, these results suggest that fANP activates ENaC through increases in cGMP production and activation of PKA.

  14. A Localized Surface Plasmon Resonance Sensing Method for Simultaneous Determination of Atenolol and Amiloride in Pharmaceutical Dosage Forms and Urine Samples

    Directory of Open Access Journals (Sweden)

    Marwa R. El-Zahry

    2018-01-01

    Full Text Available This contribution describes a simple, fast, and sensitive application of localized surface plasmon resonance effect of silver nanoparticles for simultaneous determination of antihypertensive drugs’ mixture atenolol and amiloride in both pharmaceutical dosage forms and in biological samples (urine. Silver nanoparticles were prepared by chemical reduction of silver nitrate using hydroxylamine HCL in an alkaline medium. Application of silver-hydroxylamine nanoparticles (SH NPs provides many advantages including reproducibility, sensitivity, and cost effective way of analyte determination. Amiloride has four amino groups which act as attachment points on the surface of silver nanoparticles resulting in a synergistic effect on the absorption intensity of atenolol, leading to increase the sensitivity of the determination of both compounds. This method shows excellent advantages comparing with the previously reported methods, including accuracy, precision, and selectivity. The linear range of atenolol is 1 × 10−5–1 × 10−4 mol·L−1 and of amiloride is 1 × 10−6–1 × 10−5 mol·L−1. The limit of detection (LOD values of atenolol and amiloride are 0.89 × 10−5 and 0.42 × 10−6 mol·L−1, respectively.

  15. Assignment of the human amiloride-sensitive Na{sup +} channel {delta} isoform to chromosome 1p36.3-p36.2

    Energy Technology Data Exchange (ETDEWEB)

    Waldmann, R.; Bassilana, F.; Voilley, N. [Institut de Pharmacologie Moleculaire et Cellulaire, Valbonne (France)] [and others

    1996-06-01

    This report describes the localization of the human amiloride-sensitive Na{sup +} channel {delta} isoform to human chromosome 1p36.3-p36.2 using in situ hybridization. Mutations in this group of ion channels have been implicated in various hereditary diseases. 18 refs., 1 fig.

  16. A genome-wide study of panic disorder suggests the amiloride-sensitive cation channel 1 as a candidate gene

    DEFF Research Database (Denmark)

    Gregersen, Noomi; Dahl, Hans A.; Buttenschön, Henriette N.

    2012-01-01

    Panic disorder (PD) is a mental disorder with recurrent panic attacks that occur spontaneously and are not associated to any particular object or situation. There is no consensus on what causes PD. However, it is recognized that PD is influenced by environmental factors, as well as genetic factors....... Despite a significant hereditary component, genetic studies have only been modestly successful in identifying genes of importance for the development of PD. In this study, we conducted a genome-wide scan using microsatellite markers and PD patients and control individuals from the isolated population...... of the Faroe Islands. Subsequently, we conducted a fine mapping, which revealed the amiloride-sensitive cation channel 1 (ACCN1) located on chromosome 17q11.2-q12 as a potential candidate gene for PD. The further analyses of the ACCN1 gene using single-nucleotide polymorphisms (SNPs) revealed significant...

  17. A study of pH-dependent photodegradation of amiloride by a multivariate curve resolution approach to combined kinetic and acid-base titration UV data.

    Science.gov (United States)

    De Luca, Michele; Ioele, Giuseppina; Mas, Sílvia; Tauler, Romà; Ragno, Gaetano

    2012-11-21

    Amiloride photostability at different pH values was studied in depth by applying Multivariate Curve Resolution Alternating Least Squares (MCR-ALS) to the UV spectrophotometric data from drug solutions exposed to stressing irradiation. Resolution of all degradation photoproducts was possible by simultaneous spectrophotometric analysis of kinetic photodegradation and acid-base titration experiments. Amiloride photodegradation showed to be strongly dependent on pH. Two hard modelling constraints were sequentially used in MCR-ALS for the unambiguous resolution of all the species involved in the photodegradation process. An amiloride acid-base system was defined by using the equilibrium constraint, and the photodegradation pathway was modelled taking into account the kinetic constraint. The simultaneous analysis of photodegradation and titration experiments revealed the presence of eight different species, which were differently distributed according to pH and time. Concentration profiles of all the species as well as their pure spectra were resolved and kinetic rate constants were estimated. The values of rate constants changed with pH and under alkaline conditions the degradation pathway and photoproducts also changed. These results were compared to those obtained by LC-MS analysis from drug photodegradation experiments. MS analysis allowed the identification of up to five species and showed the simultaneous presence of more than one acid-base equilibrium.

  18. A new member of the amiloride-sensitive sodium channel family in Drosophila melanogaster peripheral nervous system.

    Science.gov (United States)

    Darboux, I; Lingueglia, E; Pauron, D; Barbry, P; Lazdunski, M

    1998-05-08

    Amiloride sensitivity is a common characteristic of structurally related cationic channels that are associated with a wide range of physiological functions. In Caenorhabditis elegans, neuronal and muscular degenerins are involved in mechanoperception. In animal epithelia, a Na(+)-selective channel participates in vectorial Na+ transport. In the snail nervous system, an ionotropic receptor for the peptide FMRFamide forms a Na(+)-selective channel. In mammalian brain and/or in sensory neurons, ASIC channels form H(+)-activated cation channels involved in nociception linked to acidosis. We have now cloned a new member of this family from Drosophila melanogaster. The corresponding protein displays low sequence identity with the previously cloned members of the super-family but it has the same structural organization. Its mRNA was detected from late embryogenesis (14-17 hours) and was present in the dendritic arbor subtype of the Drosophila peripheral nervous system multiple dendritic (md) sensory neurons. While the origin and specification of md neurons are well documented, their roles are still poorly understood. They could function as stretch or touch receptors, raising the possibility that this Drosophila gene product, called dmdNaC1, could also be involved in mechanotransduction.

  19. Frog atrial natriuretic peptide and cGMP activate amiloride-sensitive Na(+) channels in urinary bladder cells of Japanese tree frog, Hyla japonica.

    Science.gov (United States)

    Yamada, Toshiki; Konno, Norifumi; Matsuda, Kouhei; Uchiyama, Minoru

    2007-07-01

    In our previous study, it was suggested that ANP and cGMP may increase Na(+) absorption in the urinary bladder of the Japanese tree frog, Hyla japonica. Thus, Na(+) transport activated by ANP was investigated electrophysiologically by using a cell-attached patch-clamp technique in freshly isolated cells from the urinary bladder. A predominant channel expressed was a low conductance Na(+) channel in the epithelial cells. The channel exhibited conductance for inward currents of 4.9 +/- 0.2 pS, long open and closed times (c.a. 190 ms), and positive reversal potential. The channel activity was decreased under the pipette solution including 10(-6) M amiloride. These characteristics were similar to those of amiloride-sensitive Na(+) channels (ENaC). Addition of 10(-9) M ANP activated and significantly increased the ENaC activity from 0.58 +/- 0.09 to 1.47 +/- 0.34. On the other hand, mean amplitudes and conductance of single channel did not change significantly after the addition of ANP. Addition of 10(-5) M 8-Br-cGMP also activated the ENaC and significantly increased the channel activity from 0.56 +/- 0.10 to 2.00 +/- 0.33. The addition of ANP failed to activate the ENaC in the presence of 10(-6) M amiloride. These results suggested that ANP and cGMP activate Na(+) transport via ENaC in the epithelial cells of frog urinary bladder.

  20. Changes in selected metabolic parameters in patients over 65 receiving hydrochlorothiazide plus amiloride, atenolol or placebo in the MRC elderly trial.

    Science.gov (United States)

    Damian, Damian J; McNamee, Roseanne; Carr, Matthew

    2016-10-04

    Treatment of hypertension reduces incidence of stroke, myocardial infarction and heart failure perhaps partly by controlling different metabolic parameters. There is limited information regarding the changes in potassium, sodium, weight, cholesterol and glucose levels in patients using anti-hypertensives. This study aimed to determine changes in potassium, sodium, glucose, cholesterol, weight, urea and urate levels in patients using anti-hypertensives. Furthermore, to describe these changes and differences between the atenolol, hydrochlorothiazide plus amiloride and placebo arms of the Medical Research Council (MRC) elderly randomised controlled trial. Patients were randomly allocated to one of the three treatment arms. Measurements were taken at baseline, end of year one and end of year two in 4396 subjects. Linear Mixed Models (LMM) were used to determine the longitudinal profiles of sodium, potassium, weight, cholesterol, glucose, urea and urate. Estimates of changes within groups and difference between groups were obtained. Patients randomised to receive hydrochlorothiazide + amiloride experienced a significantly greater mean reduction in potassium, sodium and weight compared to placebo at end of year one - mean differences in change -0.18 mmol/L, (95 % CI: -0.21, -0.15); -1.45 mmol/L, (95 % CI: -1.62, -1.29) and -0.46 kgs (95 % CI: -0.73, -0.20) respectively, and greater increases in cholesterol, urea and urate - mean differences in change 0.16 mmol/L, (95 % CI: 0.10,0.22); 0.77 mmol/L, (95 % CI: 0.68, 0.87) and 53.10 μmol/L, (95 % CI: 49.35, 56.85) respectively. Changes were in the same direction but smaller in the atenololarm except for potassium and weight (increases). No group differences in glucose were found. Results were in line with expectation except for lack of change in glucose in the hydrochlorothiazide + amiloride arms.

  1. Amiloride and Hydrochlorothiazide

    Science.gov (United States)

    ... in a class of medications called diuretics ('water pills'). They work by causing the kidneys to get ... skip the missed dose and continue your regular dosing schedule. Do not take a double dose to ...

  2. Amiloride but not memantine reduces neurodegeneration, seizures and myoclonic jerks in rats with cardiac arrest-induced global cerebral hypoxia and reperfusion.

    Science.gov (United States)

    Tai, Kwok Keung; Truong, Daniel D

    2013-01-01

    It has been reported that both activation of N-methyl-D-aspartate receptors and acid-sensing ion channels during cerebral ischemic insult contributed to brain injury. But which of these two molecular targets plays a more pivotal role in hypoxia-induced brain injury during ischemia is not known. In this study, the neuroprotective effects of an acid-sensing cation channel blocker and an N-methyl-D-aspartate receptor blocker were evaluated in a rat model of cardiac arrest-induced cerebral hypoxia. We found that intracisternal injection of amiloride, an acid-sensing ion channel blocker, dose-dependently reduced cerebral hypoxia-induced neurodegeneration, seizures, and audiogenic myoclonic jerks. In contrast, intracisternal injection of memantine, a selective uncompetitive N-methyl-D-aspartate receptor blocker, had no significant effect on cerebral hypoxia-induced neurodegeneration, seizure and audiogenic myoclonic jerks. Intracisternal injection of zoniporide, a specific sodium-hydrogen exchanger inhibitor, before cardiac arrest-induced cerebral hypoxia, also did not reduce cerebral hypoxia-induced neurodegeneration, seizures and myoclonic jerks. These results suggest that acid-sensing ion channels play a more pivotal role than N-methyl-D-aspartate receptors in mediating cerebral hypoxia-induced brain injury during ischemic insult.

  3. Amiloride but not memantine reduces neurodegeneration, seizures and myoclonic jerks in rats with cardiac arrest-induced global cerebral hypoxia and reperfusion.

    Directory of Open Access Journals (Sweden)

    Kwok Keung Tai

    Full Text Available It has been reported that both activation of N-methyl-D-aspartate receptors and acid-sensing ion channels during cerebral ischemic insult contributed to brain injury. But which of these two molecular targets plays a more pivotal role in hypoxia-induced brain injury during ischemia is not known. In this study, the neuroprotective effects of an acid-sensing cation channel blocker and an N-methyl-D-aspartate receptor blocker were evaluated in a rat model of cardiac arrest-induced cerebral hypoxia. We found that intracisternal injection of amiloride, an acid-sensing ion channel blocker, dose-dependently reduced cerebral hypoxia-induced neurodegeneration, seizures, and audiogenic myoclonic jerks. In contrast, intracisternal injection of memantine, a selective uncompetitive N-methyl-D-aspartate receptor blocker, had no significant effect on cerebral hypoxia-induced neurodegeneration, seizure and audiogenic myoclonic jerks. Intracisternal injection of zoniporide, a specific sodium-hydrogen exchanger inhibitor, before cardiac arrest-induced cerebral hypoxia, also did not reduce cerebral hypoxia-induced neurodegeneration, seizures and myoclonic jerks. These results suggest that acid-sensing ion channels play a more pivotal role than N-methyl-D-aspartate receptors in mediating cerebral hypoxia-induced brain injury during ischemic insult.

  4. In Acute IgA Nephropathy, Proteinuria and Creatinine Are in the Spot, but Podocyturia Operates in Silence: Any Place for Amiloride?

    Directory of Open Access Journals (Sweden)

    H. Trimarchi

    2017-01-01

    Full Text Available IgA nephropathy is the most frequent cause of primary glomerulonephritis, portends erratic patterns of clinical presentation, and lacks specific treatment. In general, it slowly progresses to end-stage renal disease. The clinical course and the response to therapy are usually assessed with proteinuria and serum creatinine. Validated biomarkers have not been identified yet. In this report, we present a case of acute renal injury with proteinuria and microscopic hematuria in a young male. A kidney biopsy disclosed IgA nephropathy. Podocyturia was significantly elevated compared to normal subjects. Proteinuria, renal function, and podocyturia improved promptly after steroids and these variables remained normal after one year of follow-up, when steroids had already been discontinued and patient continued on valsartan and amiloride. Our report demonstrates that podocyturia is critically elevated during an acute episode of IgA nephropathy, and its occurrence may explain the grim long-term prognosis of this entity. Whether podocyturia could be employed in IgA nephropathy as a trustable biomarker for treatment assessment or even for early diagnosis of IgA nephropathy relapses should be further investigated.

  5. Severe hyponatraemia in an amiloride/hydrochlorothiazide-treated patient

    NARCIS (Netherlands)

    Van Assen, S.; Mudde, A.H.

    1999-01-01

    A 85-year-old woman treated with, among other drugs, a thiazide diuretic presented with a severe hyponatraemia. She met several of the criteria for SIADH and, besides drugs, no cause for SIADH was found. After stopping the thiazide diuretic and restricting fluid intake the patient recovered fully.

  6. Untersuchungen zur Farbreaktion von Amilorid, Chlorhexidin und Proguanil mit Hypobromit

    OpenAIRE

    Huth, Silke

    2004-01-01

    Bei der Reaktion von Amiloridhydrochlorid (1· HCl) mit Brom in alkalischer Lösung wird ein gelbbraunes Dehydrierungsprodukt erhalten, daß als 3-(3-Amino-1,2,4-oxadiazol-5-yl)-5-chlor-2,6-pyrazindiamin (6) identifiziert werden konnte. Durch Vergleich von Massen- und NMR-Spektren der Verbindung 6 mit Spektren von 1· HCl und Referenzsubstanzen I-III konnte auf die verbindung 6 geschlossen werden. Die Struktur wurde durch eine Röntgenstrukturanalyse abgesichert. Erhitzen von Amiloridhydrochlorid ...

  7. Amiloride (Am) dissociates human neutrophil (N) activation events

    International Nuclear Information System (INIS)

    Berkow, R.L.; Dodson, R.; Kraft, A.S.

    1986-01-01

    Human N can be stimulated to release granule contents and superoxide anion (O/sub 2 - /). These events are associated with an Am sensitive Na + /H + exchange and N alkalinization. Am has been reported to inhibit protein kinase C (PKC) in HL-60 cells. Due to the central role of PKC in N activation they assessed the effect of prolonged exposure of N to Am. When N were treated with 10 -6 to 10 -3 M Am at 37 0 C for 15 min a dose dependent inhibition of O/sub 2 - / release was seen upon N stimulation with FMLP (10 -6 M), A23187 (10 -5 M), or serum treated Zymosan (Z) (2.5 mg/ml). Maximal inhibition depended on the time of exposure of N to Am prior to stimulation and remained after removal of Am by washing. N treated with 10 -3 M Am had a decreased influx of 45 Ca ++ upon stimulation with FMLP. Phorbol myristate acetate induced release of N O/sub 2 - / was unaffected by pretreatment with Am. Similarly, Am did not inhibit stimulated N lysozyme release or the incorporation of 32 P into proteins. Monensin (a Na + /H + ionophore) did not correct the Am induced inhibition of O/sub 2 - / suggesting that cell acidification alone can not explain the Am effect. In conclusion: (1) Na + /H + exchange modulates N O/sub 2 - / release upon stimulation with FMLP, A23187, and Z. PMA induced N responses are not affected by cell acidification; (2) N granule release is under separate cellular control than O/sub 2 - /; (3) Am does not inhibit PKC or protein phosphorylation in N; and (4) decreased 45 Ca ++ influx may partially explain the Am effect on FMLP induced O/sub 2 - / release

  8. Drug: D10271 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available D10271 Mixture ... Drug Amiloride hydrochloride - hydrochlorothiazide mixt; Amiloride hydrochloride and hydro...chlorothiazide (TN) Amiloride hydrochloride [DR:D00649], Hydrochlorothiazide [DR:D00340] ... ATC code: C03EA01 ... PubChem: 163312302 ...

  9. Amiloride but Not Memantine Reduces Neurodegeneration, Seizures and Myoclonic Jerks in Rats with Cardiac Arrest-Induced Global Cerebral Hypoxia and Reperfusion

    OpenAIRE

    Tai, Kwok Keung; Truong, Daniel D.

    2013-01-01

    It has been reported that both activation of N-methyl-D-aspartate receptors and acid-sensing ion channels during cerebral ischemic insult contributed to brain injury. But which of these two molecular targets plays a more pivotal role in hypoxia-induced brain injury during ischemia is not known. In this study, the neuroprotective effects of an acid-sensing cation channel blocker and an N-methyl-D-aspartate receptor blocker were evaluated in a rat model of cardiac arrest-induced cerebral hypoxi...

  10. Fluid absorption related to ion transport in human airway epithelial spheroids

    DEFF Research Database (Denmark)

    Pedersen, P S; Holstein-Rathlou, N H; Larsen, P L

    1999-01-01

    in non-CF than in CF spheroids, 3) Cl(-)-channel inhibitors increased fluid absorption in amiloride-treated non-CF spheroids to a level equal to that of amiloride-treated CF spheroids, 4) hydrochlorothiazide reduced the amiloride-insensitive fluid absorption in both non-CF and CF spheroids, and 5...

  11. Structural changes and effect of denopamine on alveolar fluid ...

    African Journals Online (AJOL)

    GREGORY

    2010-09-13

    Sep 13, 2010 ... The epithelial cellular structure of control rat. (×12000). stimulatory effects of terbutaline on AFC (Gu et al., 2001). To determine whether hypoxia altered the effects of denopamine on amiloride-sensitive and amiloride- insensitive sodium channels, an isomolar albumin solution in the presence of 10-5 mol/L ...

  12. Plasmin in Nephrotic Urine Activates the Epithelial Sodium Channel

    DEFF Research Database (Denmark)

    Svenningsen, Per; Bistrup, Claus; Friis, Ulla G

    2009-01-01

    stimulated amiloride-sensitive transepithelial sodium transport in M-1 cells and increased amiloride-sensitive whole-cell currents in Xenopus laevis oocytes heterologously expressing ENaC. Activation of ENaC by plasmin involved cleavage and release of an inhibitory peptide from the ENaC gamma subunit...

  13. Ion exchange mechanisms on the erythrocyte membrane of the aquatic salamander, Amphiuma tridactylum

    DEFF Research Database (Denmark)

    Tufts, B L; Nikinmaa, M; Steffensen, J F

    1987-01-01

    that could be abolished by amiloride, ouabain or removal of sodium from the incubation medium. In addition, amiloride and DIDS both caused a decrease in cell water content. The data indicate that sodium/proton and chloride/bicarbonate exchangers are present on the membrane of Amphiuma erythrocytes...

  14. Fluid absorption related to ion transport in human airway epithelial spheroids

    DEFF Research Database (Denmark)

    Pedersen, P S; Holstein-Rathlou, N H; Larsen, P L

    1999-01-01

    , and amiloride inhibited both values. Fluid transport rates were calculated from repeated measurements of spheroid diameters. The results showed that 1) non-CF and CF spheroids absorbed fluid at identical rates (4.4 microl x cm(-2) x h(-1)), 2) amiloride inhibited fluid absorption to a lower residual level...... in non-CF than in CF spheroids, 3) Cl(-)-channel inhibitors increased fluid absorption in amiloride-treated non-CF spheroids to a level equal to that of amiloride-treated CF spheroids, 4) hydrochlorothiazide reduced the amiloride-insensitive fluid absorption in both non-CF and CF spheroids, and 5......) osmotic water permeabilities were equal in non-CF and CF spheroids ( approximately 27 x 10(-7) cm x s(-1) x atm(-1))....

  15. Comparison of ion transport by cultured secretory and absorptive canine airway epithelia

    DEFF Research Database (Denmark)

    Boucher, R C; Larsen, Erik Hviid

    1988-01-01

    preparation exhibited an equivalent short-circuit circuit (Ieq) that was sensitive to inhibitors of Cl- transport (bumetanide, diphenylamine carboxylic acid) but was insensitive to an inhibitor of Na+ transport, amiloride. In contrast, the bronchial preparation, like the native tissue, exhibited an Ieq...... sensitive to amiloride but insensitive to bumetanide. As compared with the trachea, the bronchial (absorptive) epithelium is characterized by 1) a large amiloride-sensitive cellular conductance and 2) a relatively depolarized basolateral membrane. We conclude that this primary cell culture technique...... provides epithelial preparations comparable to the native tissue and suitable for quantitative studies of regional differences in ion transport function....

  16. Potent synergistic in vitro interaction between nonantimicrobial membrane-active compounds and itraconazole against clinical isolates of Aspergillus fumigatus resistant to itraconazole.

    NARCIS (Netherlands)

    Afeltra, J.; Vitale, R.G.; Mouton, J.W.; Verweij, P.E.

    2004-01-01

    To develop new approaches for the treatment of invasive infections caused by Aspergillus fumigatus, the in vitro interactions between itraconazole (ITZ) and seven different nonantimicrobial membrane-active compounds--amiodarone (AMD), amiloride, lidocaine, lansoprazole (LAN), nifedipine (NIF),

  17. Modulation of KCNQ1 alternative splicing regulates cardiac IKs and action potential repolarization.

    Science.gov (United States)

    Lee, Hsiang-Chun; Rudy, Yoram; Po-Yuan, Phd; Sheu, Sheng-Hsiung; Chang, Jan-Gowth; Cui, Jianmin

    2013-08-01

    Slow delayed-rectifier potassium current (IKs) channels, made of the pore-forming KCNQ1 and auxiliary KCNE1 subunits, play a key role in determining action potential duration (APD) in cardiac myocytes. The consequences of drug-induced KCNQ1 splice alteration remain unknown. To study the modulation of KCNQ1 alternative splicing by amiloride and the consequent changes in IKs and action potentials (APs) in ventricular myocytes. Canine endocardial, midmyocardial, and epicardial ventricular myocytes were isolated. Levels of KCNQ1a and KCNQ1b as well as a series of splicing factors were quantified by using the reverse transcriptase-polymerase chain reaction and Western blot. The effect of amiloride-induced changes in the KCNQ1b/total KCNQ1 ratio on AP was measured by using whole-cell patch clamp with and without isoproterenol. With 50 μmol/L of amiloride for 6 hours, KCNQ1a at transcriptional and translational levels increased in midmyocardial myocytes but decreased in endo- and epicardial myocytes. Likewise, changes in splicing factors in midmyocardial were opposite to that in endo- and epicardial myocytes. In midmyocardial myocytes amiloride shortened APD and decreased isoproterenol-induced early afterdepolarizations significantly. The same amiloride-induced effects were demonstrated by using human ventricular myocyte model for AP simulations under beta-adrenergic stimulation. Moreover, amiloride reduced the transmural dispersion of repolarization in pseudo-electrocardiogram. Amiloride regulates IKs and APs with transmural differences and reduces arrhythmogenicity through the modulation of KCNQ1 splicing. We suggested that the modulation of KCNQ1 splicing may help prevent arrhythmia. Copyright © 2013 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

  18. Glutaraldehyde fixation of sodium transport in dog red blood cells

    International Nuclear Information System (INIS)

    Parker, J.C.

    1984-01-01

    The large increase in passive Na flux that occurs when dog red blood cells are caused to shrink is amiloride sensitive and inhibited when Cl is replaced by nitrate or thiocyanate. Activation and deactivation of this transport pathway by manipulation of cell volume is reversible. Brief treatment of the cells with 0.01-0.03% glutaraldehyde can cause the shrinkage-activated transporter to become irreversibly activated or inactivated, depending on the volume of the cells at the time of glutaraldehyde exposure. Thus, if glutaraldehyde is applied when the cells are shrunken, the amiloride-sensitive Na transporter is activated and remains so regardless of subsequent alterations in cell volume. If the fixative is applied to swollen cells, no amount of subsequent shrinkage will turn on the Na pathway. In its fixed state, the activated transporter is fully amiloride sensitive, but it is no longer inhibited when Cl is replaced by thiocyanate. The action of glutaraldehyde thus allows one to dissect the response to cell shrinkage into two phases. Activation of the pathway is affected by anions and is not prevented by amiloride. Once activated and fixed, the anion requirement disappears. Amiloride inhibits movement of Na through the activated transporter. These experiments demonstrate how a chemical cross-linking agent may be used to study the functional properties of a regulable transport pathway

  19. Aldosterone induction of electrogenic sodium transport in the apical membrane vesicles of rat distal colon

    International Nuclear Information System (INIS)

    Rajendran, V.M.; Kashgarian, M.; Binder, H.J.

    1989-01-01

    Na-H exchange is present in apical membrane vesicles (AMV) isolated from distal colon of normal rats. Because in intact tissue aldosterone both induces amiloride-sensitive electrogenic sodium transport and inhibits electroneutral sodium absorption, these studies with AMV were designed to establish the effect of aldosterone on sodium transport. An outward-directed proton gradient stimulated 22Na uptake in AMV isolated from distal colon of normal and dietary sodium depleted (with elevated aldosterone levels) experimental rats. Unlike normal AMV, proton gradient-dependent 22Na uptake in experimental AMV was inhibited when uptake was measured under voltage-clamped conditions. 10 microM amiloride inhibited the initial rate of proton gradient-dependent 22Na uptake in AMV of normal and experimental rats by 30 and 75%, respectively. In contrast, 1 mM amiloride produced comparable inhibition (90 and 80%) of 22Na uptake in normal and experimental AMV. Intravesicular-negative potential stimulated 22Na uptake in experimental but not in normal AMV. This increase was inhibited by 90% by 10 microM amiloride. An analogue of amiloride, 5-(N-ethylisopropyl) amiloride (1 microM), a potent inhibitor of electroneutral Na-H exchange in AMV of normal rat distal colon, did not alter potassium diffusion potential-dependent 22Na uptake. Increasing sodium concentration saturated proton gradient-dependent 22Na uptake in normal AMV. However, in experimental AMV, 22Na uptake stimulated by both proton gradient and potassium diffusion potential did not saturate as a function of increasing sodium concentration. We conclude from these results that an electrically sensitive conductive channel, not electroneutral Na-H exchange, mediates 22Na uptake in AMV isolated from the distal colon of aldosterone rats

  20. Urinary serine proteases and activation of ENaC in kidney-implications for physiological renal salt handling and hypertensive disorders with albuminuria

    DEFF Research Database (Denmark)

    Svenningsen, Per; Andersen, Henrik; Nielsen, Lise H

    2015-01-01

    and diabetic nephropathy; blood pressure is salt-sensitive in conditions with microalbuminuria/proteinuria; and extracellular volume is expanded, plasma atrial natriuretic peptide (ANP) concentration is increased, and diuretics, like amiloride and spironolactone, are effective blood pressure-reducing add......-ons. Active plasmin in urine has been demonstrated in diabetes, preeclampsia, and nephrosis. Urine from these patients activates, plasmin-dependently, amiloride-sensitive inward current in vitro. The concept predicts that patients with albuminuria may benefit particularly from reduced salt intake with RAS...

  1. UniProt search blastx result: AK289104 [KOME

    Lifescience Database Archive (English)

    Full Text Available ensing ion channel 3) (ASIC3) (hASIC3) (Testis sodium channel 1) (hTNaC1) - Homo sapiens (Human) 0 ... ...AK289104 J090096G16 Q9UHC3|ACCN3_HUMAN Amiloride-sensitive cation channel 3 (Acid-s

  2. Transport of ascorbic acid and dehydroascorbic acid by pancreatic islet cells from neonatal rats

    DEFF Research Database (Denmark)

    Zhou, A; Farver, O; Thorn, N A

    1991-01-01

    a saturable process (estimated Km 17.6 microM). Transport was inhibited by ouabain, phloridzin, cytochalasin B, amiloride and probenecid. Glucose inhibited or stimulated uptake, depending on the length of incubation time of the cells. The uptake of dehydroascorbic acid was linearly dependent on concentration...

  3. Expression of NHE1 and NHE4 in rat pancreatic zymogen granule membranes.

    Science.gov (United States)

    Anderie, I; Blum, R; Haase, W; Grinstein, S; Thévenod, F

    1998-05-19

    We previously characterized a Na+/H+ exchange activity in rat pancreatic zymogen granules [Anderie, I., and Thévenod, F. (1996) J. Membrane Biol, 152, 195-205]. Here we have identified the Na+/H+ exchanger (NHE) isoforms present in zymogen granules by functional studies with NHE inhibitors. The NHE1 specific blocker HOE 694 [3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate] inhibited zymogen granule Na+/H+ exchange in a concentration dependent manner, maximally to 53 +/- 5% of controls at 100nM. The remaining Na+/H+ exchange activity was inhibitable by EIPA [5-(N-ethyl-N-isopropyl)amiloride] (EC50 approximately 25 microM) or benzamil (EC50 approximately 100 microM). Amiloride inhibited weakly suggesting that "amiloride-resistant" and "amiloride-sensitive" NHE are expressed in zymogen granules. cDNA sequences encoding NHE1- and NHE4-specific transmembrane domains were detected by RT-PCR in rat pancreatic tissue and in the rat pancreatic acinar cell line AR4-2J. The presence of NHE1 and NHE4 in zymogen granule membranes was confirmed by immunoblots of zymogen granule membranes and by pre-embedding immunogold labeling of purified rat pancreatic zymogen granules with polyclonal NHE1 and NHE4 antibodies. Therefore, we propose that NHE1 and NHE4 are expressed in zymogen granule membranes of rat exocrine pancreas.

  4. Transcellular sodium transport in cultured cystic fibrosis human nasal epithelium

    DEFF Research Database (Denmark)

    Willumsen, Niels J.; Boucher, Richard C.

    1991-01-01

    Cystic fibrosis (CF) airway epithelia exhibit raised transepithelial Na+ transport rates, as determined by open-circuit isotope fluxes and estimates of the amiloride-sensitive equivalent short-circuit current (Ieq). To study the contribution of apical and basolateral membrane paths to raised Na+ ...

  5. Two independent anion transport systems in rabbit mandibular salivary glands

    DEFF Research Database (Denmark)

    Novak, I; Young, J A

    1986-01-01

    in the following sequence: Br = greater than Cl greater than I = greater than NO3 greater than isethionate. Furosemide, 1.0 and 0.1 mmol/l, inhibited Cl-supported secretion by 97-99% and 70-78%, respectively. SITS, 0.1 mmol/l, had no effect and amiloride, 1.0 mmol/l, caused a 55-65% inhibition. Addition of SITS...... to amiloride-treated glands produced no further effect. In glands perfused with Cl-free solutions, but containing 25 mM HCO3, amiloride, 1.0 mmol/l, inhibited secretion by 95% and methazolamide, 0.1 mmol/l, by 55%. In glands perfused with solutions containing both HCO3 and Cl, furosemide had smaller effects...... stimulated secretion by about 30%, but when infused in addition to furosemide (0.1 mmol/l), it inhibited by about 20%. Amiloride (1.0 mmol/l) caused no inhibition. The results suggest that there are at least three distinct carriers in the rabbit mandibular gland. One is a furosemide-sensitive Na-coupled Cl...

  6. West African Journal of Pharmacology and Drug Research - Vol 17 ...

    African Journals Online (AJOL)

    Effect of baseline lipid levels I: influence on plasma total cholesterol and triglyceride changes during treatment of hypertension with a combination of hydrochlorothiazide and amiloride [Moduretic] · EMAIL FULL TEXT EMAIL FULL TEXT DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT. J.E Ahneku, G.O Taylor, E.O ...

  7. Sequence Classification: 791090 [

    Lifescience Database Archive (English)

    Full Text Available resistant FLR-1, ion channel similar to amiloride-sensitive sodium channel, modulates defecation rhythm, pace of development and fluo...ride sensitivity (72.3 kD) (flr-1) || http://www.ncbi.nlm.nih.gov/protein/17568351 ...

  8. Transport of ascorbic acid and dehydroascorbic acid by pancreatic islet cells from neonatal rats

    DEFF Research Database (Denmark)

    Zhou, A; Farver, O; Thorn, N A

    1991-01-01

    a saturable process (estimated Km 17.6 microM). Transport was inhibited by ouabain, phloridzin, cytochalasin B, amiloride and probenecid. Glucose inhibited or stimulated uptake, depending on the length of incubation time of the cells. The uptake of dehydroascorbic acid was linearly dependent on concentration...... contained a fairly low concentration of iron but a high concentration of copper....

  9. The rise of [Na(+)] (i) during ischemia and reperfusion in the rat heart-underlying mechanisms.

    Science.gov (United States)

    Williams, Iwan A; Xiao, Xiao-hui; Ju, Yue-kun; Allen, David G

    2007-09-01

    Intracellular Na(+) concentration ([Na(+)](i)) rises in the heart during ischemia, and on reperfusion, there is a transient rise followed by a return toward control. These changes in [Na(+)](i) contribute to ischemic and reperfusion damage through their effects on Ca(2+) overload. Part of the rise of [Na(+)](i) during ischemia may be caused by increased activity of the cardiac Na(+)/H(+) exchanger (NHE1), activated by the ischemic rise in [H(+)](i). In support of this view, NHE1 inhibitors reduce the [Na(+)](i) rise during ischemia. Another possibility is that the rise of [Na(+)](i) during ischemia is caused by Na(+) influx through channels. We have reexamined these issues by use of two different NHE1 inhibitors, amiloride, and zoniporide, in addition to tetrodotoxin (TTX), which blocks voltage-sensitive Na(+) channels. All three drugs produced cardioprotection after ischemia, but amiloride (100 microM) and TTX (300 nM) prevented the rise in [Na(+)](i) during ischemia, whereas zoniporide (100 nM) did not. Both amiloride and zoniporide prevented the rise of [Na(+)](i) on reperfusion, whereas TTX was without effect. In an attempt to explain these differences, we measured the ability of the three drugs to block Na(+) currents. At the concentrations used, TTX reduced the transient Na(+) current (I (Na)) by 11 +/- 2% while amiloride and zoniporide were without effect. In contrast, TTX largely eliminated the persistent Na(+) current (I (Na,P)) and amiloride was equally effective, whereas zoniporide had a substantially smaller effect reducing I (Na,P) to 41 +/- 8%. These results suggest that part of the effect of NHE1 inhibitors on the [Na(+)](i) during ischemia is by blockade of I (Na,P). The fact that a low concentration of TTX eliminated the rise of [Na(+)](i) during ischemia suggests that I (Na,P) is a major source of Na(+) influx in this model of ischemia.

  10. Acetazolamide Attenuates Lithium-Induced Nephrogenic Diabetes Insipidus.

    Science.gov (United States)

    de Groot, Theun; Sinke, Anne P; Kortenoeven, Marleen L A; Alsady, Mohammad; Baumgarten, Ruben; Devuyst, Olivier; Loffing, Johannes; Wetzels, Jack F; Deen, Peter M T

    2016-07-01

    To reduce lithium-induced nephrogenic diabetes insipidus (lithium-NDI), patients with bipolar disorder are treated with thiazide and amiloride, which are thought to induce antidiuresis by a compensatory increase in prourine uptake in proximal tubules. However, thiazides induced antidiuresis and alkalinized the urine in lithium-NDI mice lacking the sodium-chloride cotransporter, suggesting that inhibition of carbonic anhydrases (CAs) confers the beneficial thiazide effect. Therefore, we tested the effect of the CA-specific blocker acetazolamide in lithium-NDI. In collecting duct (mpkCCD) cells, acetazolamide reduced the cellular lithium content and attenuated lithium-induced downregulation of aquaporin-2 through a mechanism different from that of amiloride. Treatment of lithium-NDI mice with acetazolamide or thiazide/amiloride induced similar antidiuresis and increased urine osmolality and aquaporin-2 abundance. Thiazide/amiloride-treated mice showed hyponatremia, hyperkalemia, hypercalcemia, metabolic acidosis, and increased serum lithium concentrations, adverse effects previously observed in patients but not in acetazolamide-treated mice in this study. Furthermore, acetazolamide treatment reduced inulin clearance and cortical expression of sodium/hydrogen exchanger 3 and attenuated the increased expression of urinary PGE2 observed in lithium-NDI mice. These results show that the antidiuresis with acetazolamide was partially caused by a tubular-glomerular feedback response and reduced GFR. The tubular-glomerular feedback response and/or direct effect on collecting duct principal or intercalated cells may underlie the reduced urinary PGE2 levels with acetazolamide, thereby contributing to the attenuation of lithium-NDI. In conclusion, CA activity contributes to lithium-NDI development, and acetazolamide attenuates lithium-NDI development in mice similar to thiazide/amiloride but with fewer adverse effects. Copyright © 2016 by the American Society of Nephrology.

  11. [Effect of Scalp-acupuncture Stimulation on Neurological Function and Expression of ASIC 1 a and ASIC 2 b of Hippocampal CA 1 Region in Cerebral Ischemia Rats].

    Science.gov (United States)

    Tian, Liang; Wang, Jin-Hai; Zhao, Min; Bao, Ying-Cun; Shang, Jun-Fang; Yan, Qi; Zhang, Zhen-Chang; Du, Xiao-Zheng; Jiang, Hua; Sun, Run-Jie; Yuan, Bo; Zhang, Xing-Hua; Zhang, Ting-Zhuo; Li, Xing-Lan

    2016-10-25

    To observe the influence of scalp-acupuncture on the expression of acid-sensing ion channels (ASICs) 1 a and 2 b of hippocampal CA 1 region in cerebral ischemia (CI) rats, so as to investigate its mechanism underlying improvement of ischemic stroke. Thirty-two male SD rats were randomly allocated to normal control, model, scalp-acupuncture and Amiloride group ( n =8 in each group). The model of focal CI was established by middle cerebral artery occlusion (MCAO). Scalp acupuncture stimulation was applied to bilateral Dingnieqianxiexian (MS 6) and Dingniehouxiexian (MS 7), once daily for 7 days. Rats of the Amiloride group were fed with Amiloride solution, twice a day for 7 days, and those of the normal control and model groups were grabbled and fixed in the same way with the acupuncture and Amiloride groups. The neurological deficit score was given according to Longa's method. The expression of hippocampal ASIC 1 a and ASIC 2 b was detected by immunohistochemistry, and the Ca 2+ concentration in the hippocampal tissue assayed using flowing cytometry. After the intervention, the neurological deficit score of both the scalp-acupuncture and Amiloride groups were significantly decreased in comparison with pre-treatment ( P ASIC 1 a and ASIC 2 b in the hippocampal CA 1 region and hip-pocampal Ca 2+ concentration were significantly up-regulated in the model group compared with the normal control group ( P ASIC 1 a and ASIC 2 b expression and Ca 2+ concentration ( P >0.05). Scalp-acupuncture stimulation can improve neurological function in CI rats, which may be related to its effects in suppressing the increased expression of hippocampal ASIC 1 a and ASIC 2 b proteins and in reducing calcium overload in hip-pocampal neurocytes.

  12. Structure and inhibition of the SARS coronavirus envelope protein ion channel.

    Directory of Open Access Journals (Sweden)

    Konstantin Pervushin

    2009-07-01

    Full Text Available The envelope (E protein from coronaviruses is a small polypeptide that contains at least one alpha-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA, but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV that the transmembrane domain of E protein (ETM forms pentameric alpha-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular alpha-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293 cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA, but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target.

  13. Sodium selectivity of semicircular canal duct epithelial cells

    Directory of Open Access Journals (Sweden)

    Harbidge Donald G

    2011-09-01

    Full Text Available Abstract Background Sodium absorption by semicircular canal duct (SCCD epithelial cells is thought to contribute to the homeostasis of the volume of vestibular endolymph. It was previously shown that the epithelial cells could absorb Na+ under control of a glucocorticoid hormone (dexamethasone and the absorptive transepithelial current was blocked by amiloride. The most commonly-observed target of amiloride is the epithelial sodium channel (ENaC, comprised of the three subunits α-, β- and γ-ENaC. However, other cation channels have also been observed to be sensitive in a similar concentration range. The aim of this study was to determine whether SCCD epithelial cells absorb only Na+ or also K+ through an amiloride-sensitive pathway. Parasensory K+ absorption could contribute to regulation of the transduction current through hair cells, as found to occur via vestibular transitional cells [S. H. Kim and D. C. Marcus. Regulation of sodium transport in the inner ear. Hear.Res. doi:10.1016/j.heares.2011.05.003, 2011]. Results We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6197, whole-cell patch clamp and transepithelial recordings in primary cultures of rat SCCD. α-, β- and γ-ENaC were all previously reported as present. The selectivity of the amiloride-sensitive transepithelial and cell membrane currents was observed in Ussing chamber and whole-cell patch clamp recordings. The cell membrane currents were carried by Na+ but not K+, but the Na+ selectivity disappeared when the cells were cultured on impermeable supports. Transepithelial currents across SCCD were also carried exclusively by Na+. Conclusions These results are consistent with the amiloride-sensitive absorptive flux of SCCD mediated by a highly Na+-selective channel, likely αβγ-ENaC. These epithelial cells therefore absorb only Na+ via the amiloride-sensitive pathway and do not provide a parasensory K+ efflux from the

  14. Normal Coagulation

    Science.gov (United States)

    2014-09-04

    fibrinolytic process and replaced by cellular reconstruction of the perfo- rated vessel. Fibrinolysis Proteins Clot formation is integrated with clot...Res 50:1488–1494, 1990. 226. Evans DM, et al: Time and dose dependency of the suppression of pulmonary metastases of rat mammary cancer by amiloride...Suppression of breast cancer growth and metastasis by a serpin myoepithelium-derived serine proteinase inhibitor expressed in the mammary

  15. Novel Therapeutic Targets for Chronic Migraine

    Science.gov (United States)

    2014-11-01

    Francisco (UCSF) or the University of Utah. They then underwent physical and neuro- logical examinations and structured interviews related to self...Amiloride was found to block CSD in rats when delivered acutely. Ongoing studies are examining the effects of this and other ASIC blockers on CSD thresholds...Interventional Cardiology Meeting, Boston November 2011 Invited speaker American Headache Society Teaching Course, Scottsdale April 2012 Speaker, American

  16. Serumelektrolytter og medikamentel behandling hos patienter indlagt på en geriatrisk afdeling

    DEFF Research Database (Denmark)

    Sørensen, I J; Matzen, L E

    1993-01-01

    The distributions of sodium and potassium in the serum on admission, and the types of medicine used were studied retrospectively in hospitalized geriatric patients (n = 1418). Sodium concentrations below 130 mmol/l were found in 7.4% and potassium concentrations below 3.0 mmol/l in 5.0% of patien......+amiloride was frequently withdrawn. 38% were given benzodiazepines on discharge. In 40.4% of these, treatment had been started during the hospital stay, most often on account of insomnia. Udgivelsesdato: 1993-Nov-29....... Risk factors for low sodium concentrations were treatment with the combination of thiazide+amiloride, potassium-sparing diuretics, thiazides, emergency hospitalization and low body weight. Risk factors for low potassium concentrations were treatment with the combination of thiazide+amiloride, thiazides...... and female gender. On an average, patients were given two different drugs from specified groups both on admission and on discharge, but changes in medical treatment were often performed during the hospital stay. Prehospital treatment with thiazide diuretics and the combination of thiazide...

  17. Vascular permeability in the RG2 glioma model can be mediated by macropinocytosis and be independent of the opening of the tight junction.

    Science.gov (United States)

    Pernet-Gallay, Karin; Jouneau, Pierre-Henri; Bertrand, Anne; Delaroche, Julie; Farion, Régine; Rémy, Chantal; Barbier, Emmanuel L

    2017-04-01

    This study evaluates the extravasation pathways of circulating macromolecules in a rat glioma model (RG2) which was observed by both magnetic resonance imaging using ultrasmall superparamagnetic iron oxide and electron microscopy. Although magnetic resonance imaging signal enhancement was observed as soon as 10 min after injection (9.4% 2 h after injection), electron microscopy showed that endothelial cells were still tightly sealed. However, circulating immunoglobulin G and ultrasmall superparamagnetic iron oxide were found in large membrane compartments of endothelial cells, in the basal lamina (7.4 ± 1.2 gold particles/µm 2 in the tumor versus 0.38 ± 0.17 in healthy tissue, p = 1.4.10 -5 ) and between tumoral cells. Altogether, this strongly suggests an active transport mediated by macropinocytosis. To challenge this transport mechanism, additional rats were treated with amiloride, an inhibitor of macropinocytosis, leading to a reduction of membrane protrusions (66%) and of macropinosomes. Amiloride however also opened tumoral tight junctions allowing a larger extravasation of ultrasmall superparamagnetic iron oxide (magnetic resonance imaging signal enhancement of 35.7% 2 h after injection). Altogether, these results suggest that ultrasmall superparamagnetic iron oxide and immunoglobulin G in the RG2 glioma model follow an active extravasation pathway mediated by a macropinocytosis process. Amiloride also appears as a potential strategy to facilitate the extravasation of chemotherapeutic drugs in glioma.

  18. Inhibition of epithelial Na+ transport by atriopeptin, protein kinase c, and pertussis toxin

    International Nuclear Information System (INIS)

    Mohrmann, M.; Cantiello, H.F.; Ausiello, D.A.

    1987-01-01

    The authors have recently shown the selective inhibition of an amiloride-sensitive, conductive pathway for Na + by atrial natriuretic peptide and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) in the renal epithelial cell line, LLC-PK i . Using 22 Na + fluxes, they further investigated the modulation of Na + transport by atrial natriuretic peptide and by agents that increase cGMP production, activate protein kinase c, or modulate guanine nucleotide regulatory protein function. Sodium nitroprusside increases intracellular cGMP concentrations without affecting cAMP concentrations and completely inhibits amiloride-sensitive Na + uptake in a time- and concentration-dependent manner. Oleoyl 2-acetylglycerol and phorbol 12-myristate 13-acetate, activators of protein kinase c, inhibit Na + uptake by 93 ± 13 and 51 ± 10%, respectively. Prolonged incubation with phorbol ester results in the downregulation of protein kinase c activity and reduces the inhibitory effect of atrial natriuretic peptide, suggesting that the action of this peptide involves stimulation of protein kinase c. Pertussis toxin, which induces the ADP-ribosylation of a 41-kDa guanine nucleotide regulatory protein in LLC-PK i cells, inhibits 22 Na + influx to the same extent as amiloride. Thus, increasing cGMP, activating protein kinase c, and ADP-ribosylating a guanine nucleotide regulatory protein all inhibit Na + uptake. These events may be sequentially involved in the action of atrial natriuretic peptide

  19. Effect of endocytosis inhibitors on Coxiella burnetii interaction with host cells

    International Nuclear Information System (INIS)

    Tujulin, E.; Macellaro, A.; Norlander, L.; Liliehoeoek, B.

    1998-01-01

    The obligate intracellular rickettsia Coxiella burnetii has previously been reported to reach the intra-vacuolar compartment of host cells by phagocytosis. With the aim to further examine the mechanisms of C. burnetii internalisation, macrophage monolayers were treated with well characterised inhibitors of endocytosis. The treatment with two general inhibitors, colchicine and methylamine, resulted in a pronounced dose-dependent decrease of radiolabelled phase II rickettsiae retained from the intracellular fraction. A third inhibitor used, amiloride, has been reported to reduce effectively clathrin-independent pinocytic pathways. The internalisation of C. burnetii was shown to be substantially reduced also by amiloride and the effect was dependent on its concentration. The passive role of C. burnetii in the internalisation was verified by using heat-killed C. burnetii. Host cells treated with either of the three inhibitors (amiloride, colchicine and methylamine) showed a similar reduction of intracellular C. burnetii after exposure to killed as weal as live organisms. The data presented indicate that different endocytic mechanisms, pinocytosis as well as phagocytosis, may mediate the uptake of C. burnetii by a host cell. Key words: Coxiella burnetii; internalisation; endocytosis (authors)

  20. Parathyroid hormone depresses cytosolic pH and DNA synthesis in osteoblast-like cells

    International Nuclear Information System (INIS)

    Reid, I.R.; Civitelli, R.; Avioli, L.V.; Hruska, K.A.

    1988-01-01

    It has recently become apparent that a number of hormones and growth factors modulate cytosolic pH (pH i ) and there is some evidence that this in turn may influence cell growth. The authors have examined the effects of parathyroid hormone (PTH) on both these parameters in an osteoblast-like cell line, UMR 106. Preliminary studies, using the pH-sensitive fluorescent probe 2',7'-bis(2-carboxyethyl)-5,(6)-carboxyfluorescein indicated that these cells regulate pH i by means of an amiloride-inhibitable Na + -H + exchanger. Rat PTH-(1-34) (rPTH) caused a progressive dose-related decrease in pH i with a half-maximal effect at 10 -11 M. The diacylglycerol analogue, phorbol 12-myristate 13-acetate, increased both pH i and [ 3 H]thymidine incorporation, and amiloride reduced both indexes. However, rPTH remained a potent inhibitor of [ 3 H]thymidine incorporation in the presence of amiloride, even though it did not affect pH i in these circumstances. It is concluded that PTH decreases pH i and growth in UMR 106 cells but that these changes can be dissociated. Depression of pH i may have other important effects on bone metabolism, such as reducing cell-cell communication, and may be associated with alkalinization of the bone fluid compartment

  1. Identification of Functionally Distinct Na-HCO3 Co-Transporters in Colon

    Science.gov (United States)

    Barmeyer, Christian; Ye, Jeff Huaqing; Soroka, Carol; Geibel, Peter; Hingsammer, Lukas M.; Weitgasser, Laurence; Atway, Danny; Geibel, John P.; Binder, Henry J.; Rajendran, Vazhaikkurichi M.

    2013-01-01

    Na-HCO3 cotransport (NBC) regulates intracellular pH (pHi) and HCO3 secretion in rat colon. NBC has been characterized as a 5,5′-diisothiocyanato-2-2′-stilbene (DIDS)-sensitive transporter in several tissues, while the colonic NBC is sensitive to both amiloride and DIDS. In addition, the colonic NBC has been identified as critical for pHi regulation as it is activated by intravesicular acid pH. Molecular studies have identified several characteristically distinct NBC isoforms [i.e. electrogenic (NBCe) and electroneutral (NBCn)] that exhibit tissue specific expression. This study was initiated to establish the molecular identity and specific function of NBC isoforms in rat colon. Northern blot and reverse transcriptase PCR (RT-PCR) analyses revealed that electrogenic NBCe1B or NBCe1C (NBCe1B/C) isoform is predominantly expressed in proximal colon, while electroneutral NBCn1C or NBCn1D (NBCn1C/D) is expressed in both proximal and distal colon. Functional analyses revealed that amiloride-insensitive, electrogenic, pH gradient-dependent NBC activity is present only in basolateral membranes of proximal colon. In contrast, amiloride-sensitive, electroneutral, [H+]-dependent NBC activity is present in both proximal and distal colon. Both electrogenic and electroneutral NBC activities are saturable processes with an apparent Km for Na of 7.3 and 4.3 mM, respectively; and are DIDS-sensitive with apparent Ki of 8.9 and 263.8 µM, respectively. In addition to Na-H exchanger isoform-1 (NHE1), pHi acidification is regulated by a HCO3-dependent mechanism that is HOE694-insensitive in colonic crypt glands. We conclude from these data that electroneutral, amiloride-sensitive NBC is encoded by NBCn1C/D and is present in both proximal and distal colon, while NBCe1B/C encodes electrogenic, amiloride-insensitive Na-HCO3 cotransport in proximal colon. We also conclude that NBCn1C/D regulates HCO3-dependent HOE694-insensitive Na-HCO3 cotransport and plays a critical role in p

  2. The role of distal tubule and collecting duct sodium reabsorption in sunitinib-induced hypertension.

    Science.gov (United States)

    Witte, Jeannine; Lampe, Josephine; Koenen, Anna; Urbaneck, Ines; Steinbach, Antje; Rettig, Rainer; Grisk, Olaf

    2018-04-01

    Antiangiogenic receptor tyrosine kinase inhibitors (RTKI) induce arterial hypertension which may limit their use. Renal fractional sodium excretion (FENa) is reduced in early RTKI-induced hypertension, whereas fractional lithium excretion is unaltered. Therefore, we tested the hypothesis that activated distal tubule and collecting duct sodium reabsorption contributes to RTKI-induced hypertension. Amiloride-sensitive and hydrochlorothiazide (HCTZ)-sensitive fractional sodium reabsorption (FRNa) and renal epithelial sodium channel (ENaC) as well as sodium chloride cotransporter (NCC) abundances were determined in sunitinib-treated and control rats. The antihypertensive effects of amiloride and HCTZ were investigated by radiotelemery. After 4 days of treatment, mean arterial pressure was 20 mmHg higher, FENa was lower (0.32 ± 0.08% vs. 0.65 ± 0.14%; P < 0.05), and renal medullary-ENaC protein abundance was higher in sunitinib-treated rats than in controls. Amiloride-sensitive FRNa was 2.37 ± 0.52% in sunitinib-treated rats vs. 2.66 ± 0.44% in controls (n.s.). HCTZ increased FENa by a similar magnitude without affecting amiloride-sensitive FRNa in both groups. After 14 days of treatment, renal medullary β-ENaC protein abundance was higher in rats that received sunitinib than in controls, whereas α-ENaC, γ-ENaC, and NCC abundances were similar in both groups. Amiloride and HCTZ reduced the sunitinib-induced mean arterial pressure rise by 8 ± 3 mmHg (P < 0.05) and 12 ± 2 mmHg (P < 0.05), respectively, without additive effects when combined. ENaC-dependent and thiazide-sensitive sodium-retaining mechanisms are not overactive in sunitinib-induced hypertension but ENaC blockers and in particular thiazides may be suitable for its treatment.

  3. Cholera toxin enhances Na+ absorption across MCF10A human mammary epithelia

    Science.gov (United States)

    Wang, Qian

    2013-01-01

    Cellular mechanisms to account for the low Na+ concentration in human milk are poorly defined. MCF10A cells, which were derived from human mammary epithelium and grown on permeable supports, exhibit amiloride- and benzamil-sensitive short-circuit current (Isc; a sensitive indicator of net ion transport), suggesting activity of the epithelial Na+ channel ENaC. When cultured in the presence of cholera toxin (Ctx), MCF10A cells exhibit greater amiloride-sensitive Isc at all time points tested (2 h to 7 days), an effect that is not reduced with Ctx washout for 12 h. Amiloride-sensitive Isc remains elevated by Ctx in the presence of inhibitors for PKA (H-89, Rp-cAMP), PI3K (LY294002), and protein trafficking (brefeldin A). Additionally, the Ctx B subunit, alone, does not replicate these effects. RT-PCR and Western blot analyses indicate no significant increase in either the mRNA or protein expression for α-, β-, or, γ-ENaC subunits. Ctx increases the abundance of both β- and γ-ENaC in the apical membrane. Additionally, Ctx increases both phosphorylated and nonphosphorylated Nedd4-2 expression. These results demonstrate that human mammary epithelia express ENaC, which can account for the low Na+ concentration in milk. Importantly, the results suggest that Ctx increases the expression but reduces the activity of the E3 ubiquitin ligase Nedd4-2, which would tend to reduce the ENaC retrieval and increase steady-state membrane residency. The results reveal a novel mechanism in human mammary gland epithelia by which Ctx regulates ENaC-mediated Na+ transport, which may have inferences for epithelial ion transport regulation in other tissues throughout the body. PMID:24371040

  4. Cholera toxin enhances Na(+) absorption across MCF10A human mammary epithelia.

    Science.gov (United States)

    Wang, Qian; Schultz, Bruce D

    2014-03-01

    Cellular mechanisms to account for the low Na(+) concentration in human milk are poorly defined. MCF10A cells, which were derived from human mammary epithelium and grown on permeable supports, exhibit amiloride- and benzamil-sensitive short-circuit current (Isc; a sensitive indicator of net ion transport), suggesting activity of the epithelial Na(+) channel ENaC. When cultured in the presence of cholera toxin (Ctx), MCF10A cells exhibit greater amiloride-sensitive Isc at all time points tested (2 h to 7 days), an effect that is not reduced with Ctx washout for 12 h. Amiloride-sensitive Isc remains elevated by Ctx in the presence of inhibitors for PKA (H-89, Rp-cAMP), PI3K (LY294002), and protein trafficking (brefeldin A). Additionally, the Ctx B subunit, alone, does not replicate these effects. RT-PCR and Western blot analyses indicate no significant increase in either the mRNA or protein expression for α-, β-, or, γ-ENaC subunits. Ctx increases the abundance of both β- and γ-ENaC in the apical membrane. Additionally, Ctx increases both phosphorylated and nonphosphorylated Nedd4-2 expression. These results demonstrate that human mammary epithelia express ENaC, which can account for the low Na(+) concentration in milk. Importantly, the results suggest that Ctx increases the expression but reduces the activity of the E3 ubiquitin ligase Nedd4-2, which would tend to reduce the ENaC retrieval and increase steady-state membrane residency. The results reveal a novel mechanism in human mammary gland epithelia by which Ctx regulates ENaC-mediated Na(+) transport, which may have inferences for epithelial ion transport regulation in other tissues throughout the body.

  5. An overview of inhibitors of Na(+)/H(+) exchanger.

    Science.gov (United States)

    Masereel, B; Pochet, L; Laeckmann, D

    2003-06-01

    The Na(+)/H(+) exchanger (NHE) is involved in intracellular pH homeostasis of many mammalian cell types. To date seven NHE isoforms (NHE1-NHE7) have been identified. NHE1 is the most predominant isoform expressed in heart where it contributes to cardiomyocyte pH homeostasis. Although the NHE activation is essential for the restoration of physiological pH, hyperactivation of NHE1 during ischemia-reperfusion episodes disrupts the intracellular ion balance, leading to cardiac dysfunction and damage. Beside its ability to inhibit a conductive Na(+) channel and the Na(+)/Ca(++) exchanger, amiloride was the first drug described as NHE inhibitor. Double substitution of the nitrogen of the 5-amino group of amiloride gave DMA, EIPA, MIBA and HMA. Later, several acylguanidines were prepared to selectively inhibit NHE1. The replacement of the pyrazine ring of amiloride by a pyridine ring or by a phenyl increased the potency and the NHE selectivity. The simultaneous replacement of the pyrazine ring by a phenyl, of the 6-chloro by a sulfomethyl led to drugs such as HOE-694, cariporide, eniporide and BIIB-513 which also selectively inhibited NHE1. In the last decade several bicyclic guanidines were prepared: zoniporide, MS-31038, SM-20220, SM-20550, SMP-300, KB-R9032, BMS-284640, T-162559, TY-12533, S-3226 or SL-591227. Extensive pre-clinical studies indicated that NHE inhibitors afford substantial protection in different animal models of myocardial ischemia (MI) and reperfusion, but the results of clinical trials involving eniporide and cariporide were mixed.

  6. Cross-reactivity of acid-sensing ion channel and Na+–H+ exchanger antagonists with nicotinic acetylcholine receptors

    Science.gov (United States)

    Santos-Torres, Julio; Ślimak, Marta A; Auer, Sebastian; Ibañez-Tallon, Inés

    2011-01-01

    Abstract Nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the mammalian central and peripheral nervous systems, where they contribute to neuronal excitability and synaptic communication. It has been reported that nAChRs are modulated by BK channels and that BK channels, in turn, are inhibited by acid-sensing ion channels (ASICs). Here we investigate the possible functional interaction between these channels in medial habenula (MHb) neurones. We report that selective antagonists of large-conductance calcium-activated potassium channels and ASIC1a channels, paxilline and psalmotoxin 1, respectively, did not induce detectable changes in nicotine-evoked currents. In contrast, the non-selective ASIC and Na+–H+ exchanger (NHE1) antagonists, amiloride and its analogues, suppressed nicotine-evoked responses in MHb neurones of wild-type and ASIC2 null mice, excluding a possible involvement of ASIC2 in the nAChR inhibition by amiloride. Zoniporide, a more selective inhibitor of NHE1, reversibly inhibited α3β4-, α7- and α4-containing (*) nAChRs in Xenopus oocytes and in brain slices, as well as in PS120 cells deficient in NHE1 and virally transduced with nAChRs, suggesting a generalized effect of zoniporide in most neuronal nAChR subtypes. Independently from nAChR antagonism, zoniporide profoundly blocked synaptic transmission onto MHb neurones without affecting glutamatergic and GABA receptors. Taken together, these results indicate that amiloride and zoniporide, which are clinically used to treat hypertension and cardiovascular disease, have an inhibitory effect on neuronal nAChRs when used experimentally at high doses. The possible cross-reactivity of these compounds with nAChRs in vivo will require further investigation. PMID:21911609

  7. Cross-reactivity of acid-sensing ion channel and Na⁺-H⁺ exchanger antagonists with nicotinic acetylcholine receptors.

    Science.gov (United States)

    Santos-Torres, Julio; Ślimak, Marta A; Auer, Sebastian; Ibañez-Tallon, Inés

    2011-11-01

    Nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the mammalian central and peripheral nervous systems, where they contribute to neuronal excitability and synaptic communication. It has been reported that nAChRs are modulated by BK channels and that BK channels, in turn, are inhibited by acid-sensing ion channels (ASICs). Here we investigate the possible functional interaction between these channels in medial habenula (MHb) neurones. We report that selective antagonists of large-conductance calcium-activated potassium channels and ASIC1a channels, paxilline and psalmotoxin 1, respectively, did not induce detectable changes in nicotine-evoked currents. In contrast, the non-selective ASIC and Na(+)-H(+) exchanger (NHE1) antagonists, amiloride and its analogues, suppressed nicotine-evoked responses in MHb neurones of wild-type and ASIC2 null mice, excluding a possible involvement of ASIC2 in the nAChR inhibition by amiloride. Zoniporide, a more selective inhibitor of NHE1, reversibly inhibited α3β4-, α7- and α4-containing (*) nAChRs in Xenopus oocytes and in brain slices, as well as in PS120 cells deficient in NHE1 and virally transduced with nAChRs, suggesting a generalized effect of zoniporide in most neuronal nAChR subtypes. Independently from nAChR antagonism, zoniporide profoundly blocked synaptic transmission onto MHb neurones without affecting glutamatergic and GABA receptors. Taken together, these results indicate that amiloride and zoniporide, which are clinically used to treat hypertension and cardiovascular disease, have an inhibitory effect on neuronal nAChRs when used experimentally at high doses. The possible cross-reactivity of these compounds with nAChRs in vivo will require further investigation.

  8. Transport characteristics and morphology of the colon and coprodeum in two wild birds of different habitats, the rock ptarmigan (Lagopus mutus) and the common murre (Uria aalge).

    Science.gov (United States)

    Árnason, Sighvatur S; Elbrønd, Vibeke S; Laverty, Gary

    2015-09-01

    Dietary salt intake in domestic fowl affects epithelial transport and morphology of the lower intestine (colon and coprodeum). This study investigated lower intestinal morphology and transport activity in two wild bird species with natural diets containing either low or high salt. Tissues from rock ptarmigan (Lagopus mutus) and common murres (Uria aalge) were sampled for histology and electrophysiological analyses. The ptarmigan exists on a low salt diet, while the murre lives on a high protein and high salt diet. The ptarmigan colon and coprodeum had villi/folds and crypts and the epithelium contained absorptive epithelial cells, mitochondria-rich cells and goblet cells. The colon had significant amiloride-inhibitable Isc, 5-15 μA/cm(2), with no glucose-stimulated Isc, and no significant phloridzin inhibition. The coprodeum also had high amiloride-inhibitable Isc. This transport pattern corresponded to that of chickens on low-salt diets. However, the ptarmigan colon also had a significant lysine/leucine-stimulated Isc of 3±1.0 μA/cm(2). The short U. aalge colon was similar to that of ptarmigans, but with no villi. It demonstrated a significant lysine/leucine-stimulated Isc (11±3.5 μA/cm(2)) with no amiloride-inhibitable Isc, similar to the high-salt chicken colon, but with no Na(+)-glucose cotransport. The murre coprodeum was inert to all substances and showed high resistance (1000 Ω·cm(2)), with a multilayered squamous epithelium. Despite some variations possibly associated with dietary protein intake, we conclude that natural high and low salt diets in different avian species are associated with different lower intestinal transport patterns, providing for post-renal adjustments in ion and water excretion. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Coupled in silico platform: Computational fluid dynamics (CFD) and physiologically-based pharmacokinetic (PBPK) modelling.

    Science.gov (United States)

    Vulović, Aleksandra; Šušteršič, Tijana; Cvijić, Sandra; Ibrić, Svetlana; Filipović, Nenad

    2018-02-15

    One of the critical components of the respiratory drug delivery is the manner in which the inhaled aerosol is deposited in respiratory tract compartments. Depending on formulation properties, device characteristics and breathing pattern, only a certain fraction of the dose will reach the target site in the lungs, while the rest of the drug will deposit in the inhalation device or in the mouth-throat region. The aim of this study was to link the Computational fluid dynamics (CFD) with physiologically-based pharmacokinetic (PBPK) modelling in order to predict aerolisolization of different dry powder formulations, and estimate concomitant in vivo deposition and absorption of amiloride hydrochloride. Drug physicochemical properties were experimentally determined and used as inputs for the CFD simulations of particle flow in the generated 3D geometric model of Aerolizer® dry powder inhaler (DPI). CFD simulations were used to simulate air flow through Aerolizer® inhaler and Discrete Phase Method (DPM) was used to simulate aerosol particles deposition within the fluid domain. The simulated values for the percent emitted dose were comparable to the values obtained using Andersen cascade impactor (ACI). However, CFD predictions indicated that aerosolized DPI have smaller particle size and narrower size distribution than assumed based on ACI measurements. Comparison with the literature in vivo data revealed that the constructed drug-specific PBPK model was able to capture amiloride absorption pattern following oral and inhalation administration. The PBPK simulation results, based on the CFD generated particle distribution data as input, illustrated the influence of formulation properties on the expected drug plasma concentration profiles. The model also predicted the influence of potential changes in physiological parameters on the extent of inhaled amiloride absorption. Overall, this study demonstrated the potential of the combined CFD-PBPK approach to model inhaled drug

  10. Determination of diuretics and laxatives as adulterants in herbal formulations for weight loss.

    Science.gov (United States)

    Moreira, Ana Paula Lançanova; Motta, Monique Jung; Dal Molin, Thaís Ramos; Viana, Carine; de Carvalho, Leandro Machado

    2013-01-01

    A new method is described for the determination of the most common diuretic and laxative adulterants found in formulations of anorexics and antidepressants. The method is based on the separation of furosemide, hydrochlorothiazide, chlorthalidone and amiloride (diuretics), phenolphthalein (laxative), amfepramone (anorexic) and fluoxetine and paroxetine (antidepressants) by capillary zone electrophoresis with capacitively coupled contactless conductivity detection. The method showed a precision ranging from 1.9% to 6.9% for a concentration of 25 mg/L, 0.6% to 5.3% for a concentration of 50 mg/L and 1.6% to 6.0% for a concentration of 100 mg/L for all analytes. The accuracy was 99% for amiloride, 102% for chlorthalidone, 101% for hydrochlorothiazide, 101% for furosemide, 94% for phenolphthalein, 105% for fluoxetine, 114% for paroxetine and 117% for amfepramone. The method allowed the drugs to be determined in the formulations at concentrations higher than 5.1 mg/kg for amiloride, 7.7 mg/kg for chlorthalidone, 6.8 mg/kg for hydrochlorothiazide, 10.7 mg/kg for furosemide, 8.4 mg/kg for phenolphthalein, 11.0 mg/kg for fluoxetine, 9.4 mg/kg for paroxetine and 11.0 mg/kg for amfepramone. Three of the 26 analysed herbal formulations were found to be adulterated (not declared on the label) with the diuretic hydrochlorothiazide. Five other samples contained diuretics declared on the label on the formulation. Thus, a total of eight samples, which were marketed as natural products, contained diuretics (declared or not) on the formulation.

  11. Potential drug-drug interactions among elderly patients on anti-hypertensive medications in two tertiary healthcare facilities in Ekiti State, South-West Nigeria

    Directory of Open Access Journals (Sweden)

    Joseph Olusesan Fadare

    2016-01-01

    Full Text Available Introduction: Drug-drug interactions remain a major cause of adverse drug reactions with great consequences such as increased morbidity and increased healthcare cost. In elderly patients with systemic hypertension, there is a tendency for them to be prescribed multiple medications and this may expose them to some drug-drug interactions (DDIs especially in the context of physiological changes of ageing. The objective of this study was to evaluate potential drug-drug interaction among some Nigerian elderly hypertension. Methods: A cross-sectional study involving elderly hypertensive patients attending the general outpatient clinic of two tertiary healthcare facilities located in Ekiti State, South-West Nigeria. The information collected from the patients′ medical records included their ages, gender, diagnosis and list of prescribed anti-hypertensive medications. Potential drug-drug interactions were checked for using the Multi-Drug Interaction Checker (Medscape Reference and Epocrates Drug Interaction Checker (San Mateo CA, USA. Results: A total of 350 elderly patients attended the clinics during the study period of which 208 (59.4% hypertensive patients were identified and their records used for analysis. The fixed-dose combination drug Moduretic® (Amiloride /Hydrochlorothiazide-25.7% was the most commonly prescribed antihypertensive followed by Lisinopril (16.6%, Amlodipine (13.2% and Nifedipine (12.6%. The anti-platelet Acetyl-salicylic acid (ASA was prescribed for 100 (48.1% patients and represented 19.8% of all prescribed medications. A total of 231 potential DDIs were found among the patients giving a mean of 1.3 interactions per patient. The most common identified drug pairs with potential interactions were ACE inhibitors - Amiloride, followed by ACE inhibitors - Hydrochlorothiazide, ACE inhibitors - ASA and ARB - Amiloride. Conclusion: Potential drug-drug interactions, though common in this study comprised mainly of minor and moderate

  12. Sodium Carbonate is Saltier Than Sodium Chloride to Sodium-Depleted Rats.

    Science.gov (United States)

    St John, Steven J; McBrayer, Anya M; Krauskopf, Erin E

    2017-10-01

    In a series of behavioral experiments in the 1960s, G.R. Morrison identified several unique features of the taste of Na2CO3 to rats; namely, it is 1) considerably more intense than NaCl at isomolar concentrations, 2) avoided at 10 times lower concentrations than NaCl to thirsty rats, 3) preferred at 10 times lower concentrations than NaCl in sodium-depleted rats. He also demonstrated its qualitatively similarity to NaCl. In Experiment 1, we confirmed and extended many of Morrison's observations. Rats were injected with furosemide on 3 occasions to stimulate a sodium appetite. After each depletion, rats were given a brief-access taste test in a lickometer presenting, in random order, water and 7 concentrations of salt. One test used NaCl (0.028-0.89 M, quarter log steps), another used Na2CO3, and the third used Na2CO3, but at a tenfold lower concentration range (0.0028-0.089 M). Rats licked NaCl in an inverted-U shaped concentration-response function peaking at 0.158-0.281 M. As Morrison's results predicted, rats licked Na2CO3 in nearly identical fashion, but at a tenfold lower concentration range (peak at 0.0158-0.028 M). In a second experiment, furosemide-treated rats were repeatedly tested with the lower Na2CO3 range but mixed in the epithelial sodium channel blocker amiloride at various concentrations (3-300 μM, half log steps). Amiloride reduced licking for Na2CO3 and shifted the peak response rightward up to about half a log unit. Thus, this "super-saltiness" of Na2CO3 to rats is at least partly amiloride-dependent. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Salty taste deficits in CALHM1 knockout mice.

    Science.gov (United States)

    Tordoff, Michael G; Ellis, Hillary T; Aleman, Tiffany R; Downing, Arnelle; Marambaud, Philippe; Foskett, J Kevin; Dana, Rachel M; McCaughey, Stuart A

    2014-07-01

    Genetic ablation of calcium homeostasis modulator 1 (CALHM1), which releases adenosine triphosphate from Type 2 taste cells, severely compromises the behavioral and electrophysiological responses to tastes detected by G protein-coupled receptors, such as sweet and bitter. However, the contribution of CALHM1 to salty taste perception is less clear. Here, we evaluated several salty taste-related phenotypes of CALHM1 knockout (KO) mice and their wild-type (WT) controls: 1) In a conditioned aversion test, CALHM1 WT and KO mice had similar NaCl avoidance thresholds. 2) In two-bottle choice tests, CALHM1 WT mice showed the classic inverted U-shaped NaCl concentration-preference function but CALHM1 KO mice had a blunted peak response. 3) In brief-access tests, CALHM1 KO mice showed less avoidance than did WT mice of high concentrations of NaCl, KCl, NH(4)Cl, and sodium lactate (NaLac). Amiloride further ameliorated the NaCl avoidance of CALHM1 KO mice, so that lick rates to a mixture of 1000 mM NaCl + 10 µM amiloride were statistically indistinguishable from those to water. 4) Relative to WT mice, CALHM1 KO mice had reduced chorda tympani nerve activity elicited by oral application of NaCl, NaLac, and sucrose but normal responses to HCl and NH(4)Cl. Chorda tympani responses to NaCl and NaLac were amiloride sensitive in WT but not KO mice. These results reinforce others demonstrating that multiple transduction pathways make complex, concentration-dependent contributions to salty taste perception. One of these pathways depends on CALHM1 to detect hypertonic NaCl in the mouth and signal the aversive taste of concentrated salt. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Angiotensin II stimulates renin in inner medullary collecting duct cells via protein kinase C and independent of epithelial sodium channel and mineralocorticoid receptor activity.

    Science.gov (United States)

    Gonzalez, Alexis A; Liu, Liu; Lara, Lucienne S; Seth, Dale M; Navar, L Gabriel; Prieto, Minolfa C

    2011-03-01

    Collecting duct (CD) renin is stimulated by angiotensin (Ang) II, providing a pathway for Ang I generation and further conversion to Ang II. Ang II stimulates the epithelial sodium channel via the Ang II type 1 receptor and increases mineralocorticoid receptor activity attributed to increased aldosterone release. Our objective was to determine whether CD renin augmentation is mediated directly by Ang II type 1 receptor or via the epithelial sodium channel and mineralocorticoid receptor. In vivo studies examined the effects of epithelial sodium channel blockade (amiloride; 5 mg/kg per day) on CD renin expression and urinary renin content in Ang II-infused rats (80 ng/min, 2 weeks). Ang II infusion increased systolic blood pressure, medullary renin mRNA, urinary renin content, and intrarenal Ang II levels. Amiloride cotreatment did not alter these responses despite a reduction in the rate of progression of systolic blood pressure. In primary cultures of inner medullary CD cells, renin mRNA and (pro)renin protein levels increased with Ang II (100 nmol/L), and candesartan (Ang II type 1 receptor antagonist) prevented this effect. Aldosterone (10(-10) to 10(-7) mol/L) with or without amiloride did not modify the upregulation of renin mRNA in Ang II-treated cells. However, inhibition of protein kinase C with calphostin C prevented the Ang II-mediated increases in renin mRNA and (pro)renin protein levels. Furthermore, protein kinase C activation with phorbol 12-myristate 13-acetate increased renin expression to the same extent as Ang II. These data indicate that an Ang II type 1 receptor-mediated increase in CD renin is induced directly by Ang II via the protein kinase C pathway and that this regulation is independent of mineralocorticoid receptor activation or epithelial sodium channel activity.

  15. Ion transport mechanisms in the mesonephric collecting duct system of the toad Bufo bufo: microelectrode recordings from isolated and perfused tubules

    DEFF Research Database (Denmark)

    Møbjerg, Nadja; Larsen, Erik Hviid; Novak, Ivana

    2004-01-01

    It is not clear how and whether terrestrial amphibians handle NaCl transport in the distal nephron. Therefore, we studied ion transport in isolated perfused collecting tubules and ducts from toad, Bufo bufo, by means of microelectrodes. No qualitative difference in basolateral cell membrane...... and amiloride application showed a small apical Na+ conductance. Arginine vasotocin depolarized Vbl. The small apical Na+ conductance indicates that the collecting duct system contributes little to NaCl reabsorption when compared to aquatic amphibians. In contrast, Vbl rapidly depolarized upon lowering of [Na...

  16. Beta-adrenergic activation of solute coupled water uptake by toad skin epithelium results in near-isosmotic transport

    DEFF Research Database (Denmark)

    Nielsen, Robert; Larsen, Erik Hviid

    2007-01-01

    .2+/-0.3 to 2.4+/-0.4 mS x cm(-2) and slower increases in equivalent short circuit current, I(SC)(Eqv) = -G(T) x V(T), from 12.7+/-3.2 to 33.1+/-6.8 microA cm(-2), and J(V) from 0.72+/-0.17 to 3.01+/-0.49 nL cm(-2) s(-1). Amiloride in the outside solution abolished I(SC)(Eqv) (-1.6+/-0.1 microA cm(-2)) while J...

  17. Acid sensing ion channel (ASIC) inhibitors exhibit anxiolytic-like activity in preclinical pharmacological models.

    Science.gov (United States)

    Dwyer, Jason M; Rizzo, Stacey J Sukoff; Neal, Sarah J; Lin, Qian; Jow, Flora; Arias, Robert L; Rosenzweig-Lipson, Sharon; Dunlop, John; Beyer, Chad E

    2009-03-01

    Acid sensing ion channels (ASICs) are proton-gated ion channels located in the central and peripheral nervous systems. Of particular interest is ASIC1a, which is located in areas associated with fear and anxiety behaviors. Recent reports suggest a role for ASIC1a in preclinical models of fear conditioning and anxiety. The present experiments evaluated various ASIC inhibitors in preclinical models of autonomic and behavioral parameters of anxiety. In addition, neurochemical studies evaluated the effects of an ASIC inhibitor (A-317567) on neurotransmitter levels in the amygdala. In electrophysiological studies using hippocampal primary neuronal cultures, three ASIC inhibitors (PcTX-1, A-317567, and amiloride) produced concentration-dependent inhibition of acid-evoked currents. In the stress-induced hyperthermia model, acute administration of psalmotoxin 1 (PcTX-1; 10-56 ng, i.c.v.), A-317567 (0.1-1.0 mg/kg, i.p.), and amiloride (10-100 mg/kg, i.p.) prevented stress-induced elevations in core body temperature. In the four-plate test, acute treatment with PcTX-1 (10-56 ng, i.c.v.) and A-317567 (0.01-0.1 mg/kg, i.p.), but not amiloride (3-100 mg/kg, i.p.), produced dose-dependent and significant increases in the number of punished crossings relative to vehicle-treated animals. Additionally, PcTX-1 (56-178 ng, i.c.v.), A-317567 (0.1-10 mg/kg, i.p.), and amiloride (10-100 mg/kg, i.p.) lacked significant anxiolytic-like activity in the elevated zero maze. In neurochemical studies, an infusion of A-317567 (100 microM) into the amygdala significantly elevated the extracellular levels of GABA, but not glutamate, in this brain region. These findings demonstrate that ASIC inhibition produces anxiolytic-like effects in some behavioral models and indicate a potential role for GABAergic mechanisms to underlie these anxiolytic-like effects.

  18. Screening of pharmacologic adulterant classes in herbal formulations using voltammetry of microparticles.

    Science.gov (United States)

    Doménech-Carbó, Antonio; Martini, Mariele; de Carvalho, Leandro Machado; Viana, Carine; Doménech-Carbó, María Teresa; Silva, Miguel

    2013-02-23

    A solid state electrochemical method for screening different families of adulterant chemicals illegally added to commercial phytotherapuetic formulations is described. The proposed method, based on the voltammetry of microparticles approach, permits a fast and sensitive way to distinguish between anorexics (amfepramone, fenproporex, sibutramine), benzozodiazepinic anxiolytics (clonazepam, flurazepam, alprazolam, midazolam, medazepam, chlordiazepoxide, diazepam), antidepressants (bupropione, fluoxetine, sertraline, paroxetine), diuretics (hydrochlorothiazide, furosemide, chlortalidone, amiloride, spironolactone), and hypoglycemics (glimepiride, chlorpropamide, glibenclamide) based on characteristic voltammetric signals recorded on solid micro- or nanosamples attached to graphite electrodes immersed into aqueous electrolytes. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Altered ion transport in normal human bronchial epithelial cells following exposure to chemically distinct metal welding fume particles.

    Science.gov (United States)

    Fedan, Jeffrey S; Thompson, Janet A; Meighan, Terence G; Zeidler-Erdely, Patti C; Antonini, James M

    2017-07-01

    Welding fume inhalation causes pulmonary toxicity, including susceptibility to infection. We hypothesized that airway epithelial ion transport is a target of fume toxicity, and investigated the effects of fume particulates from manual metal arc-stainless steel (MMA-SS) and gas metal arc-mild steel (GMA-MS) on ion transport in normal human bronchial epithelium (NHBE) cultured in air-interface. MMA-SS particles, more soluble than GMA-MS particles, contain Cr, Ni, Fe and Mn; GMA-MS particles contain Fe and Mn. MMA-SS or GMA-MS particles (0.0167-166.7μg/cm 2 ) were applied apically to NHBEs. After 18h transepithelial potential difference (V t ), resistance (R t ), and short circuit current (I sc ) were measured. Particle effects on Na + and Cl¯ channels and the Na + ,K + ,2Cl¯-cotransporter were evaluated using amiloride (apical), 5-nitro-2-[(3-phenylpropyl)amino]benzoic acid (NPPB, apical), and bumetanide (basolateral), respectively. MMA-SS (0.0167-16.7μg/cm 2 ) increased basal V t . Only 16.7μg/cm 2 GMA-MS increased basal V t significantly. MMA-SS or GMA-MS exposure potentiated I sc responses (decreases) to amiloride and bumetanide, while not affecting those to NPPB, GMA-MS to a lesser degree than MMA-SS. Variable effects on R t were observed in response to amiloride, and bumetanide. Generally, MMA-SS was more potent in altering responses to amiloride and bumetanide than GMA-MS. Hyperpolarization occurred in the absence of LDH release, but decreases in V t , R t , and I sc at higher fume particulate doses accompanied LDH release, to a greater extent for MMA-SS. Thus, Na + transport and Na + ,K + ,2Cl¯-cotransport are affected by fume exposure; MMA-MS is more potent than GMA-MS. Enhanced Na + absorption and decreased airway surface liquid could compromise defenses against infection. Published by Elsevier Inc.

  20. Nephrogenic diabetes insipidus: treat with caution.

    Science.gov (United States)

    Boussemart, Thierry; Nsota, Jacqueline; Martin-Coignard, Dominique; Champion, Gérard

    2009-09-01

    Current therapy for congenital nephrogenic diabetes insipidus consists of appropriate water intake coupled with decreased urine output obtained by means of a low-sodium diet and a combination of thiazide diuretics with renal prostaglandins inhibitors or amiloride. We report a case of congenital nephrogenic diabetes insipidus that was complicated by paradoxical water intoxication secondary to liberal water intake and the initiation of hydrochlorothiazide and indomethacin combination therapy. This report emphasizes the importance of evaluating the water balance and of a quick response with strict protocols following the initiation of indomethacin and thiazide diuretics in nephrogenic diabetes insipidus.

  1. Thermal diversities of two Na+/H+ exchanges in guinea pig red cells.

    Science.gov (United States)

    Ji, H L

    2001-09-01

    To test the effect of hypothermia on Na+/H+ exchange, activated by shrinkage and cytoplasmic acidosis. Amiloride-sensitive Na+ influx in guinea pig red cells was traced with isotope 22Na and intracellular Na+ concentration was measured by emission flame photometry. Amiloride-sensitive Na+ influx decreased linearly as a function of temperatures (about 37 degrees C) in shrunken cells, but increased in acidified cells. The up-regulation of acid-induced Na+/H+ exchange by elevated temperature was enhanced by hypo-osmolarity. Less sensitivity of intracellular H+ site at 41 degrees C may be the mechanism for the inhibition of shrinkage-induced Na+/H+ exchange by elevated temperature. Heating-mediated explosive increase in the activity of acid-induced Na+/H+ exchange may be due to enhanced extracellular Na+ sensitivity and lower intracellular pH caused by acidic metabolites. Acid-induced Na+/H+ exchange contributes to cytoplasmic Na+ accumulation. These two modes of Na+/H+ exchange with different response to elevated temperature may play different roles in the cellular pathogenesis of heatstroke.

  2. A Ca-activated K channel from rabbit renal brush-border membrane vesicles in planar lipid bilayers.

    Science.gov (United States)

    Zweifach, A; Desir, G V; Aronson, P S; Giebisch, G H

    1991-07-01

    Rabbit renal brush-border membranes were fused to planar lipid bilayers to gain insight into the nature and properties of ion channels from the luminal membrane of the proximal tubule. Fusion was obtained using osmotic gradients. A large conductance channel was commonly observed. Measurements of reversal potentials indicated that the channel was selective for K over Rb, Na, and Cl. Channel open probability was increased by membrane depolarization and by increased Ca activity on the intracellular face of the channel. The channel was inhibited by charybdotoxin (CTX), a protein from leiurus venom, from the external side of the channel. The channel was also blocked by Ba and quinidine added to the intracellular bathing solution. Na added to the intracellular bathing solution reduced current amplitude in a voltage-dependent fashion. In addition, methylisobutyl amiloride, an analogue of the K-sparing diuretic amiloride, inhibited channel activity when added to the external solution. The possible physiological role of the channel is discussed. The usefulness to the study of renal ion channels of the technique of fusing membrane vesicles to planar lipid bilayers is evaluated.

  3. Electrolyte transport in distal colon of sodium-depleted rats: Effect of sodium repletion

    International Nuclear Information System (INIS)

    Turnamian, S.G.; Binder, H.J.

    1988-01-01

    Dietary sodium depletion increases plasma aldosterone level and, as a result, induces amiloride-sensitive electrogenic sodium absorption and electrogenic potassium secretion and stimulates Na + -K + -ATPase activity in rat distal colon, while inhibiting electroneutral sodium chloride absorption. To assess the events that occur as the aldosterone-stimulated colon reverts to normal, unidirectional 22 Na and 36 Cl fluxes were measured under voltage-clamp conditions across isolated distal colonic mucosa of rats that were initially dietary sodium depleted for 7 days and then sodium repleted for varying periods of time before the study. Within 8 h of dietary sodium repletion, plasma aldosterone level and Na + -K + -ATPase activity declined to normal, amiloride-sensitive electrogenic sodium absorption decreased by >90%, and active electrogenic potassium secretion also decreased markedly. In contrast, electroneutral sodium chloride absorption did not completely return to levels seen in normal animals until ∼64-68 h. These results demonstrate that maintenance of electrogenic sodium absorption and potassium secretion are directly dependent on elevated plasma aldosterone levels. The inhibition of electroneutral sodium absorption, although initiated by excess aldosterone, persists after normalization of the plasma aldosterone level, thereby implying that the inhibition is dependent on additional factor(s)

  4. Effect of NSAIDs on Na⁺/H⁺ exchanger activity in rat colonic crypts.

    Science.gov (United States)

    Roginiel, Aliya C; Kohut, Daniel L; Kaur, Sumanpreet; Saleh, Ahmad M A; Weber, Theresa; Geibel, Peter; Singh, Harmeet; Geibel, John P

    2013-09-01

    Nonsteroidal anti-inflammatory drugs (NSAIDs; 1) are widely recommended for several acute and chronic conditions. For example, both indomethacin and aspirin are taken for pain relief. Aspirin is also used for prevention of myocardial infarction, and indomethacin can be administered orally or as a suppository for patients with rheumatoid disease and other chronic inflammatory states. However, use of NSAIDs can cause damage to the mucosal barrier surrounding the gastrointestinal (GI) tract, increasing the risk of ulcer formation. While microencapsulation of NSAIDs has been shown to reduce upper GI injury, sustained release in the lower GI tract and colon may cause epithelial erosion due to increased acidification. The use of suppositories has also been linked to rectal and lower GI bleeding. In this study, we investigated the role of NSAIDs aspirin and indomethacin on Na⁺/H⁺ exchanger (NHE) activity in rat colonic crypts. By comparing average rates of pH recovery between control and NSAID perfusion runs, we were able to determine that both aspirin and indomethacin increase hydrogen extrusion into the colonic lumen. Through treatment with 5-ethylisopropyl amiloride (EIPA), amiloride, and zoniporide dihydrochloride, we further demonstrated that indomethacin specifically enhances proton excretion through regulation of apical NHE-3 and NHE-2 and to a lesser extent on basolateral NHE-1 and NHE-4. Our results suggest that clinical exposure to NSAIDs may affect colonic tissue at the site of selected NHE isoforms, resulting in modulation of transport and barrier function.

  5. Hormonal regulation of Na+-K+-ATPase in cultured epithelial cells

    International Nuclear Information System (INIS)

    Johnson, J.P.; Jones, D.; Wiesmann, W.P.

    1986-01-01

    Aldosterone and insulin stimulate Na + transport through mechanisms involving protein synthesis. Na + -K + -ATPase has been implicated in the action of both hormones. The authors examined the effect of aldosterone and insulin on Na + -K + -ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (I/sub sc/) in TB6C cells. Aldosterone increases Na + -K + -[ 32 P]ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in I/sub sc/, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I/sub sc/ in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na + -K + -ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na + entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na + -K + -ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on I/sub sc/

  6. ASIC1a Deficient Mice Show Unaltered Neurodegeneration in the Subacute MPTP Model of Parkinson Disease.

    Science.gov (United States)

    Komnig, Daniel; Imgrund, Silke; Reich, Arno; Gründer, Stefan; Falkenburger, Björn H

    2016-01-01

    Inflammation contributes to the death of dopaminergic neurons in Parkinson disease and can be accompanied by acidification of extracellular pH, which may activate acid-sensing ion channels (ASIC). Accordingly, amiloride, a non-selective inhibitor of ASIC, was protective in an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson disease. To complement these findings we determined MPTP toxicity in mice deficient for ASIC1a, the most common ASIC isoform in neurons. MPTP was applied i.p. in doses of 30 mg per kg on five consecutive days. We determined the number of dopaminergic neurons in the substantia nigra, assayed by stereological counting 14 days after the last MPTP injection, the number of Nissl positive neurons in the substantia nigra, and the concentration of catecholamines in the striatum. There was no difference between ASIC1a-deficient mice and wildtype controls. We are therefore not able to confirm that ASIC1a are involved in MPTP toxicity. The difference might relate to the subacute MPTP model we used, which more closely resembles the pathogenesis of Parkinson disease, or to further targets of amiloride.

  7. Efficacy of Precordial Percussion Pacing Assessed in a Cardiac Standstill Microminipig Model.

    Science.gov (United States)

    Wada, Takeshi; Ohara, Hiroshi; Nakamura, Yuji; Cao, Xin; Izumi-Nakaseko, Hiroko; Ando, Kentaro; Honda, Mitsuru; Yoshihara, Katsunori; Nakazato, Yuji; Lurie, Keith G; Sugiyama, Atsushi

    2017-07-25

    Potential cardiovascular benefits of precordial percussion pacing (PPP) during cardiac standstill are unknown.Methods and Results:A cardiac standstill model in amicrominipigwas created by inducing complete atrioventricular block with a catheter ablation technique (n=7). Next, the efficacy of cardiopulmonary resuscitation by standard chest compressions (S-CPR), PPP and ventricular electrical pacing in this model were analyzed in series (n=4). To assess the mechanism of PPP, a non-selective, stretch-activated channel blocker, amiloride, was administered during PPP (n=3). Peak systolic and diastolic arterial pressures during S-CPR, PPP and ventricular electrical pacing were statistically similar. However, the duration of developed arterial pressure with PPP was comparable to that with ventricular electrical pacing, and significantly greater than that with S-CPR. Amiloride decreased the induction rate of ventricular electrical activity by PPP in a dose-related manner. Each animal survived without any neurological deficit at 24, 48 h and 1 week, even with up to 2 h of continuous PPP. In amicrominipigmodel of cardiac standstill, PPP can become a novel means to significantly improve physiological outcomes after cardiac standstill or symptomatic bradyarrhythmias in the absence of cardiac pacing. Activation of the non-selective stretch-activated channels may mediate some of the mechanophysiological effects of PPP. Further study of PPP by itself and together with S-CPR is warranted using cardiac arrest models of atrioventricular block and asystole.

  8. Thyroid hormones increase Na -H exchange activity in renal brush border membranes

    Energy Technology Data Exchange (ETDEWEB)

    Kinsella, J.; Sacktor, B.

    1985-06-01

    Na -H exchange activity, i.e., amiloride-sensitive Na and H flux, in renal proximal tubule brush border (luminal) membrane vesicles was increased in the hyperthyroid rat and decreased in the hypothyroid rat, relative to the euthyroid animal. A positive correlation was found between Na -H exchange activity and serum concentrations of thyroxine (T4) and triiodothyronine (T3). The thyroid status of the animal did not alter amiloride-insensitive Na uptake. The rate of passive pH gradient dissipation was higher in membrane vesicles from hyperthyroid rats compared to the rate in vesicles from hypothyroid animals, a result which would tend to limit the increase in Na uptake in vesicles from hyperthyroid animals. Na -dependent phosphate uptake was increased in membrane vesicles from hyperthyroid rats; Na -dependent D-glucose and L-proline uptakes were not changed by the thyroid status of the animal. The effect of thyroid hormones in increasing the uptake of Na in the brush border membrane vesicle is consistent with the action of the hormones in enhancing renal Na reabsorption.

  9. Nasal potential difference outcomes support diagnostic decisions in cystic fibrosis.

    Science.gov (United States)

    Tridello, Gloria; Menin, Laura; Pintani, Emily; Bergamini, Gabriella; Assael, Baroukh Maurice; Melotti, Paola

    2016-09-01

    When cystic fibrosis (CF) is suspected Nasal Potential Difference (NPD) measurements are proposed to support controversial diagnosis: we investigated appropriate outcomes at the CF Centre of Verona. NPD were measured in 196 subjects: 50 non-CF, 65 classical CF (the reference group) and 81 with uncertain CF (case group). Discriminating power was determined by comparison between several outcomes from the CF reference group versus non-CF: basal, amiloride, 0Cl, isoproterenol, ATP, Delta-amiloride, Delta-0Cl, Delta-isoproterenol, Delta-ATP, Delta-isoproterenol+Delta-0Cl, Wilschanski Index (WI) and Sermet score (SS). The most appropriate cut-off values for variables with the best discriminating power were then applied to the case group. Descriptive statistics, logistic regression models and ROC curve analysis were applied. WI and SS were the most powerful in discriminating CF from non-CF subjects. In the reference group sensitivity of the 0.82 WI cut-off was 98%, specificity 96%; both sensitivity and specificity of the -0.44 SS cut-off value were 100%. For the case group, WI and SS were, respectively, consistent with CF diagnosis in 94% and 92% of the cases. Formulae have the highest discriminating power and can support the diagnosis in uncertain cases; they should be utilized for standardized interpretation of NPD for diagnosis and possibly for clinical research. Copyright © 2016. Published by Elsevier B.V.

  10. Multivariate curve resolution of incomplete fused multiset data from chromatographic and spectrophotometric analyses for drug photostability studies

    Energy Technology Data Exchange (ETDEWEB)

    Luca, Michele De, E-mail: michele.deluca@unical.it [Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Via P. Bucci, Rende, CS 87036 (Italy); Ragno, Gaetano; Ioele, Giuseppina [Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Via P. Bucci, Rende, CS 87036 (Italy); Tauler, Romà [Department of Environmental Chemistry, IDAEA-CSIC, C/Jordi Girona, 18-26, Barcelona 08034 (Spain)

    2014-07-21

    Highlights: • A new MCR-ALS algorithm is proposed for the analysis of incomplete fused multiset. • Resolution of the data allowed the description of amiloride kinetic photodegradation. • The new MCR-ALS algorithm can be easily applied to other drugs and chemicals. - Abstract: An advanced and powerful chemometric approach is proposed for the analysis of incomplete multiset data obtained by fusion of hyphenated liquid chromatographic DAD/MS data with UV spectrophotometric data from acid–base titration and kinetic degradation experiments. Column- and row-wise augmented data blocks were combined and simultaneously processed by means of a new version of the multivariate curve resolution-alternating least squares (MCR-ALS) technique, including the simultaneous analysis of incomplete multiset data from different instrumental techniques. The proposed procedure was applied to the detailed study of the kinetic photodegradation process of the amiloride (AML) drug. All chemical species involved in the degradation and equilibrium reactions were resolved and the pH dependent kinetic pathway described.

  11. Clathrin-mediated entry and cellular localization of chlorotoxin in human glioma

    Directory of Open Access Journals (Sweden)

    Johnson Joseph O

    2011-08-01

    Full Text Available Abstract Background Chlorotoxin (TM601, a scorpion venom- derived 36-AA peptide, is an experimental drug against recurrent glioma with tumor specificity but unknown route of intracellular distribution. The aim of this study was to evaluate the route of entry and cellular localization of TM601 in glioma cells. Results We have found that in human gliomas, lung carcinoma and normal vascular endothelial cells, TM601 localizes near trans-Golgi while in normal human dermal fibroblasts (NHDF and astrocytes it is dispersed in the cytoplasm. The uptake of TM601 by U373 glioma cells is rapid, concentration and time dependent, not affected by inhibitors such as filipin (caveolae-dependent endocytosis and amiloride (non-selective macropinocytosis, but significantly affected by chlorpromazine (clathrin-dependent intracellular transport of coated pits resulting in intracellular build-up of the drug and clathrin near the Golgi. In contrast, TM601 uptake by NHDF cells was significantly affected by amiloride indicating that macropinocytosis is the dominant uptake route of TM601 in these cells. Conclusions In conclusion, we found a distinct cellular localization pattern and uptake of TM601 by glioma cells differing from that found in normal cells. Further insight into the cellular processing of TM601 should assist in the development of effective anti-glioma therapeutic modalities.

  12. ASIC1a Deficient Mice Show Unaltered Neurodegeneration in the Subacute MPTP Model of Parkinson Disease.

    Directory of Open Access Journals (Sweden)

    Daniel Komnig

    Full Text Available Inflammation contributes to the death of dopaminergic neurons in Parkinson disease and can be accompanied by acidification of extracellular pH, which may activate acid-sensing ion channels (ASIC. Accordingly, amiloride, a non-selective inhibitor of ASIC, was protective in an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP mouse model of Parkinson disease. To complement these findings we determined MPTP toxicity in mice deficient for ASIC1a, the most common ASIC isoform in neurons. MPTP was applied i.p. in doses of 30 mg per kg on five consecutive days. We determined the number of dopaminergic neurons in the substantia nigra, assayed by stereological counting 14 days after the last MPTP injection, the number of Nissl positive neurons in the substantia nigra, and the concentration of catecholamines in the striatum. There was no difference between ASIC1a-deficient mice and wildtype controls. We are therefore not able to confirm that ASIC1a are involved in MPTP toxicity. The difference might relate to the subacute MPTP model we used, which more closely resembles the pathogenesis of Parkinson disease, or to further targets of amiloride.

  13. Angiotensin II and taste sensitivity

    Directory of Open Access Journals (Sweden)

    Noriatsu Shigemura, DDS, PhD

    2015-05-01

    Full Text Available The sense of taste plays a major role in evaluating the quality of food components in the oral cavity. Sweet, salty, umami, sour and bitter taste are generally accepted as five basic taste qualities. Among them, salty taste is attractive to animals and influences sodium intake. Angiotensin II (ANG II and aldosterone (ALDO, which is stimulated by ANG II are key hormones that regulate sodium homeostasis and water balance. At the peripheral gustatory organs, it has been reported that ALDO increases the amiloride-sensitivity of the rat gustatory neural responses to NaCl in a time course of several hours. A recent study demonstrated that ANG II suppresses amiloride-sensitivity of the mouse gustatory and behavioral responses to NaCl via its receptor AT1 within an hour. Moreover, ANG II enhances sweet taste sensitivity without affecting umami, sour and bitter tastes. These results suggest that the reciprocal and sequential regulatory mechanisms by ANG II (as an acute suppressor together with ALDO (as a slow enhancer on the salt taste sensitivity may exist in peripheral taste organs, contribute to salt intake, and play an important role in sodium homeostasis. Furthermore, the linkage between salty and sweet taste modulations via the ANG II signaling may optimize sodium and calorie intakes.

  14. Use of acetazolamide in lithium-induced nephrogenic diabetes insipidus: a case report.

    Science.gov (United States)

    Macau, Ricardo A; da Silva, Tiago Nunes; Silva, Joana Rego; Ferreira, Ana Gonçalves; Bravo, Pedro

    2018-01-01

    Lithium-induced nephrogenic diabetes insipidus (Li-NDI) is a rare and difficult-to-treat condition. A study in mice and two recent papers describe the use of acetazolamide in Li-NDI in 7 patients (a case report and a 6 patient series). We describe the case of a 63-year-old woman with bipolar disorder treated with lithium and no previous history of diabetes insipidus. She was hospitalized due to a bowel obstruction and developed severe dehydration after surgery when she was water deprived. After desmopressin administration and unsuccessful thiazide and amiloride treatment, acetazolamide was administrated to control polyuria and hydroelectrolytic disorders without significant side effects. To our knowledge, this is the third publication on acetazolamide use in Li-NDI patients. Treatment of lithium-induced nephrogenic diabetes insipidus might be challenging.Vasopressin, amiloride and thiazide diuretics have been used in lithium-induced nephrogenic diabetes insipidus treatment.Acetazolamide might be an option to treat lithium-induced nephrogenic diabetes insipidus patients who fail to respond to standard treatment.The use of acetazolamide in lithium-induced nephrogenic diabetes insipidus must be monitored, including its effects on glomerular filtration rate.

  15. The prevalence of major potential drug-drug interactions at a University health centre pharmacy in Jamaica

    Directory of Open Access Journals (Sweden)

    Kennedy-Dixon T

    2015-12-01

    Full Text Available Objective: To identify major potential drug-drug interactions (DDIs on prescriptions filled at the University Health Centre Pharmacy, Mona Campus, Jamaica. Methods: This investigation utilised a cross-sectional analysis on all prescriptions with more than one drug that were filled at the Health Centre Pharmacy between November 2012 and February 2013. Potential DDIs were identified using the online Drug Interactions Checker database of Drugs.com. Results: During the period of the study, a total of 2814 prescriptions were analysed for potential DDIs. The prevalence of potential DDIs found during the study period was 49.82%. Major potential DDIs accounted for 4.7 % of the total number of interactions detected, while moderate potential DDIs and minor potential DDIs were 80.8 % and 14.5 % respectively. The three most frequently occurring major potential DDIs were amlodipine and simvastatin (n=46, amiloride and losartan (n=27 and amiloride and lisinopril (n=16. Conclusion: This study has highlighted the need for educational initiatives to ensure that physicians and pharmacists collaborate in an effort to minimise the risks to the patients. These interactions are avoidable for the most part, as the use of online tools can facilitate the selection of therapeutic alternatives or guide decisions for closer patient monitoring and thus reduce the risks of adverse events.

  16. Multivariate curve resolution of incomplete fused multiset data from chromatographic and spectrophotometric analyses for drug photostability studies

    International Nuclear Information System (INIS)

    Luca, Michele De; Ragno, Gaetano; Ioele, Giuseppina; Tauler, Romà

    2014-01-01

    Highlights: • A new MCR-ALS algorithm is proposed for the analysis of incomplete fused multiset. • Resolution of the data allowed the description of amiloride kinetic photodegradation. • The new MCR-ALS algorithm can be easily applied to other drugs and chemicals. - Abstract: An advanced and powerful chemometric approach is proposed for the analysis of incomplete multiset data obtained by fusion of hyphenated liquid chromatographic DAD/MS data with UV spectrophotometric data from acid–base titration and kinetic degradation experiments. Column- and row-wise augmented data blocks were combined and simultaneously processed by means of a new version of the multivariate curve resolution-alternating least squares (MCR-ALS) technique, including the simultaneous analysis of incomplete multiset data from different instrumental techniques. The proposed procedure was applied to the detailed study of the kinetic photodegradation process of the amiloride (AML) drug. All chemical species involved in the degradation and equilibrium reactions were resolved and the pH dependent kinetic pathway described

  17. Impaired sodium excretion and salt-sensitive hypertension in corin-deficient mice.

    Science.gov (United States)

    Wang, Wei; Shen, Jianzhong; Cui, Yujie; Jiang, Jingjing; Chen, Shenghan; Peng, Jianhao; Wu, Qingyu

    2012-07-01

    Corin is a protease that activates atrial natriuretic peptide, a cardiac hormone important in the control of blood pressure and salt-water balance. Here we examined the role of corin in regulating blood pressure and sodium homeostasis upon dietary salt challenge. Radiotelemetry-tracked blood pressure in corin knockout mice on a high-salt diet (4% sodium chloride) was significantly increased; however, there was no such change in similarly treated wild-type mice. In the knockout mice on the high-salt diet there was an impairment of urinary sodium excretion and an increase in body weight, but no elevation of plasma renin or serum aldosterone levels. When the knockout mice on the high-salt diet were treated with amiloride, an epithelial sodium channel blocker that inhibits renal sodium reabsorption, the impaired urinary sodium excretion and increased body weight were normalized. Amiloride treatment also reduced high blood pressure caused by the high-salt diet in these mice. Thus, the lack of corin in mice impairs their adaptive renal response to high dietary salt, suggesting that corin deficiency may represent an important mechanism underlying salt-sensitive hypertension.

  18. Hormonal regulation of Na -K -ATPase in cultured epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, J.P.; Jones, D.; Wiesmann, W.P.

    1986-08-01

    Aldosterone and insulin stimulate Na transport through mechanisms involving protein synthesis. Na -K -ATPase has been implicated in the action of both hormones. The authors examined the effect of aldosterone and insulin on Na -K -ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (I/sub sc/) in TB6C cells. Aldosterone increases Na -K -(TSP)ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in I/sub sc/, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I/sub sc/ in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na -K -ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na -K -ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on I/sub sc/.

  19. Long-term alterations in peripheral taste responses to NaCl in adult rats following neonatal chorda tympani transection.

    Science.gov (United States)

    Martin, Louis J; Sollars, Suzanne I

    2015-02-01

    The peripheral taste system of the adult rodent is highly resilient against damage, with morphological, behavioral, and functional recovery evident after regeneration of a transected nerve. If chorda tympani transection (CTX) occurs at early postnatal ages however, the nerve fails to regenerate and effects on tongue morphology and behavior are more severe and longer-lasting compared to adult denervation. To examine whether neonatal CTX induces functional changes in intact nerves, whole-nerve electrophysiology was performed on the glossopharyngeal (GL) and chorda tympani (CT) nerves of adult rats that received CTX at P10. Attenuation of NaCl-elicited GL responses were observed in CTX rats 2 months after surgery, with bilateral denervation causing the largest decreases in responses. When assessed 1 year after neonatal CTX, amiloride-sensitive responses to NaCl in the contralateral CT increased while amiloride-insensitive responses decreased. Responses to other tastants were consistent with control animals. This is the first evidence of long-term functional changes to the peripheral taste system after injury in rats fed a normal diet. This study further characterizes the developing peripheral taste system as highly susceptible to change following neural injury. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  20. Ca-sensitive sodium absorption in the colon of Xenopus laevis.

    Science.gov (United States)

    Krattenmacher, R; Voigt, R; Clauss, W

    1990-01-01

    Transepithelial electrogenic Na transport (INa) was investigated in the colon of the frog Xenopus laevis with electrophysiological methods in vitro. The short circuit current (Isc) of the voltage-clamped tissue was 24.2 +/- 1.8 microA.cm-2 (n = 10). About 60% of this current was generated by electrogenic Na transport. Removal of Ca2+ from the mucosal Ringer solution stimulated INa by about 120%. INa was not blockable by amiloride (0.1 mmol.l-1), a specific Na-channel blocker in epithelia, but a fully and reversible inhibition was achieved by mucosal application of 1 mmol.l-1 lanthanum (La3+). No Na-self-inhibition was found, because INa increased linearly with the mucosal Na concentration. A stimulation of INa by antidiuretic hormones was not possible. The analysis of fluctuations in the short circuit current (noise analysis) indicated that Na ions pass the apical cell membrane via a Ca-sensitive ion channel. The results clearly demonstrate that in the colon of Xenopus laevis Na ions are absorbed through Ca-sensitive apical ion channels. They differ considerably in their properties and regulation from the amiloride-sensitive Na channel which is "typically" found in the colon of vertebrates.

  1. Double-stranded RNA induces similar pulmonary dysfunction to respiratory syncytial virus in BALB/c mice.

    Science.gov (United States)

    Aeffner, Famke; Traylor, Zachary P; Yu, Erin N Z; Davis, Ian C

    2011-07-01

    Both respiratory syncytial virus (RSV) and influenza A virus induce nucleotide/P2Y purinergic receptor-mediated impairment of alveolar fluid clearance (AFC), which contributes to formation of lung edema. Although genetically dissimilar, both viruses generate double-stranded RNA replication intermediates, which act as Toll-like receptor (TLR)-3 ligands. We hypothesized that double-stranded RNA/TLR-3 signaling underlies nucleotide-mediated inhibition of amiloride-sensitive AFC in both infections. We found that addition of the synthetic double-stranded RNA analog poly-inosinic-cytidylic acid [poly(I:C)] (500 ng/ml) to the AFC instillate resulted in nucleotide/P2Y purinergic receptor-mediated inhibition of amiloride-sensitive AFC in BALB/c mice but had no effect on cystic fibrosis transmembrane regulator (CFTR)-mediated Cl(-) transport. Poly(I:C) also induced acute keratinocyte cytokine-mediated AFC insensitivity to stimulation by the β-adrenergic agonist terbutaline. Inhibitory effects of poly(I:C) on AFC were absent in TLR-3(-/-) mice and were not replicated by addition to the AFC instillate of ligands for other TLRs except TLR-2. Intranasal poly(I:C) administration (250 μg/mouse) similarly induced nucleotide-dependent AFC inhibition 2-3 days later, together with increased lung water content and neutrophilic inflammation. Intranasal treatment of BALB/c mice with poly(I:C) did not induce airway hyperresponsiveness at day 2 but did result in insensitivity to airway bronchodilation by β-adrenergic agonists. These findings suggest that viral double-stranded RNA replication intermediates induce nucleotide-mediated impairment of amiloride-sensitive AFC in both infections, together with β-adrenergic agonist insensitivity. Both of these effects also occur in RSV infection. However, double-stranded RNA replication intermediates do not appear to be sufficient to induce either adenosine-mediated, CFTR-dependent Cl(-) secretion in the lung or severe, lethal hypoxemia, both

  2. DSC, FT-IR, NIR, NIR-PCA and NIR-ANOVA for determination of chemical stability of diuretic drugs: impact of excipients

    Directory of Open Access Journals (Sweden)

    Gumieniczek Anna

    2018-03-01

    Full Text Available It is well known that drugs can directly react with excipients. In addition, excipients can be a source of impurities that either directly react with drugs or catalyze their degradation. Thus, binary mixtures of three diuretics, torasemide, furosemide and amiloride with different excipients, i.e. citric acid anhydrous, povidone K25 (PVP, magnesium stearate (Mg stearate, lactose, D-mannitol, glycine, calcium hydrogen phosphate anhydrous (CaHPO4 and starch, were examined to detect interactions. High temperature and humidity or UV/VIS irradiation were applied as stressing conditions. Differential scanning calorimetry (DSC, FT-IR and NIR were used to adequately collect information. In addition, chemometric assessments of NIR signals with principal component analysis (PCA and ANOVA were applied.

  3. Where have all the Na+ channels gone? In search of functional ENaC in exocrine pancreas

    DEFF Research Database (Denmark)

    Novak, Ivana; Hansen, Mette R

    2002-01-01

    was to investigate if pancreatic ducts express functional ENaC. Membrane voltages (V) of ducts isolated from rat pancreas were measured with microelectrodes or whole-cell patch-clamp technique. Amiloride and benzamil given from bath or luminal sides did not hyperpolarize V. Lowering of extracellular Na...... with glucocorticoids had no effect on pancreatic fluid secretion evoked from ducts, or from acini. Hence, our study shows that pancreas especially pancreatic ducts do not express functional ENaC.......(+) concentrations had effects that were not consistent with a simple Na(+) conductance, but rather with a Na(+)/Ca(2+) exchange. Acute or long-lasting treatment of pancreatic ducts with mineralocorticoids had no effect on V of unstimulated or secretin-stimulated preparations. Furthermore, pre-treatment of animals...

  4. Steady-state sodium absorption and chloride secretion of colon and coprodeum, and plasma levels of osmoregulatory hormones in hens in relation to sodium intake.

    Science.gov (United States)

    Arnason, S S; Skadhauge, E

    1991-01-01

    The plasma levels of four osmoregulatory hormones and their target ion-transport systems in the lower intestines of the domestic fowl were determined in order to elucidate their interrelationship and their setpoints in relation to NaCl intake. White Plymouth Rock hens were adapted to six intake levels of NaCl (0.20 +/- 0.02-24.7 +/- 1.9 mmoles Na+.kg bw-1.day-1) for 6 weeks. The Na+ absorption and the Cl- secretion of colon and coprodeum were characterized in vitro by the effects of hexoses, amino acids, amiloride, and theophylline on the short-circuit current (SCC) and electrical potential difference (PD). The NaCl-conserving system of the adult chicken is set at low intake levels of NaCl as the 80% range (quantized by non-linear, logistic regression analyses) of the change in the plasma [ALDO], the amiloride-inhibitable Na+ absorption of coprodeum and colon (delta SCC), occurred from 0.18 to 2.3, from 0.9 to 4.3, and from 1.2 to 7.3 mmoles Na+.kg bw-1.day-1, respectively. These results demonstrate that the amiloride-inhibitable Na+ absorption of coprodeum is more closely linked to plasma [ALDO] than that of colon. The aminoacid-Na+ coabsorption of colon increased over exactly the same range of Na+ intake as the colonic amiloride-inhibitable Na+ absorption decreased, whereas the hexose-Na+ coabsorption increased at higher levels of Na+ coabsorption increased at higher levels of Na+ intake, from 2 to 11 mmoles Na+.kg bw-1.day-1. Both these Na+ absorption types had reached their maximums at 24.7 mmoles Na+.kg bw-1.day-1, whereas the plasma [AVT] and plasma [PRL], although significantly increased, apparently had not; their 80% range of change occurred from 9.9 to 99 mmoles Na+.kg bw-1.day-1, and the main changes in plasma osmolality were predicted to occur from 5.4 to 107 mmoles Na+.kg bw-1.day-1. These results suggest that these colonic and hormonal variables conserve osmotically-free water and operate at high NaCl intake. The theophylline-induced colonic Cl

  5. Electrophysiological study of transport systems in isolated perfused pancreatic ducts: properties of the basolateral membrane

    DEFF Research Database (Denmark)

    Novak, I; Greger, R

    1988-01-01

    short circuit current (Isc) under similar conditions was 26 and 50 microA . cm-2. The specific transepithelial resistance (Rte) was 88 omega cm2. In control solutions the PD across the basolateral membrane (PDbl) was -63 +/- 1 mV (n = 314). Ouabain (3 mmol/l) depolarized PDbl by 4.8 +/- 1.1 mV (n = 6.......4 +/- 1.4 mV (n = 19), while another K+ channel blocker TEA+ (50 mmol/l) depolarized PDbl only by 7.7 +/- 2.0 mV (n = 9). Addition of amiloride (1 mmol/l) to the bath caused 3-4 mV depolarization of PDbl. Furosemide (0.1 mmol/l) and SITS (0.1 mmol/l) had no effect on PDbl. An increase in the bath HCO3...

  6. Mechanisms of renal NaCl retention in proteinuric disease

    DEFF Research Database (Denmark)

    Svenningsen, Per; Friis, Ulla G; Versland, Jostein B

    2013-01-01

    In diseases with proteinuria, for example nephrotic syndrome and pre-eclampsia, there often are suppression of plasma renin-angiotensin-aldosterone system components, expansion of extracellular volume and avid renal sodium retention. Mechanisms of sodium retention in proteinuria are reviewed....... In animal models of nephrotic syndrome, the amiloride-sensitive epithelial sodium channel ENaC is activated while more proximal renal Na(+) transporters are down-regulated. With suppressed plasma aldosterone concentration and little change in ENaC abundance in nephrotic syndrome, the alternative modality...... of proteolytic activation of ENaC has been explored. Proteolysis leads to putative release of an inhibitory peptide from the extracellular domain of the gamma ENaC subunit. This leads to full activation of the channel. Plasminogen has been demonstrated in urine from patients with nephrotic syndrome and pre...

  7. Cation transport by sweat ducts in primary culture. Ionic mechanism of cholinergically evoked current oscillations

    DEFF Research Database (Denmark)

    Larsen, Erik Hviid; Novak, I; Pedersen, P S

    1990-01-01

    V and a fractional resistance of the apical membrane, fR = 0.59 +/- 0.01 (n = 115 cells). 4. The Na+ channel blocker amiloride (mucosal bath, 10 microM) abolished Isc -0.8 +/- 0.6 microA cm-2), the cells hyperpolarized to -61.0 +/- 1.2 mV, and fR increased to 0.85 +/- 0.01 (n = 44). These effects were fully...... with methacholine an increase in mucosal potassium concentration ([K+]m) from 5 to 25 mM had no significant effect, while a similar increase in the serosal K+ concentration ([K+]s) produced a change in Vc of 44 +/- 3 mV per log10[K+]s (n = 9). In non-stimulated preparations this change was only 16 +/- 2 mV per log...

  8. Multivariate curve resolution of incomplete fused multiset data from chromatographic and spectrophotometric analyses for drug photostability studies.

    Science.gov (United States)

    De Luca, Michele; Ragno, Gaetano; Ioele, Giuseppina; Tauler, Romà

    2014-07-21

    An advanced and powerful chemometric approach is proposed for the analysis of incomplete multiset data obtained by fusion of hyphenated liquid chromatographic DAD/MS data with UV spectrophotometric data from acid-base titration and kinetic degradation experiments. Column- and row-wise augmented data blocks were combined and simultaneously processed by means of a new version of the multivariate curve resolution-alternating least squares (MCR-ALS) technique, including the simultaneous analysis of incomplete multiset data from different instrumental techniques. The proposed procedure was applied to the detailed study of the kinetic photodegradation process of the amiloride (AML) drug. All chemical species involved in the degradation and equilibrium reactions were resolved and the pH dependent kinetic pathway described. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Flow cytometry evidence of human granulocytes interaction with polyhedral oligomeric silsesquioxanes: effect of nanoparticle charge

    Science.gov (United States)

    Renò, Filippo; Carniato, Fabio; Rizzi, Manuela; Olivero, Francesco; Pittarella, Pamela; Marchese, Leonardo

    2013-05-01

    Nanoparticles (NPs) entering the human body are immediately confronted with the innate part of human immune system. In particular, monocyte and neutrophil granulocytes readily clear particles by phagocytosis, even if in the case of NPs the uptake mechanism may be classified as macropinocytosis. Among engineered nanoparticles, in the last years, siliceous materials have emerged as promising materials for several applications ranging from catalysis to biomedical. The polyhedral oligomeric silsesquioxanes (POSS) are nanodimensional, easily synthesizable molecular compounds and POSS-based systems are promising carriers for biological molecules. In this work, the ability of human granulocytes to uptake positively and negatively charged POSS was measured using a simple flow cytometry analysis based on cell size modifications. The data obtained showed that after a 30 min exposure only positive NPs were uptaken by human granulocyte using both macropinocytosis and clathrin-mediated mechanisms as demonstrated by uptake inhibition mediated by amiloride and chlorpromazine.

  10. Na+-H+ exchange and Na+-dependent transport systems in streptozotocin diabetic rat kidneys

    International Nuclear Information System (INIS)

    El-Seifi, S.; Freiberg, J.M.; Kinsella, F.J.; Cheng, L.; Sacktor, B.

    1987-01-01

    The streptozotocin-induced diabetic rat was used to test the hypothesis that Na + -H + exchange activity in the proximal tubule luminal membrane would be increased in association with renal hypertrophy, altered glomerular hemodynamics, enhanced filtered load and tubular reabsorption of 22 Na + , and stimulated 22 Na= pump activity in the basolateral membrane, previously reported characteristics of this experimental animal model. Amiloride-sensitive H + gradient-dependent Na + uptake and Na + gradient-dependent H + flux were increased in brush-border membrane vesicles from the streptozotocin-treated animals. Na + gradient-dependent uptakes of phosphate, D-glucose, L-proline, and myoinositol were decreased in the drug-induced diabetic animals. These membrane transport alterations were not found when the streptozotocin-diabetic animals were treated with insulin

  11. Regulation of electrolyte transport with IL-1β in rabbit distal colon

    Directory of Open Access Journals (Sweden)

    F. R. Homaidan

    1995-01-01

    Full Text Available Interletrkin-1β levels are elevated in inflammatory bowel disease. In this study the mechanism by which interleukin-1β affects electrolyte transport in the rabbit distal colon, was investigated. Interleukin-1β caused a delayed increase in short-circuit current (Isc which was attributed to protein synthesis since the effect was inhibited by cycloheximide. The interleukin-1β induced increase in Isc was not affected by amiloride treatment but was completely inhibited by bumetanide or in chloride-free buffer and by indomethacin. Prostaglandin E2 levels increased in tissue treated with interleukin-1β, but this increase was reversed by cycloheximide. These data suggest that interleukin-1β causes its effect via a yet to be identified second messenger, by increasing chloride secretion through a prostaglandin E2 mediated mechanism.

  12. Influence of bicarbonate on the sensitivity of renin release to sodium chloride

    DEFF Research Database (Denmark)

    Skøtt, O; Jensen, B L

    1989-01-01

    glomeruli treated with bicarbonate/chloride exchange inhibitor (DNDS), NaCl/KCl cotransport inhibitor (bumetanide), or Na+/H+ antiport inhibitor (amiloride) in the presence or absence of bicarbonate. In addition, the sensitivity to increases in osmolality by addition of sucrose was tested in the presence...... or absence of bicarbonate. Renin release from time controls superfused with a bicarbonate-free Ringer was identical to release from glomeruli superfused with a bicarbonate Ringer. DNDS (0.11 or 1.1 mM) had no effect on renin release in a bicarbonate Ringer. 30 mM sucrose inhibited renin release independently...... of bicarbonate. 15 mM NaCl stimulated renin release when bicarbonate was absent, while it caused an inhibition in the presence of bicarbonate. When bicarbonate/chloride exchange was inhibited, addition of NaCl stimulated renin release even when bicarbonate was present. The effect of NaCl on renin release...

  13. Use of Ion-Channel Modulating Agents to Study Cyanobacterial Na+ - K+ Fluxes

    Directory of Open Access Journals (Sweden)

    Pomati Francesco

    2004-01-01

    Full Text Available Here we describe an experimental design aimed to investigate changes in total cellular levels of Na+ and K+ ions in cultures of freshwater filamentous cyanobacteria. Ion concentrations were measured in whole cells by flame photometry. Cellular Na+ levels increased exponentially with rising alkalinity, with K+ levels being maximal for optimal growth pH (~8. At standardized pH conditions, the increase in cellular Na+, as induced by NaCl at 10 mM, was coupled by the two sodium channel-modulating agents lidocaine hydrochloride at 1 &mgr;M and veratridine at 100 &mgr;M. Both the channel-blockers amiloride (1 mM and saxitoxin (1 &mgr;M, decreased cell-bound Na+ and K+ levels. Results presented demonstrate the robustness of well-defined channel blockers and channel-activators in the study of cyanobacterial Na+- K+ fluxes.

  14. Perspectives on edema in childhood nephrotic syndrome.

    Science.gov (United States)

    Teoh, Chia Wei; Robinson, Lisa A; Noone, Damien

    2015-10-01

    There have been two major theories surrounding the development of edema in nephrotic syndrome (NS), namely, the under- and overfill hypotheses. Edema is one of the cardinal features of NS and remains one of the principal reasons for admission of children to the hospital. Recently, the discovery that proteases in the glomerular filtrate of patients with NS are activating the epithelial sodium channel (ENaC), resulting in intrarenal salt retention and thereby contributing to edema, might suggest that targeting ENaC with amiloride might be a suitable strategy to manage the edema of NS. Other potential agents, particularly urearetics and aquaretics, might also prove useful in NS. Recent evidence also suggests that there may be other areas involved in salt storage, especially the skin, and it will be intriguing to study the implications of this in NS. Copyright © 2015 the American Physiological Society.

  15. Two independent anion transport systems in rabbit mandibular salivary glands

    DEFF Research Database (Denmark)

    Novak, I; Young, J A

    1986-01-01

    Cholinergically stimulated Cl and HCO3 transport in perfused rabbit mandibular glands has been studied with extracellular anion substitution and administration of transport inhibitors. In glands perfused with HCO3-free solutions, replacement of Cl with other anions supported secretion in the foll......Cholinergically stimulated Cl and HCO3 transport in perfused rabbit mandibular glands has been studied with extracellular anion substitution and administration of transport inhibitors. In glands perfused with HCO3-free solutions, replacement of Cl with other anions supported secretion...... stimulated secretion by about 30%, but when infused in addition to furosemide (0.1 mmol/l), it inhibited by about 20%. Amiloride (1.0 mmol/l) caused no inhibition. The results suggest that there are at least three distinct carriers in the rabbit mandibular gland. One is a furosemide-sensitive Na-coupled Cl...

  16. An Ursolic Acid Derived Small Molecule Triggers Cancer Cell Death through Hyperstimulation of Macropinocytosis.

    Science.gov (United States)

    Sun, Lin; Li, Bin; Su, Xiaohui; Chen, Ge; Li, Yaqin; Yu, Linqian; Li, Li; Wei, Wanguo

    2017-08-10

    Macropinocytosis is a transient endocytosis that internalizes extracellular fluid and particles into vacuoles. Recent studies suggest that hyperstimulation of macropinocytosis can induce a novel nonapoptotic cell death, methuosis. In this report, we describe the identification of an ursolic acid derived small molecule (compound 17), which induces cancer cell death through hyperstimulation of macropinocytosis. 17 causes the accumulation of vacuoles derived from macropinosomes based on transmission electron microscopy, time-lapse microscopy, and labeling with extracellular fluid phase tracers. The vacuoles induced by 17 separate from other cytoplasmic compartments but acquire some characteristics of late endosomes and lysosomes. Inhibiting hyperstimulation of macropinocytosis with the specific inhibitor amiloride blocks cell death, implicating that 17 leads to cell death via macropinocytosis, which is coincident with methuosis. Our results uncovered a novel cell death pathway involved in the activity of 17, which may provide a basis for further development of natural-product-derived scaffolds for drugs that trigger cancer cell death by methuosis.

  17. [Elective caesarean: impact of evolution neonatal respiration].

    Science.gov (United States)

    Jonguitud Aguilar, Adriana

    2011-04-01

    The number of Caesarean births has increased worldwide. Respiratory distress syndrome is associated with caesarean delivery, especially in the absence of labor. During the latter stages of pregnancy physiologic changes occur that are accelerated with the onset of labor, which is accompanied by changes in hormones and mediators in the mother and fetus. An acceleration in the evacuation of lung liquid is held in this period, largely dependent sodium channels sensitive to amiloride than are found in the alveolar epithelium. The failure of these mechanisms can lead to newborn severe respiratory difficulty and require intensive care, mechanical ventilation and surfactant. We need to develop preventive and therapeutic strategies to improve outcomes in this vulnerable population.

  18. Potassium transport across guinea pig distal colon

    International Nuclear Information System (INIS)

    Rechkemmer, G.; Halm, D.R.; Frizzell, R.A.

    1986-01-01

    Active absorption and secretion of K was studied by measuring bidirectional 42 K fluxes across short-circuited guinea pig distal colon. Tissues were pretreated with mucosal (m) and serosal (s) indomethacin (1 μM) and amiloride (0.1 mM, m) to suppress spontaneous, electrogenic Cl secretion and Na absorption. Under these conditions, the short-circuit current (I/sub sc/) was 0.4 μeq/cm 2 h while electroneutral K absorption was 2.8 μeq/cm 2 h. Epinephrine (5 μM, s) stimulated electrogenic K secretion, reducing net K absorption to 1.3 μeq/cm 2 h. Bumetanide (0.1 mM, s) abolished this K secretion and restored K absorption to control values, suggesting mechanistic similarities between K and Cl secretion. K absorption was inhibited 40% by the gastric H/K ATPase inhibitor, omeprazole (0.1 mM, m), and was abolished by ouabain (0.1 mM, m). Neutral K absorption does not appear to be mediated by an apical membrane Na/K pump since: the effect of mucosal ouabain on K absorption does not require the presence of mucosal or serosal Na, unidirectional Na fluxes are not influenced by mucosal ouabain, and K absorption is not affected when Na absorption is abolished by amiloride. Net K transport is determined by the balance between electroneutral K absorption and electrogenic K secretion. The ouabain sensitivity of K absorption suggests that colonic H/K ATPase differs from its gastric counterpart

  19. Mechanism of norepinephrine release elicited by renal nerve stimulation, veratridine and potassium chloride in the isolated rat kidney

    International Nuclear Information System (INIS)

    el-Din, M.M.; Malik, K.U.

    1987-01-01

    We have investigated the mechanism by which renal nerve stimulation (RNS), veratridine (Vt) and KCl promote release of norepinephrine in the isolated rat kidney perfused with Tyrode's solution and prelabeled with [ 3 H]norepinephrine by examining the overflow of tritium elicited by these stimuli during 1) extracellular Ca++ depletion, 2) alterations in extracellular Na+ concentration and 3) administration of tetrodotoxin, amiloride, LiCl and calcium channel blockers. RNS (1-4 Hz), Vt (15-90 nmol) and KCl (150-500 mumol) produced renal vasoconstriction and enhanced the tritium overflow in a frequency- and concentration-dependent manner, respectively. Omission of Ca++ (1.8 mM) from the perfusion fluid abolished the renal vasoconstriction and the increase in tritium overflow elicited by RNA and KCl and substantially reduced that caused by Vt. Lowering the Na+ concentration in the perfusion medium (from 150 to 25 mM) reduced the overflow of tritium and the renal vasoconstriction caused by RNS (2 Hz) or Vt (45 nmol); the increase in tritium overflow in response to these stimuli was positively correlated with extracellular Na+ (25-150 mM). In contrast, KCl-induced tritium overflow was negatively correlated with extracellular Na+ concentration. Tetrodotoxin (0.3 microM) abolished the effect of RNS and Vt, but not that of KCl, to increase overflow of tritium and to produce renal vasoconstriction. Administration of amiloride (180 microM) enhanced the overflow of tritium but attenuated the associated renal vasoconstriction produced by RNS, Vt and KCl. Replacement of NaCl (75 mM) with equimolar concentration of LiCl enhanced the overflow of tritium elicited by RNS, Vt and KCl; the associated renal vasoconstriction remained unaltered

  20. Different modes of electrogenic Na+ absorption in the coprodeum of the chicken embryo: role of extracellular Ca2+.

    Science.gov (United States)

    Heinz, M; Krattenmacher, R; Hoffmann, B; Clauss, W

    1991-01-01

    Transepithelial electrogenic Na+ transport (INa) was investigated in the coprodeum of 20-days-old chicken embryos in Ussing chambers. Short circuit current (Isc) and transepithelial resistance (Rt) were 14.7 +/- 4.8 microA.cm-2 (n = 12) and 0.53 +/- 0.09 k omega.cm-2 (n = 12), respectively. INa was calculated from changes in Isc by substitution of mucosal Na+ by (N-methyl-D-glucamine) (NMDG). Isc inversed during Na+ removal, and INa was found to be 27.8 +/- 4.7 microA.cm-2 (n = 12). Amiloride (100 mumol.l-1) inhibited only about 60% of INa. Analysis of Isc fluctuations revealed a Lorentzian component in the power density spectrum with a corner frequency of about 57 Hz. This component was not correlated to INa, and its origin is still unclear. Removal of mucosal Ca2+ increased INa about 2.5-fold due to an increase of the amiloride-insensitive component of INa in additionally investigated adult tissues. The results clearly show that this is due to a non-selective cation channel with an "apparent" order of selectivity Cs+ greater than Na+ = K+ greater than Rb+ greater than Li+. The Ca2+ concentration required to block 50% of the Isc was about 18 mumol.l-1. The IscCa could also be suppressed by other divalent cations such as Mg2+ and Ba2+. Additionally, an INa-linked Lorentzian component occurred which dominated the control spectrum with a significantly higher corner frequency (about 88 Hz). The results indicate that Na+ absorption in the coprodeum of the chicken embryo is more complex than in adult hens.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. The plasminogen activation system modulates differently adipogenesis and myogenesis of embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Ola Hadadeh

    Full Text Available Regulation of the extracellular matrix (ECM plays an important functional role either in physiological or pathological conditions. The plasminogen activation (PA system, comprising the uPA and tPA proteases and their inhibitor PAI-1, is one of the main suppliers of extracellular proteolytic activity contributing to tissue remodeling. Although its function in development is well documented, its precise role in mouse embryonic stem cell (ESC differentiation in vitro is unknown. We found that the PA system components are expressed at very low levels in undifferentiated ESCs and that upon differentiation uPA activity is detected mainly transiently, whereas tPA activity and PAI-1 protein are maximum in well differentiated cells. Adipocyte formation by ESCs is inhibited by amiloride treatment, a specific uPA inhibitor. Likewise, ESCs expressing ectopic PAI-1 under the control of an inducible expression system display reduced adipogenic capacities after induction of the gene. Furthermore, the adipogenic differentiation capacities of PAI-1(-/- induced pluripotent stem cells (iPSCs are augmented as compared to wt iPSCs. Our results demonstrate that the control of ESC adipogenesis by the PA system correspond to different successive steps from undifferentiated to well differentiated ESCs. Similarly, skeletal myogenesis is decreased by uPA inhibition or PAI-1 overexpression during the terminal step of differentiation. However, interfering with uPA during days 0 to 3 of the differentiation process augments ESC myotube formation. Neither neurogenesis, cardiomyogenesis, endothelial cell nor smooth muscle formation are affected by amiloride or PAI-1 induction. Our results show that the PA system is capable to specifically modulate adipogenesis and skeletal myogenesis of ESCs by successive different molecular mechanisms.

  2. The plasminogen activation system modulates differently adipogenesis and myogenesis of embryonic stem cells.

    Science.gov (United States)

    Hadadeh, Ola; Barruet, Emilie; Peiretti, Franck; Verdier, Monique; Bernot, Denis; Hadjal, Yasmine; Yazidi, Claire El; Robaglia-Schlupp, Andrée; De Paula, Andre Maues; Nègre, Didier; Iacovino, Michelina; Kyba, Michael; Alessi, Marie-Christine; Binétruy, Bernard

    2012-01-01

    Regulation of the extracellular matrix (ECM) plays an important functional role either in physiological or pathological conditions. The plasminogen activation (PA) system, comprising the uPA and tPA proteases and their inhibitor PAI-1, is one of the main suppliers of extracellular proteolytic activity contributing to tissue remodeling. Although its function in development is well documented, its precise role in mouse embryonic stem cell (ESC) differentiation in vitro is unknown. We found that the PA system components are expressed at very low levels in undifferentiated ESCs and that upon differentiation uPA activity is detected mainly transiently, whereas tPA activity and PAI-1 protein are maximum in well differentiated cells. Adipocyte formation by ESCs is inhibited by amiloride treatment, a specific uPA inhibitor. Likewise, ESCs expressing ectopic PAI-1 under the control of an inducible expression system display reduced adipogenic capacities after induction of the gene. Furthermore, the adipogenic differentiation capacities of PAI-1(-/-) induced pluripotent stem cells (iPSCs) are augmented as compared to wt iPSCs. Our results demonstrate that the control of ESC adipogenesis by the PA system correspond to different successive steps from undifferentiated to well differentiated ESCs. Similarly, skeletal myogenesis is decreased by uPA inhibition or PAI-1 overexpression during the terminal step of differentiation. However, interfering with uPA during days 0 to 3 of the differentiation process augments ESC myotube formation. Neither neurogenesis, cardiomyogenesis, endothelial cell nor smooth muscle formation are affected by amiloride or PAI-1 induction. Our results show that the PA system is capable to specifically modulate adipogenesis and skeletal myogenesis of ESCs by successive different molecular mechanisms.

  3. Uncovering the Molecular Mechanism of Actions between Pharmaceuticals and Proteins on the AD Network.

    Directory of Open Access Journals (Sweden)

    Shujuan Cao

    Full Text Available This study begins with constructing the mini metabolic networks (MMNs of beta amyloid (Aβ and acetylcholine (ACh which stimulate the Alzheimer's Disease (AD. Then we generate the AD network by incorporating MMNs of Aβ and ACh, and other MMNs of stimuli of AD. The panel of proteins contains 49 enzymes/receptors on the AD network which have the 3D-structure in PDB. The panel of drugs is formed by 5 AD drugs and 5 AD nutraceutical drugs, and 20 non-AD drugs. All of these complexes formed by these 30 drugs and 49 proteins are transformed into dyadic arrays. Utilizing the prior knowledge learned from the drug panel, we propose a statistical classification (dry-lab. According to the wet-lab for the complex of amiloride and insulin degrading enzyme, and the complex of amiloride and neutral endopeptidase, we are confident that this dry-lab is reliable. As the consequences of the dry-lab, we discover many interesting implications. Especially, we show that possible causes of Tacrine, donepezil, galantamine and huperzine A cannot improve the level of ACh which is against to their original design purpose but they still prevent AD to be worse as Aβ deposition appeared. On the other hand, we recommend Miglitol and Atenolol as the safe and potent drugs to improve the level of ACh before Aβ deposition appearing. Moreover, some nutrients such as NADH and Vitamin E should be controlled because they may harm health if being used in wrong way and wrong time. Anyway, the insights shown in this study are valuable to be developed further.

  4. Renal Dysfunction Induced by Kidney-Specific Gene Deletion of Hsd11b2 as a Primary Cause of Salt-Dependent Hypertension.

    Science.gov (United States)

    Ueda, Kohei; Nishimoto, Mitsuhiro; Hirohama, Daigoro; Ayuzawa, Nobuhiro; Kawarazaki, Wakako; Watanabe, Atsushi; Shimosawa, Tatsuo; Loffing, Johannes; Zhang, Ming-Zhi; Marumo, Takeshi; Fujita, Toshiro

    2017-07-01

    Genome-wide analysis of renal sodium-transporting system has identified specific variations of Mendelian hypertensive disorders, including HSD11B2 gene variants in apparent mineralocorticoid excess. However, these genetic variations in extrarenal tissue can be involved in developing hypertension, as demonstrated in former studies using global and brain-specific Hsd11b2 knockout rodents. To re-examine the importance of renal dysfunction on developing hypertension, we generated kidney-specific Hsd11b2 knockout mice. The knockout mice exhibited systemic hypertension, which was abolished by reducing salt intake, suggesting its salt-dependency. In addition, we detected an increase in renal membrane expressions of cleaved epithelial sodium channel-α and T53-phosphorylated Na + -Cl - cotransporter in the knockout mice. Acute intraperitoneal administration of amiloride-induced natriuresis and increased urinary sodium/potassium ratio more in the knockout mice compared with those in the wild-type control mice. Chronic administration of amiloride and high-KCl diet significantly decreased mean blood pressure in the knockout mice, which was accompanied with the correction of hypokalemia and the resultant decrease in Na + -Cl - cotransporter phosphorylation. Accordingly, a Na + -Cl - cotransporter blocker hydrochlorothiazide significantly decreased mean blood pressure in the knockout mice. Chronic administration of mineralocorticoid receptor antagonist spironolactone significantly decreased mean blood pressure of the knockout mice along with downregulation of cleaved epithelial sodium channel-α and phosphorylated Na + -Cl - cotransporter expression in the knockout kidney. Our data suggest that kidney-specific deficiency of 11β-HSD2 leads to salt-dependent hypertension, which is attributed to mineralocorticoid receptor-epithelial sodium channel-Na + -Cl - cotransporter activation in the kidney, and provides evidence that renal dysfunction is essential for developing the

  5. Effects of sodium ions on rat thyrocyte (FRTL-5 cells) swelling- and thyrotropin-activated taurine efflux dependent on cAMP and Epac.

    Science.gov (United States)

    Fugelli, Kjell

    2016-03-01

    Cellular osmolyte release is important in preventing water accumulation and swelling. However, the signaling pathways that detect volume increase and activate solute efflux are still not fully understood. We investigated efflux activation of the osmolyte taurine which is actively accumulated in rat thyrocytes (FRTL-5). Efflux of accumulated [(3)H]taurine was stimulated by cellular swelling and thyrotropin (TSH). These effects were significantly diminished in cells having reduced TSH receptor concentrations. Phosphodiesterase inhibitors (IBMX, Rolipram) enhanced both responses. An analog of forskolin (FSK; 7-deacetyl-7-[O-(N-methylpiperazino)-γ-butyryl] dihydrochloride) and an analog of cAMP, specific for activating exchange protein activated directly by cAMP (Epac; 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate, acetoxymethyl ester), significantly stimulated [(3)H]taurine efflux. A cAMP analog specific for activating protein kinase A (PKA; N6-benzoyladenosine-3',5'-cyclic monophosphate, acetoxymethyl ester) had no significant stimulatory effect on [(3)H]taurine efflux rate. The amiloride analog, 5-(N-ethyl-N-isopropyl)-amiloride, which inhibits a TSH-stimulated Na(+)/H(+) exchanger, enhanced (100 %) and ouabain inhibited (50 %) the TSH-stimulated [(3)H]taurine efflux rate. The effect of FSK on efflux was strongly potentiated by Na(+)-free iso-osmotic conditions and by osmolality/cell volume that affected also the db-cAMP-stimulated efflux. The TSH receptors and downstream elements of the signaling pathway comprising adenylyl cyclase, cAMP and Epac appeared to mediate the hormone-induced signal for [(3)H]taurine efflux from FRTL-5 cells. With less evidence, the cell volume/osmolality-induced [(3)H]taurine efflux cascade appeared to share some of the hormone signaling elements and to modulate the hormone signaling pathway at two levels through cellular Na(+).

  6. ROMK inhibitor actions in the nephron probed with diuretics.

    Science.gov (United States)

    Kharade, Sujay V; Flores, Daniel; Lindsley, Craig W; Satlin, Lisa M; Denton, Jerod S

    2016-04-15

    Diuretics acting on specific nephron segments to inhibit Na + reabsorption have been used clinically for decades; however, drug interactions, tolerance, and derangements in serum K + complicate their use to achieve target blood pressure. ROMK is an attractive diuretic target, in part, because its inhibition is postulated to indirectly inhibit the bumetanide-sensitive Na + -K + -2Cl - cotransporter (NKCC2) and the amiloride- and benzamil-sensitive epithelial Na + channel (ENaC). The development of small-molecule ROMK inhibitors has created opportunities for exploring the physiological responses to ROMK inhibition. The present study evaluated how inhibition of ROMK alone or in combination with NKCC2, ENaC, or the hydrochlorothiazide (HCTZ) target NCC alter fluid and electrolyte transport in the nephron. The ROMK inhibitor VU591 failed to induce diuresis when administered orally to rats. However, another ROMK inhibitor, termed compound A, induced a robust natriuretic diuresis without kaliuresis. Compound A produced additive effects on urine output and Na + excretion when combined with HCTZ, amiloride, or benzamil, but not when coadministered with bumetanide, suggesting that the major diuretic target site is the thick ascending limb (TAL). Interestingly, compound A inhibited the kaliuretic response induced by bumetanide and HCTZ, an effect we attribute to inhibition of ROMK-mediated K + secretion in the TAL and CD. Compound A had no effect on heterologously expressed flow-sensitive large-conductance Ca 2+ -activated K + channels (Slo1/β1). In conclusion, compound A represents an important new pharmacological tool for investigating the renal consequences of ROMK inhibition and therapeutic potential of ROMK as a diuretic target. Copyright © 2016 the American Physiological Society.

  7. Diuretics for Hypertension: A Review and Update.

    Science.gov (United States)

    Roush, George C; Sica, Domenic A

    2016-10-01

    This review and update focuses on the clinical features of hydrochlorothiazide (HCTZ), the thiazide-like agents chlorthalidone (CTDN) and indapamide (INDAP), potassium-sparing ENaC inhibitors and aldosterone receptor antagonists, and loop diuretics. Diuretics are the second most commonly prescribed class of antihypertensive medication, and thiazide-related diuretics have increased at a rate greater than that of antihypertensive medications as a whole. The latest hypertension guidelines have underscored the importance of diuretics for all patients, but particularly for those with salt-sensitive and resistant hypertension. HCTZ is 4.2-6.2 systolic mm Hg less potent than CTDN, angiotensin-converting enzyme inhibitors, beta blockers, and calcium channel blockers by 24-hour measurements and 5.1mm Hg systolic less potent than INDAP by office measurements. For reducing cardiovascular events (CVEs), HCTZ is less effective than enalapril and amlodipine in randomized trials, and, in network analysis of trials, it is less effective than CTDN and HCTZ-amiloride. Combined with thiazide-type diuretics, potassium-sparing agents decrease ventricular ectopy and reduce the risk for sudden cardiac death relative to thiazide-type diuretics used alone. A recent synthesis of 44 trials has shown that the relative potencies in milligrams among spironolactone (SPIR), amiloride, and eplerenone (EPLER) are approximately from 25 to 10 to 100, respectively, which may be important when SPIR is poorly tolerated. SPIR reduces proteinuria beyond that provided by other renin angiotensin aldosterone inhibitors. EPLER also reduces proteinuria and has beneficial effects on endothelial function. While guidelines often do not differentiate among specific diuretics, this review demonstrates that these distinctions are important for managing hypertension. © American Journal of Hypertension, Ltd 2016. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  8. Caveolae-dependent and -independent uptake of albumin in cultured rodent pulmonary endothelial cells.

    Directory of Open Access Journals (Sweden)

    Hui-Hua Li

    Full Text Available Although a critical role for caveolae-mediated albumin transcytosis in pulmonary endothelium is well established, considerably less is known about caveolae-independent pathways. In this current study, we confirmed that cultured rat pulmonary microvascular (RPMEC and pulmonary artery (RPAEC endothelium endocytosed Alexa488-labeled albumin in a saturable, temperature-sensitive mode and internalization resulted in co-localization by fluorescence microscopy with cholera B toxin and caveolin-1. Although siRNA to caveolin-1 (cav-1 in RPAEC significantly inhibited albumin uptake, a remnant portion of albumin uptake was cav-1-independent, suggesting alternative pathways for albumin uptake. Thus, we isolated and cultured mouse lung endothelial cells (MLEC from wild type and cav-1(-/- mice and noted that ~ 65% of albumin uptake, as determined by confocal imaging or live cell total internal reflectance fluorescence microscopy (TIRF, persisted in total absence of cav-1. Uptake of colloidal gold labeled albumin was evaluated by electron microscopy and demonstrated that albumin uptake in MLEC from cav-1(-/- mice was through caveolae-independent pathway(s including clathrin-coated pits that resulted in endosomal accumulation of albumin. Finally, we noted that albumin uptake in RPMEC was in part sensitive to pharmacological agents (amiloride [sodium transport inhibitor], Gö6976 [protein kinase C inhibitor], and cytochalasin D [inhibitor of actin polymerization] consistent with a macropinocytosis-like process. The amiloride sensitivity accounting for macropinocytosis also exists in albumin uptake by both wild type and cav-1(-/- MLEC. We conclude from these studies that in addition to the well described caveolar-dependent pulmonary endothelial cell endocytosis of albumin, a portion of overall uptake in pulmonary endothelial cells is cav-1 insensitive and appears to involve clathrin-mediated endocytosis and macropinocytosis-like process.

  9. Intravenous S-Ketamine Does Not Inhibit Alveolar Fluid Clearance in a Septic Rat Model

    Science.gov (United States)

    Weber, Nina C.; van der Sluijs, Koen; Hackl, Florian; Hotz, Lorenz; Dahan, Albert; Hollmann, Markus W.; Berger, Marc M.

    2014-01-01

    We previously demonstrated that intratracheally administered S-ketamine inhibits alveolar fluid clearance (AFC), whereas an intravenous (IV) bolus injection had no effect. The aim of the present study was to characterize whether continuous IV infusion of S-ketamine, yielding clinically relevant plasma concentrations, inhibits AFC and whether its effect is enhanced in acute lung injury (ALI) which might favor the appearance of IV S-ketamine at the alveolar surface. AFC was measured in fluid-instilled rat lungs. S-ketamine was administered IV over 6 h (loading dose: 20 mg/kg, followed by 20 mg/kg/h), or intratracheally by addition to the instillate (75 µg/ml). ALI was induced by IV lipopolysaccharide (LPS; 7 mg/kg). Interleukin (IL)-6 and cytokine-induced neutrophil chemoattractant (CINC)-3 were measured by ELISA in plasma and bronchoalveolar lavage fluid. Isolated rat alveolar type-II cells were exposed to S-ketamine (75 µg/ml) and/or LPS (1 mg/ml) for 6 h, and transepithelial ion transport was measured as short circuit current (ISC). AFC was 27±5% (mean±SD) over 60 min in control rats and was unaffected by IV S-ketamine. Tracheal S-ketamine reduced AFC to 18±9%. In LPS-treated rats, AFC decreased to 16±6%. This effect was not enhanced by IV S-ketamine. LPS increased IL-6 and CINC-3 in plasma and bronchoalveolar lavage fluid. In alveolar type-II cells, S-ketamine reduced ISC by 37% via a decrease in amiloride-inhibitable sodium transport. Continuous administration of IV S-ketamine does not affect rat AFC even in endotoxin-induced ALI. Tracheal application with direct exposure of alveolar epithelial cells to S-ketamine decreases AFC by inhibition of amiloride-inhibitable sodium transport. PMID:25386677

  10. ASIC channel inhibition enhances excitotoxic neuronal death in an in vitro model of spinal cord injury.

    Science.gov (United States)

    Mazzone, Graciela L; Veeraraghavan, Priyadharishini; Gonzalez-Inchauspe, Carlota; Nistri, Andrea; Uchitel, Osvaldo D

    2017-02-20

    In the spinal cord high extracellular glutamate evokes excitotoxic damage with neuronal loss and severe locomotor impairment. During the cell dysfunction process, extracellular pH becomes acid and may activate acid-sensing ion channels (ASICs) which could be important contributors to neurodegenerative pathologies. Our previous studies have shown that transient application of the glutamate analog kainate (KA) evokes delayed excitotoxic death of spinal neurons, while white matter is mainly spared. The present goal was to enquire if ASIC channels modulated KA damage in relation to locomotor network function and cell death. Mouse spinal cord slices were treated with KA (0.01 or 0.1mM) for 1h, and then washed out for 24h prior to analysis. RT-PCR results showed that KA (at 0.01mM concentration that is near-threshold for damage) increased mRNA expression of ASIC1a, ASIC1b, ASIC2 and ASIC3, an effect reversed by the ASIC inhibitor 4',6-diamidino-2-phenylindole (DAPI). A KA neurotoxic dose (0.1mM) reduced ASIC1a and ASIC2 expression. Cell viability assays demonstrated KA-induced large damage in spinal slices from mice with ASIC1a gene ablation. Likewise, immunohistochemistry indicated significant neuronal loss when KA was followed by the ASIC inhibitors DAPI or amiloride. Electrophysiological recording from ventral roots of isolated spinal cords showed that alternating oscillatory cycles were slowed down by 0.01mMKA, and intensely inhibited by subsequently applied DAPI or amiloride. Our data suggest that early rise in ASIC expression and function counteracted deleterious effects on spinal networks by raising the excitotoxicity threshold, a result with potential implications for improving neuroprotection. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  11. PAR-2 activation enhances weak acid-induced ATP release through TRPV1 and ASIC sensitization in human esophageal epithelial cells.

    Science.gov (United States)

    Wu, Liping; Oshima, Tadayuki; Shan, Jing; Sei, Hiroo; Tomita, Toshihiko; Ohda, Yoshio; Fukui, Hirokazu; Watari, Jiro; Miwa, Hiroto

    2015-10-15

    Esophageal visceral hypersensitivity has been proposed to be the pathogenesis of heartburn sensation in nonerosive reflux disease. Protease-activated receptor-2 (PAR-2) is expressed in human esophageal epithelial cells and is believed to play a role in inflammation and sensation. PAR-2 activation may modulate these responses through adenosine triphosphate (ATP) release, which is involved in transduction of sensation and pain. The transient receptor potential vanilloid receptor 1 (TRPV1) and acid-sensing ion channels (ASICs) are both acid-sensitive nociceptors. However, the interaction among these molecules and the mechanisms of heartburn sensation are still not clear. We therefore examined whether ATP release in human esophageal epithelial cells in response to acid is modulated by TRPV1 and ASICs and whether PAR-2 activation influences the sensitivity of TRPV1 and ASICs. Weak acid (pH 5) stimulated the release of ATP from primary human esophageal epithelial cells (HEECs). This effect was significantly reduced after pretreatment with 5-iodoresiniferatoxin (IRTX), a TRPV1-specific antagonist, or with amiloride, a nonselective ASIC blocker. TRPV1 and ASIC3 small interfering RNA (siRNA) transfection also decreased weak acid-induced ATP release. Pretreatment of HEECs with trypsin, tryptase, or a PAR-2 agonist enhanced weak acid-induced ATP release. Trypsin treatment led to the phosphorylation of TRPV1. Acid-induced ATP release enhancement by trypsin was partially blocked by IRTX, amiloride, or a PAR-2 antagonist. Conversely, acid-induced ATP release was augmented by PAR-2 activation through TRPV1 and ASICs. These findings suggested that the pathophysiology of heartburn sensation or esophageal hypersensitivity may be associated with the activation of PAR-2, TRPV1, and ASICs. Copyright © 2015 the American Physiological Society.

  12. Three homologous subunits form a high affinity peptide-gated ion channel in Hydra.

    Science.gov (United States)

    Dürrnagel, Stefan; Kuhn, Anne; Tsiairis, Charisios D; Williamson, Michael; Kalbacher, Hubert; Grimmelikhuijzen, Cornelis J P; Holstein, Thomas W; Gründer, Stefan

    2010-04-16

    Recently, three ion channel subunits of the degenerin (DEG)/epithelial Na(+) channel (ENaC) gene family have been cloned from the freshwater polyp Hydra magnipapillata, the Hydra Na(+) channels (HyNaCs) 2-4. Two of them, HyNaC2 and HyNaC3, co-assemble to form an ion channel that is gated by the neuropeptides Hydra-RFamides I and II. The HyNaC2/3 channel is so far the only cloned ionotropic receptor from cnidarians and, together with the related ionotropic receptor FMRFamide-activated Na(+) channel (FaNaC) from snails, the only known peptide-gated ionotropic receptor. The HyNaC2/3 channel has pore properties, like a low Na(+) selectivity and a low amiloride affinity, that are different from other channels of the DEG/ENaC gene family, suggesting that a component of the native Hydra channel might still be lacking. Here, we report the cloning of a new ion channel subunit from Hydra, HyNaC5. The new subunit is closely related to HyNaC2 and -3 and co-localizes with HyNaC2 and -3 to the base of the tentacles. Coexpression in Xenopus oocytes of HyNaC5 with HyNaC2 and -3 largely increases current amplitude after peptide stimulation and affinity of the channel to Hydra-RFamides I and II. Moreover, the HyNaC2/3/5 channel has altered pore properties and amiloride affinity, more similarly to other DEG/ENaC channels. Collectively, our results suggest that the three homologous subunits HyNaC2, -3, and -5 form a peptide-gated ion channel in Hydra that could contribute to fast synaptic transmission.

  13. 22Na+ and 86Rb+ transport in vascular smooth muscle of SHR, Wistar Kyoto, and Wistar rats

    International Nuclear Information System (INIS)

    Kuriyama, S.; Denny, T.N.; Aviv, A.

    1988-01-01

    To gain further insight into differences in cellular Na+ and K+ regulation between the spontaneously hypertensive rat (SHR), Wistar Kyoto (WKY), and American Wistar (W) rats, 22Na+ and 86Rb+ washouts were performed under steady-state conditions in cultured vascular smooth muscle cells from the three rat strains. SHR vascular smooth muscle cells showed significantly higher bumetanide sensitive 86Rb+ washout rate constant (x 10(-4)/min; mean +/- SEM) than WKY cells (-38.6 +/- 2.84 and -23.8 +/- 3.58, respectively; p less than 0.005). SHR vascular smooth muscle cells also exhibited significantly higher values than WKY cells in the total 22Na+ washout rate constant (x 10(-2)/min) (-61.0 +/- 1.57 vs. -53.8 +/- 1.24; p less than 0.005). The amiloride sensitive component of the 22Na+ washout rate constant accounted for these differences (-18.6 +/- 1.04 for SHR and -12.1 +/- 2.00 for WKY; p less than 0.05). There were no apparent differences in cellular Na+ concentrations between WKY and SHR cells. In general, the 86Rb+ and 22Na+ washout parameters of W rat cells were quite similar to those of cells from SHR. We conclude that the bumetanide-sensitive 86Rb+ washout (the Na+ K+-cotransport), the overall, and the amiloride-sensitive 22Na+ washout (the latter primarily represents the Na+/H+ antiport) are higher in SHR than WKY rat vascular smooth muscle cells. These findings indicate innate differences in cellular Na+ and K+ transport in vascular smooth muscle cells of the SHR and WKY rat. The mechanisms responsible for these differences are yet to be determined

  14. Intestinal ammonia transport in freshwater and seawater acclimated rainbow trout (Oncorhynchus mykiss): evidence for a Na+ coupled uptake mechanism.

    Science.gov (United States)

    Rubino, Julian G; Zimmer, Alex M; Wood, Chris M

    2015-05-01

    In vitro gut sac experiments were performed on freshwater and 60% seawater acclimated trout (Oncorhynchus mykiss) under treatments designed to discern possible mechanisms of intestinal ammonia transport. Seawater acclimation increased ammonia flux rate into the serosal saline (Jsamm) in the anterior intestine, however it did not alter Jsamm in the mid- or posterior intestine suggesting similar mechanisms of ammonia handling in freshwater and seawater fish. Both fluid transport rate (FTR) and Jsamm were inhibited in response to basolateral ouabain treatment, suggesting a linkage of ammonia uptake to active transport, possibly coupled to fluid transport processes via solvent drag. Furthermore, decreases in FTR and Jsamm caused by low Na(+) treatment indicated a Na(+) linked transport mechanism. Mucosal bumetanide (10(-4) M) had no impact on FTR, yet decreased Jsamm in the anterior and mid-intestine, suggesting NH4(+) substitution for K(+) on an apical NKCC, and at least a partial uncoupling of ammonia transport from fluid transport. Additional treatments (amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA), phenamil, bafilomycin, 4',6-diamidino-2-phenylindole (DAPI), high sodium) intended to disrupt alternative routes of Na(+) uptake yielded no change in FTR or Jsamm, suggesting the absence of direct competition between Na(+) and ammonia for transport. Finally, [(14)C]methylamine permeability (PMA) measurements indicated the likely presence of an intestinal Rh-mediated ammonia transport system, as increasing NH4Cl (0, 1, 5 mmol l(-1)) concentrations reduced PMA, suggesting competition for transport through Rh proteins. Overall, the data presented in this paper provide some of the first insights into mechanisms of teleost intestinal ammonia transport. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. [Venom of Latrodectus mactans from Chile (Araneae, Theridiidae): effect on smooth muscle].

    Science.gov (United States)

    Romero, Fernando; Altieri, Elena; Urrutia, Mauricio; Jara, Jorge

    2003-06-01

    The venoms of Latrodectus sp. have been reported to induce contraction probably mediated by adrenergic and cholinergic transmitters. We have demonstrated that the venom of Chilean Latrodectus mactans contains neurotoxins that induce a contraction partially independent of transmitters release. Transmembrane mobility of Na+ and Ca2+ ions and more specifically, the increase of cytoplasmic calcium concentration are responsible for tonic contraction in smooth muscle. Calcium may enter the cell by several ways, such as the voltage-dependent Ca2+ L-type channels and the Na+/Ca2+ exchanger. This study aimed to examine the participation of this exchanger in the tonic contraction of smooth muscle in vas deferent of rat induced by the venom of the Chilean spider L. mactans. Blockers of Na+ channels (amiloride) and Ca2+ L-type channels (nifedipine), and a stimulator of the exchanger (modified Tyrode, Na+ 80 mM) were used. Simultaneously, variations of the cytoplasmic concentration of Ca2+ were registered by microfluorimetry (Fura-2 indicator) in the presence of nifedipine. In presence of amiloride, dose-dependent inhibition of venom-induced contraction was observed, suggesting the participation of voltage-dependent Ca2+ L-type channels. The contraction was only partially inhibited by nifedipine and the Ca2+ cytoplasmic concentration increased, as assessed by the microfluorimetric registration. Finally, the venom-induced contraction increased in the presence of modified Tyrode, probably due to the action of the Na+/Ca2+ exchanger. Taken together, our results support the idea that the Na+/Ca2+ exchanger is active and may be, at least in part, responsible for the contraction induced by the venom of Chilean L. mactans.

  16. Low concentrations of neutrophil extracellular traps induce proliferation in human keratinocytes via NF-kB activation.

    Science.gov (United States)

    Tonello, Stelvio; Rizzi, Manuela; Migliario, Mario; Rocchetti, Vincenzo; Renò, Filippo

    2017-10-01

    Granulocytes play a pivotal role in innate immune response, as pathogen invasion activates neutrophils, a subclass of granulocytes, inducing the production of neutrophil extracellular traps (NETs). In this study, it has been evaluated how NETs could affect human keratinocytes (HaCaT cells) behaviour. HaCaT cells were treated with increasing NETs concentrations (0.01-200ng/ml) and the effect on cell proliferation was evaluated by MTT assay. Inhibition studies were performed by pre-treating cells with dexamethasone, chloropromazine or amiloride. NF-kB pathway activation was evaluated by western blot. HaCaT cells stimulation with increasing concentrations of NETs (0.01-50ng/ml) for 48h resulted in a modulation of cell proliferation with a maximum increase corresponding to 0.5-1ng/ml stimulation. NETs low concentrations not only increased cell proliferation, but were also able to induce a faster wound closure in an in vitro scratch assay. NETs scaffold, composed by histone proteins and DNA, is recognized by Toll Like Receptor 9 (TLR 9) that, in turn, activates the NF-kB pathway. In fact, NETs induced proliferation was inhibited by chloropromazine (1nM), that blocks chlatrin vesicles formation, and by amiloride (50nM) that inhibits macropinocytosis. Moreover, dexamethasone, an inhibitor of NF-kB, was able to abolish the NETs effect. This study thus demonstrates that low NETs concentrations undergo internalization finally resulting in a quick NF-kB pathway activation and HaCaT cells proliferation increase, suggesting a close relationship between first immune response and wound healing onset. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

  17. Staurosporine induces ganglion cell differentiation in part by stimulating urokinase-type plasminogen activator expression and activation in the developing chick retina

    International Nuclear Information System (INIS)

    Kim, Yeoun-Hee; Chang, Yongmin; Jung, Jae-Chang

    2012-01-01

    Highlights: ► Staurosporine mediates stimulation of RGC differentiation in vitro cultured retinal neuroblasts. ► Staurosporine mediates uPA activation during RGC differentiation in vitro. ► Inhibition of uPA blocks the staurosporine mediated RGC differentiation both in vitro and in ovo. ► Thus, uPA may play a role in the staurosporine-mediated stimulation of RGC differentiation. -- Abstract: Here, we investigated whether staurosporine-mediated urokinase-type plasminogen activator (uPA) activation is involved in retinal ganglion cell (RGC) differentiation. Retinal cells were isolated from developing chick retinas at embryonic day 6 (E6). Relatively few control cells grown in serum-free medium started to form processes by 12 h. In contrast, staurosporine-treated cells had processes within 3 h, and processes were evident at 8 h. Immunofluorescence staining showed that Tuj-1-positive cells with shorter neurites could be detected in control cultures at 18 h, whereas numerous Tuj-1 positive ganglion cells with longer neuritic extensions were seen in staurosporine-treated cultures. BrdU-positive proliferating cells were more numerous in control cultures than in staurosporine-treated cultures, and the BrdU staining was not detected in post-mitotic Tuj-1 positive ganglion cells. Western blotting of cell lysates showed that staurosporine induced high levels of the active form of uPA. The staurosporine-induced uPA signal was localized predominantly in the soma, neurites and axons of Tuj-1-positive ganglion cells. Amiloride, an inhibitor of uPA, markedly reduced staurosporine-induced Tuj-1 staining, neurite length, neurite number, and uPA staining versus controls. In developing retinas in ovo, amiloride administration remarkably reduced the staurosporine-induced uPA staining and RGC differentiation. Taken together, our in vitro and in vivo data collectively indicate that uPA plays a role in the staurosporine-mediated stimulation of RGC differentiation.

  18. Preeclampsia, migración celular y canales iónicos Preeclampsia, cellular migration and ion channels

    Directory of Open Access Journals (Sweden)

    Silvana M. del Mónaco

    2008-10-01

    Full Text Available En la placenta humana, el sinciciotrofoblasto es la barrera que regula el transporte de nutrientes, solutos y agua entre la sangre materna y fetal. Dentro de este movimiento transepitelial se encuentra el del Na+, su contribución a la presión osmótica es fundamental en la regulación del volumen de líquido extracelular. El canal epitelial de sodio sensible al amiloride (ENaC media el transporte de Na+ desde el lumen hacia el interior celular en numerosos epitelios absortivos. Está regulado por la aldosterona, vasopresina, catecolaminas, estrógenos y progesterona. Es bloqueado por el amiloride y sus análogos. Para su activación, diversas proteasas lo escinden en la membrana plasmática y esto a su vez es regulado por la aldosterona. El ENaC está expresado también en la placenta humana y aunque su función no es conocida, podría participar en la homeostasis de agua y electrolitos. El ENaC también es influenciado por el estado de las proteínas del citoesqueleto y los cambios en el volumen celular alteran a su vez a éste. De esta manera existe una relación entre el ENaC y el citoesqueleto. Además, las corrientes de Na+ por el ENaC y otros canales de sodio participan en la migración celular en células normales y cancerosas. Aquí presentamos evidencias que avalan la hipótesis que el ENaC es necesario para la migración celular en células BeWo, derivadas del trofoblasto humano, que sintetizan hormonas y expresan el ENaC. Las células BeWO han sido utilizadas como modelo experimental para estudiar el transporte en células de placenta.The syncytiotrophoblast acts in human placenta as a transporting barrier regulating the transference of nutrients, solutes and water between maternal and fetal blood. This transepithelial transport involves movement of Na+ and its contribution to the osmotic pressure is an important determinant of the extracellular fluid volume. ENaC is a channel that mediates entry of Na+ from the luminal fluid into

  19. Blockade of peripheral and spinal Na+/H+ exchanger increases formalin-induced long-lasting mechanical allodynia and hyperalgesia in rats.

    Science.gov (United States)

    Castañeda-Corral, Gabriela; Rocha-González, Héctor I; Araiza-Saldaña, Claudia I; Vidal-Cantú, Guadalupe C; Miguel Jiménez-Andrade, Juan; Murbartián, Janet; Granados-Soto, Vinicio

    2012-09-26

    The Na(+)/H(+) exchanger (NHE) is involved in the regulation of intracellular pH and volume by mediating the electroneutral transport of H(+) against an influx of Na(+) ions. Since NHE1 regulates pH in neurons and astrocytes and it is expressed in nociceptive nerve fibers, it is likely that NHE may modulate neuronal excitability and pain transmission. The purpose of this study was to assess the participation of peripheral and spinal NHE in the secondary allodynia/hyperalgesia induced by formalin. In addition, we determined whether formalin injection modifies the expression of NHE1 in lumbar dorsal root ganglia (DRG) and dorsal spinal cord. Subcutaneous injection of 0.5% formalin into the dorsal surface of the hind paw produced acute nociceptive behaviors (flinching and licking/lifting) followed by long-lasting bilateral secondary mechanical allodynia/hyperalgesia. Peripheral and intrathecal pre-treatment (-10min) with selective NHE inhibitors 5-(N,N-dimethyl)amiloride hydrochloride (DMA, 0.3-30μM), 5-(N-ethyl-N-isopropyl)amiloride (EIPA, 0.3-30μM) and [1-(quinolin-5-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl] guanidine dihydrochloride (zoniporide, 0.03-3μM) significantly increased 0.5% formalin-induced bilateral long-lasting secondary allodynia/hyperalgesia. Contrariwise, local peripheral or intrathecal post-treatment (day 6 postinjection) with these NHE inhibitors did not affect formalin-induced nociceptive behaviors. Formalin injection reduced NHE1 expression in ipsilateral and contralateral spinal dorsal horns from day 1 to 12. In addition, formalin diminished NHE1 protein expression in DRG at day 12. These results suggest that NHE1 plays a role in pain processing at peripheral and spinal levels in formalin-induced long-lasting nociceptive behaviors. Additionally, these results suggest that proteins involved in pH regulation could be targets for the development of new analgesic drugs. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Staurosporine induces ganglion cell differentiation in part by stimulating urokinase-type plasminogen activator expression and activation in the developing chick retina

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yeoun-Hee [Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Chang, Yongmin [Department of Molecular Medicine, Kyungpook National University College of Medicine, Kyungpook National University, 200 Dongduk-Ro Jung-Gu, Daegu 700-714 (Korea, Republic of); Jung, Jae-Chang, E-mail: jcjung@knu.ac.kr [Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu 702-701 (Korea, Republic of)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer Staurosporine mediates stimulation of RGC differentiation in vitro cultured retinal neuroblasts. Black-Right-Pointing-Pointer Staurosporine mediates uPA activation during RGC differentiation in vitro. Black-Right-Pointing-Pointer Inhibition of uPA blocks the staurosporine mediated RGC differentiation both in vitro and in ovo. Black-Right-Pointing-Pointer Thus, uPA may play a role in the staurosporine-mediated stimulation of RGC differentiation. -- Abstract: Here, we investigated whether staurosporine-mediated urokinase-type plasminogen activator (uPA) activation is involved in retinal ganglion cell (RGC) differentiation. Retinal cells were isolated from developing chick retinas at embryonic day 6 (E6). Relatively few control cells grown in serum-free medium started to form processes by 12 h. In contrast, staurosporine-treated cells had processes within 3 h, and processes were evident at 8 h. Immunofluorescence staining showed that Tuj-1-positive cells with shorter neurites could be detected in control cultures at 18 h, whereas numerous Tuj-1 positive ganglion cells with longer neuritic extensions were seen in staurosporine-treated cultures. BrdU-positive proliferating cells were more numerous in control cultures than in staurosporine-treated cultures, and the BrdU staining was not detected in post-mitotic Tuj-1 positive ganglion cells. Western blotting of cell lysates showed that staurosporine induced high levels of the active form of uPA. The staurosporine-induced uPA signal was localized predominantly in the soma, neurites and axons of Tuj-1-positive ganglion cells. Amiloride, an inhibitor of uPA, markedly reduced staurosporine-induced Tuj-1 staining, neurite length, neurite number, and uPA staining versus controls. In developing retinas in ovo, amiloride administration remarkably reduced the staurosporine-induced uPA staining and RGC differentiation. Taken together, our in vitro and in vivo data collectively indicate that

  1. Internalization of titanium dioxide nanoparticles by glial cells is given at short times and is mainly mediated by actin reorganization-dependent endocytosis.

    Science.gov (United States)

    Huerta-García, Elizabeth; Márquez-Ramírez, Sandra Gissela; Ramos-Godinez, María Del Pilar; López-Saavedra, Alejandro; Herrera, Luis Alonso; Parra, Alberto; Alfaro-Moreno, Ernesto; Gómez, Erika Olivia; López-Marure, Rebeca

    2015-12-01

    Many nanoparticles (NPs) have toxic effects on multiple cell lines. This toxicity is assumed to be related to their accumulation within cells. However, the process of internalization of NPs has not yet been fully characterized. In this study, the cellular uptake, accumulation, and localization of titanium dioxide nanoparticles (TiO2 NPs) in rat (C6) and human (U373) glial cells were analyzed using time-lapse microscopy (TLM) and transmission electron microscopy (TEM). Cytochalasin D (Cyt-D) was used to evaluate whether the internalization process depends of actin reorganization. To determine whether the NP uptake is mediated by phagocytosis or macropinocytosis, nitroblue tetrazolium (NBT) reduction was measured and the 5-(N-ethyl-N-isopropyl)-amiloride was used. Expression of proteins involved with endocytosis and exocytosis such as caveolin-1 (Cav-1) and cysteine string proteins (CSPs) was also determined using flow cytometry. TiO2 NPs were taken up by both cell types, were bound to cellular membranes and were internalized at very short times after exposure (C6, 30 min; U373, 2h). During the uptake process, the formation of pseudopodia and intracellular vesicles was observed, indicating that this process was mediated by endocytosis. No specific localization of TiO2 NPs into particular organelles was found: in contrast, they were primarily localized into large vesicles in the cytoplasm. Internalization of TiO2 NPs was strongly inhibited by Cyt-D in both cells and by amiloride in U373 cells; besides, the observed endocytosis was not associated with NBT reduction in either cell type, indicating that macropinocytosis is the main process of internalization in U373 cells. In addition, increases in the expression of Cav-1 protein and CSPs were observed. In conclusion, glial cells are able to internalize TiO2 NPs by a constitutive endocytic mechanism which may be associated with their strong cytotoxic effect in these cells; therefore, TiO2 NPs internalization and their

  2. Acidic pH facilitates peripheral αβmeATP-mediated nociception in rats: differential roles of P2X, P2Y, ASIC and TRPV1 receptors in ATP-induced mechanical allodynia and thermal hyperalgesia.

    Science.gov (United States)

    Seo, Hyoung-Sig; Roh, Dae-Hyun; Kwon, Soon-Gu; Yoon, Seo-Yeon; Kang, Suk-Yun; Moon, Ji-Young; Choi, Sheu-Ran; Beitz, Alvin J; Lee, Jang-Hern

    2011-03-01

    Peripheral ischemia is commonly associated with an increase in tissue ATP concentration and a decrease in tissue pH. Although in vitro data suggest that low tissue pH can affect ATP-binding affinities to P2 receptors, the mechanistic relationship between ATP and low pH on peripheral nociception has not been fully examined. This study was designed to investigate the potential role of an acidified environment on intraplantar αβmeATP-induced peripheral pain responses in rats. The mechanical allodynia (MA) produced by injection of αβmeATP was significantly increased in animals that received the drug diluted in pH 4.0 saline compared to those that received the drug diluted in pH 7.0 saline. Moreover, animals injected with αβmeATP (100 nmol) in pH 4.0 saline developed thermal hyperalgesia (TH), which did not occur in animals treated with αβmeATP diluted in pH 7.0 saline. To elucidate which receptors were involved in this pH-related facilitation of αβmeATP-induced MA and TH, rats were pretreated with PPADS (P2 antagonist), TNP-ATP (P2X antagonist), MRS2179 (P2Y1 antagonist), AMG9810 (TRPV1 antagonist) or amiloride (ASIC blocker). Both PPADS and TNP-ATP dose-dependently blocked pH-facilitated MA, while TH was significantly reduced by pre-treatment with MRS2179 or AMG9810. Moreover, amiloride injection significantly reduced low pH-induced facilitation of αβmeATP-mediated MA, but not TH. These results demonstrate that low tissue pH facilitates ATP-mediated MA via the activation of P2X receptors and ASICs, whereas TH induced by ATP under low pH conditions is mediated by the P2Y1 receptor and TRPV1, but not ASIC. Thus distinct mechanisms are responsible for the development of MA and TH under conditions of tissue acidosis and increased ATP. Copyright © 2010 Elsevier Ltd. All rights reserved.

  3. Quantifying Nanoparticle Internalization Using a High Throughput Internalization Assay.

    Science.gov (United States)

    Mann, Sarah K; Czuba, Ewa; Selby, Laura I; Such, Georgina K; Johnston, Angus P R

    2016-10-01

    The internalization of nanoparticles into cells is critical for effective nanoparticle mediated drug delivery. To investigate the kinetics and mechanism of internalization of nanoparticles into cells we have developed a DNA molecular sensor, termed the Specific Hybridization Internalization Probe - SHIP. Self-assembling polymeric 'pHlexi' nanoparticles were functionalized with a Fluorescent Internalization Probe (FIP) and the interactions with two different cell lines (3T3 and CEM cells) were studied. The kinetics of internalization were quantified and chemical inhibitors that inhibited energy dependent endocytosis (sodium azide), dynamin dependent endocytosis (Dyngo-4a) and macropinocytosis (5-(N-ethyl-N-isopropyl) amiloride (EIPA)) were used to study the mechanism of internalization. Nanoparticle internalization kinetics were significantly faster in 3T3 cells than CEM cells. We have shown that ~90% of the nanoparticles associated with 3T3 cells were internalized, compared to only 20% of the nanoparticles associated with CEM cells. Nanoparticle uptake was via a dynamin-dependent pathway, and the nanoparticles were trafficked to lysosomal compartments once internalized. SHIP is able to distinguish between nanoparticles that are associated on the outer cell membrane from nanoparticles that are internalized. This study demonstrates the assay can be used to probe the kinetics of nanoparticle internalization and the mechanisms by which the nanoparticles are taken up by cells. This information is fundamental for engineering more effective nanoparticle delivery systems. The SHIP assay is a simple and a high-throughput technique that could have wide application in therapeutic delivery research.

  4. Migraine with prolonged aura: phenotype and treatment.

    Science.gov (United States)

    Viana, Michele; Afridi, Shazia

    2018-01-01

    We review the published literature on migraine with prolonged aura (PA), specifically with regards to the phenotype and treatment options. PA is not uncommon. A recent study found that about 17% of migraine auras are prolonged and that 26% of patients with migraine with aura have experienced at least one PA. The characteristics of PA are similar to most typical auras with the exception of a higher number of aura symptoms (in particular sensory and/or dysphasic). There are no well-established treatments at present which target the aura component of migraine. Other than case reports, there have been open-label studies of lamotrigine and greater occipital nerve blocks. The only randomised, blinded, controlled trial to date has been of nasal ketamine showing some reduction in aura severity but not duration. A small open-labelled pilot study of amiloride was also promising. Larger randomised, controlled trials are needed to establish whether any of the existing or novel compounds mentioned are significantly effective and safe.

  5. Hypertension in the course of primary aldosteronism during pregnancy

    Directory of Open Access Journals (Sweden)

    Magdalena Wyskida

    2015-02-01

    Full Text Available Hypertension is one of the most common cardiovascular diseases during pregnancy. Primary hyperaldosteronism (PHA is the most frequent endocrinological, secondary cause of hypertension, rarely diagnosed in pregnant women. In the available literature about 50 cases of PHA in pregnant women have been described. PHA is often a cause of resistant hypertension. PHA can cause life-threatening complications both for the pregnant woman and the fetus. Diagnosis of PHA in pregnancy is difficult due to the antagonistic effect of progesterone on aldosterone, physiological increase of aldosterone release during gestation and frequent normokalaemic clinical course. Typical pharmacological treatment of PHA is limited due to the anti‑androgenic effect of spironolactone, lack of data concerning the safety of eplerenone and limited access to amiloride in Poland. Surgical treatment is a therapeutic option only in early pregnancy. This paper presents the current state of knowledge on diagnostic methods and treatment of PHA in pregnant women and a systematic review of cases described in the literature.

  6. Structure-function relationships in the integument of Salamandra salamandra during ontogenetic development.

    Science.gov (United States)

    Pederzoli, Aurora; Gambarelli, A; Gabbay, Shosh; Rozman, A; Katz, U

    2002-06-01

    Morphological, cytological and transport properties of the integument of Salamandra salamandra were investigated during natural ontogenetic development, from birth to adult. Three stages were operationally defined: I, larvae, from birth to metamorphosis; II, metamorphosis (judged externally by the colour change and loss of the gills); and III, post-metamorphosis to adult. Pieces of skin were fixed at various stages for immunocytochemical examinations, and the electrical properties were investigated on parallel pieces. Distinct cellular changes take place in the skin during metamorphosis, and lectin (PNA, WGA and ConA) binding indicates profound changes in glycoprotein composition of cell membranes, following metamorphosis. Band 3 and carbonic anhydrase I (CA I) were confined to mitochondria-rich (MR)-like cells, and were detected only in the larval stage. CA II on the other hand, was detected both in MR-like and in MR cells following metamorphosis. The electrical studies show that the skin becomes more tight (transepithelial resistance increases) upon metamorphosis, followed by manifestation of amiloride-sensitive short-circuit current (I(SC)) indicating that functional Na+ uptake has been acquired. The skin of metamorphosed adults had no finite transepithelial Cl- conductance, and band 3 was not detected in its MR cells. The functional properties of MR-like and MR cells remain to be established.

  7. Effects of insulin and epinephrine on Na+-K+ and glucose transport in soleus muscle

    International Nuclear Information System (INIS)

    Clausen, T.; Flatman, J.A.

    1987-01-01

    To identify possible cause-effect relationships between changes in active Na + -K + transport, resting membrane potential, and glucose transport, the effects of insulin and epinephrine were compared in rat soleus muscle. Epinephrine, which produced twice as large a hyperpolarization as insulin, induced only a modest increase in 14 C-labeled sugar transport. Ouabain, at a concentration (10 -3 M) sufficient to block active Na + -K + transport and the hyperpolarization induced by the two hormones, did not interfere with sugar transport stimulation. After Na + loading in K + -free buffer, the return to K + -containing standard buffer caused marked stimulation of active 22 Na + - 42 K + transport, twice the hyperpolarization produced by insulin but no change in sugar transport. The insulin-induced activation of the 22 Na + - 42 K + pump leads to decreased intracellular 22 Na + concentration and hyperpolarization, but none of these events can account for the concomitant activation of the glucose transport system. The stimulating effect of insulin on active Na + -K + transport was not suppressed by amiloride, indicating that in intact skeletal muscle it is not elicited by a primary increase in Na + influx via the Na + /H + -exchange system

  8. Kidney in potassium depletion. II. K+ handling by the isolated perfused rat kidney

    International Nuclear Information System (INIS)

    Hayashi, M.; Katz, A.I.

    1987-01-01

    In a companion paper the authors reported a large increment in Na + -K + -ATPase activity and [ 3 H]ouabain binding the inner stripe of outer medullary collecting tubules from K-depleted rats. To test the hypothesis that the increased number of Na + -K + pumps in these animals may be involved in potassium reabsorption they examined the effect of ouabain on K excretion by isolated, perfused kidneys from rats fed a K-free diet for 3 wk. Kidneys from K-depleted rats retain potassium avidly, both the fractional (FE/sub K/) and absolute K excretion being approximately fivefold lower than in control kidneys. Ouabain (5 mM) increased FE/sub K/ in kidneys from each K-depleted rat; similar results were obtained when kidneys were perfused with low and high potassium concentrations. In contrast, ouabain produced a variable effect in control kidneys, that depended on the perfusate potassium concentration. In K-depleted rats amiloride did not significantly alter K excretion and did not block the ouabain-induced kaliuresis, suggesting that the latter is not due to enhanced secretion secondary to increased distal fluid delivery. These results provide evidence for ouabain-sensitive potassium reabsorption in kidneys of chronically K-depleted rats, and suggest an explanation for the increased Na + -K + -ATPase observed in such animals

  9. Effects of non-uniform root zone salinity on water use, Na+ recirculation, and Na+ and H+ flux in cotton.

    Science.gov (United States)

    Kong, Xiangqiang; Luo, Zhen; Dong, Hezhong; Eneji, A Egrinya; Li, Weijiang

    2012-03-01

    A new split-root system was established through grafting to study cotton response to non-uniform salinity. Each root half was treated with either uniform (100/100 mM) or non-uniform NaCl concentrations (0/200 and 50/150 mM). In contrast to uniform control, non-uniform salinity treatment improved plant growth and water use, with more water absorbed from the non- and low salinity side. Non-uniform treatments decreased Na(+) concentrations in leaves. The [Na(+)] in the '0' side roots of the 0/200 treatment was significantly higher than that in either side of the 0/0 control, but greatly decreased when the '0' side phloem was girdled, suggesting that the increased [Na(+)] in the '0' side roots was possibly due to transportation of foliar Na(+) to roots through phloem. Plants under non-uniform salinity extruded more Na(+) from the root than those under uniform salinity. Root Na(+) efflux in the low salinity side was greatly enhanced by the higher salinity side. NaCl-induced Na(+) efflux and H(+) influx were inhibited by amiloride and sodium orthovanadate, suggesting that root Na(+) extrusion was probably due to active Na(+)/H(+) antiport across the plasma membrane. Improved plant growth under non-uniform salinity was thus attributed to increased water use, reduced leaf Na(+) concentration, transport of excessive foliar Na(+) to the low salinity side, and enhanced Na(+) efflux from the low salinity root.

  10. Diabetes insipidus: The other diabetes

    Science.gov (United States)

    Kalra, Sanjay; Zargar, Abdul Hamid; Jain, Sunil M.; Sethi, Bipin; Chowdhury, Subhankar; Singh, Awadhesh Kumar; Thomas, Nihal; Unnikrishnan, A. G.; Thakkar, Piya Ballani; Malve, Harshad

    2016-01-01

    Diabetes insipidus (DI) is a hereditary or acquired condition which disrupts normal life of persons with the condition; disruption is due to increased thirst and passing of large volumes of urine, even at night. A systematic search of literature for DI was carried out using the PubMed database for the purpose of this review. Central DI due to impaired secretion of arginine vasopressin (AVP) could result from traumatic brain injury, surgery, or tumors whereas nephrogenic DI due to failure of the kidney to respond to AVP is usually inherited. The earliest treatment was posterior pituitary extracts containing vasopressin and oxytocin. The synthetic analog of vasopressin, desmopressin has several benefits over vasopressin. Desmopressin was initially available as intranasal preparation, but now the oral tablet and melt formulations have gained significance, with benefits such as ease of administration and stability at room temperature. Other molecules used for treatment include chlorpropamide, carbamazepine, thiazide diuretics, indapamide, clofibrate, indomethacin, and amiloride. However, desmopressin remains the most widely used drug for the treatment of DI. This review covers the physiology of water balance, causes of DI and various treatment modalities available, with a special focus on desmopressin. PMID:26904464

  11. Active phagocytosis of Mycobacterium tuberculosis (H37Ra) by T lymphocytes (Jurkat cells).

    Science.gov (United States)

    Zhang, Min; Zhu, Qi; Shi, Ming; Liu, Yang; Ma, Lei; Yang, Yining; Feng, Dongyun; Dai, Wen; Zhang, Lin; Kang, Tao; Chen, Ping; He, Ying; Liu, Tingting; Zhao, Qing; Wang, Wenjing; Zhi, Jin; Feng, Guodong; Zhao, Gang

    2015-08-01

    This study aimed to co-culture Jurkat T lymphocytes with inactivated Mycobacterium tuberculosis (Mtb H37Ra), explore whether T lymphocytes could phagocytose H37Ra cells, and determine the underlying mechanism. Jurkat T lymphocytes were co-cultured with H37Ra cells, and confocal laser scanning microscopy, electron microscopy, and flow cytometry techniques were used to identify phagocytosis and elucidate its mechanism. After Jurkat T lymphocytes phagocytosed H37Ra cells, the cell body became larger, with abundant cytoplasm, the portion of the nucleus closest to the bacterium deformed, long and short pseudopodia were extended, and the folds of the cell membrane formed depressions that created phagocytic vesicles surrounding the bacterium. The macropinocytosis inhibitor amiloride and the cytoskeletal inhibitor cytochalasin D were found to inhibit phagocytic efficacy; serum complements might enhance phagocytosis through opsonization. Jurkat T lymphocytes could actively phagocytose inactivated Mtb via the macropinocytotic mechanism. Actin remodeling played an important role in the macropinocytotic process. Serum complements may regulate phagocytosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Early effects of aldosterone on Na-K pump in rat cortical collecting tubules

    International Nuclear Information System (INIS)

    Fujii, Y.; Takemoto, F.; Katz, A.I.

    1990-01-01

    Sustained exposure to aldosterone (Aldo) increases the abundance and activity of the Na-K pump in cortical collecting tubules (CCT). However, the onset and mechanism of the early interaction of Aldo with the CCT pump, especially in adrenal-intact animals, are unclear. We evaluated the short-term effects of the hormone on Na-K-adenosinetriphosphatase (ATPase) activity and on ouabain-sensitive 86Rb uptake, a measure of the transporting rate of the pump, in microdissected CCT from adrenal-intact rats. Incubation with Aldo (10(-8) M, 2 h) had no effect on Na-K-ATPase activity (Vmax), whereas it produced at least a twofold increase in 86Rb uptake. This effect was generated by physiological concentrations of the hormone (threshold 10(-10) M; apparent K1/2 approximately 10(-9) M), after a short lag of less than or equal to 30 min. Incubation with Aldo in the presence of amiloride or nystatin or in a Na-free medium (choline chloride) did not prevent the enhanced 86Rb uptake seen after Aldo alone; possible interpretations of these observations are discussed. We conclude that Aldo produces a rapid stimulation of pump function in CCT that precedes its induction of new pump synthesis; the physiological significance of this effect is suggested by its occurrence in tubules from adrenal-intact animals within the time frame and concentration range of the hormone's effects on electrolyte transport

  13. Flow Cytometry Analysis Reveals That Only a Subpopulation of Mouse Sperm Undergoes Hyperpolarization During Capacitation1

    Science.gov (United States)

    Escoffier, Jessica; Navarrete, Felipe; Haddad, Doug; Santi, Celia M.; Darszon, Alberto; Visconti, Pablo E.

    2015-01-01

    To gain fertilizing capacity, mammalian sperm should reside in the female tract for a period of time. The physiological changes that render the sperm able to fertilize are known as capacitation. Capacitation is associated with an increase in intracellular pH, an increase in intracellular calcium, and phosphorylation of different proteins. This process is also accompanied by the hyperpolarization of the sperm plasma membrane potential (Em). In the present work, we used flow cytometry to analyze changes in sperm Em during capacitation in individual cells. Our results indicate that a subpopulation of hyperpolarized mouse sperm can be clearly distinguished by sperm flow cytometry analysis. Using sperm bearing green fluorescent protein in their acrosomes, we found that this hyperpolarized subpopulation is composed of sperm with intact acrosomes. In addition, we show that the capacitation-associated hyperpolarization is blocked by high extracellular K+, by PKA inhibitors, and by SLO3 inhibitors in CD1 mouse sperm, and undetectable in Slo3 knockout mouse sperm. On the other hand, in sperm incubated in conditions that do not support capacitation, sperm membrane hyperpolarization can be induced by amiloride, high extracellular NaHCO3, and cAMP agonists. Altogether, our observations are consistent with a model in which sperm Em hyperpolarization is downstream of a cAMP-dependent pathway and is mediated by the activation of SLO3 K+ channels. PMID:25855261

  14. Flow cytometry analysis reveals that only a subpopulation of mouse sperm undergoes hyperpolarization during capacitation.

    Science.gov (United States)

    Escoffier, Jessica; Navarrete, Felipe; Haddad, Doug; Santi, Celia M; Darszon, Alberto; Visconti, Pablo E

    2015-05-01

    To gain fertilizing capacity, mammalian sperm should reside in the female tract for a period of time. The physiological changes that render the sperm able to fertilize are known as capacitation. Capacitation is associated with an increase in intracellular pH, an increase in intracellular calcium, and phosphorylation of different proteins. This process is also accompanied by the hyperpolarization of the sperm plasma membrane potential (Em). In the present work, we used flow cytometry to analyze changes in sperm Em during capacitation in individual cells. Our results indicate that a subpopulation of hyperpolarized mouse sperm can be clearly distinguished by sperm flow cytometry analysis. Using sperm bearing green fluorescent protein in their acrosomes, we found that this hyperpolarized subpopulation is composed of sperm with intact acrosomes. In addition, we show that the capacitation-associated hyperpolarization is blocked by high extracellular K(+), by PKA inhibitors, and by SLO3 inhibitors in CD1 mouse sperm, and undetectable in Slo3 knockout mouse sperm. On the other hand, in sperm incubated in conditions that do not support capacitation, sperm membrane hyperpolarization can be induced by amiloride, high extracellular NaHCO3, and cAMP agonists. Altogether, our observations are consistent with a model in which sperm Em hyperpolarization is downstream of a cAMP-dependent pathway and is mediated by the activation of SLO3 K(+) channels. © 2015 by the Society for the Study of Reproduction, Inc.

  15. Pseudohypoaldosteronism in a newborn male with functional polymorphisms in the mineralocorticoid receptor genes

    Directory of Open Access Journals (Sweden)

    Hyun Ah Jeong

    2015-12-01

    Full Text Available Hyponatremia and hyperkalemia in infancy can be attributed to various causes, originating from a variety of renal and genetic disorders. Pseudohypoaldosteronism type 1 (PHA1 is one of these disorders, causing mineralocorticoid resistance that results in urinary salt wasting, failure to thrive, metabolic acidosis, and dehydration. PHA1 is heterogeneous in etiology. Inactivating mutations in the NR3C2 gene (4q31.1, which encodes the mineralocorticoid receptor, causes a less severe autosomal dominant form that is restricted to the kidney, while mutations in the amiloride-sensitive epithelial sodium channel gene (alpha subunit=SCNN1A, 12p13; beta subunit=SCNN1b, 16p12.2-p12.1; gamma subunit=SCNN1G, 16p12 causes a more severe autosomal recessive form, which has systemic effects. Here we report a neonatal case of kidney restricted PHA1 (renal type of PHA1 who first showed laboratory abnormalities before obvious PHA1 manifestations, with two functional polymorphisms in the NR3C2 gene. This is the second genetically confirmed case in Korea and the first to show functional polymorphisms that have previously been reported in the literature.

  16. NH4+ secretion in the avian colon. An actively regulated barrier to ammonium permeation of the colon mucosa

    DEFF Research Database (Denmark)

    Holtug, K.; Laverty, G.; Arnason, S.S.

    2009-01-01

    -clamped to measure the short-circuit currents (I(SC)) associated with transport. Bilateral addition of NH(4)(+) caused a dose-dependent outward current (negative I(SC)), with a Km of 34+/-8 mM and a maximal current response of 311+/-47 microA cm(-2) (12+/-2 microEq cm(-2) h(-1)). A similar effect was seen...... with unilateral addition of NH(4)(+) to the serosal (s) side, but not with mucosal (m) addition. Pre-treatment with 10(-4) M amiloride exposed a net outward (negative) I(SC), and serosal NH(4)(+) addition further increased this outward current with a Km of 53+/-24 mM. Decreasing the bath pH from 7.3 to 6.0 did...... not affect the I(SC) response to NH(4)(+). Unidirectional NH(4)(+) flux measurements revealed a net secretory flux (8.8+/-3.1 micromol cm(-2) h(-1) s-m, versus 2.6+/-1.4 micromol cm(-2) h(-1) m-s). Furthermore, the secretory flux closely matched the resulting change in I(SC) with serosal NH(4)(+), showing...

  17. Correlation of open cell-attached and excised patch clamp techniques.

    Science.gov (United States)

    Filipovic, D; Hayslett, J P

    1995-11-01

    The excised patch clamp configuration provides a unique technique for some types of single channel analyses, but maintenance of stable, long-lasting preparations may be confounded by rundown and/or rapid loss of seal. Studies were performed on the amiloride-sensitive Na+ channel, located on the apical surface of A6 cells, to determine whether the nystatin-induced open cell-attached patch could serve as an alternative configuration. Compared to excised inside-out patches, stable preparations were achieved more readily with the open cell-attached patch (9% vs. 56% of attempts). In both preparations, the current voltage (I-V) relation was linear, current amplitudes were equal at opposite equivalent clamped voltages, and Erev was zero in symmetrical Na+ solutions, indicating similar Na+ activities on the cytosolic and external surfaces of the patch. Moreover, there was no evidence that nystatin altered channel activity in the patch because slope conductance (3-4pS) and Erev (75 mV), when the bath was perfused with a high K:low Na solution (ENa = 80 mV), were nearly equal in both patch configurations. Our results therefore indicate that the nystatin-induced open cell-attached patch can serve as an alternative approach to the excised inside-out patch when experiments require modulation of univalent ions in the cytosol.

  18. Diuretic activity of the aqueous extract leaves of Ficus glumosa Del. (Moraceae) in rats.

    Science.gov (United States)

    Ntchapda, Fidèle; Djedouboum Abakar; Kom, Blaise; Nana, Paulin; Bonabe, Christian; Maguirgue Kakesse; Talla, Emmanuel; Dimo, Théophile

    2014-01-01

    Experiments were carried out to validate the use of F. glumosa extract as a diuretic in the treatment of hypertension as claimed by traditional healers. The experiments were performed under the same conditions with two synthetic pharmacological diuretics considered as check (Furosemide and Amiloride hydrochlorothiazide). The aqueous extract leaves of F. glumosa accelerated the elimination of overloaded fluid. At the maximum of diuretic response, urinary osmolarity decreased significantly when compared with controls. The single dose treatment of the aqueous extract leaves of F. glumosa has significantly increased urine volume 24 h after administration of the extract. The stability of aldosterone level, the absence of correlation with the plasma levels of sodium, and the increased clearance of free water in the animals receiving the extract show that increased diuresis and natriuresis moderate elevation are tubular in origin. The increase in Na(+), K(+), and Cl(-) induced by the extract caused alkalinization of the urine and showed a strong inhibitory effect of carbonic anhydrase and saluretic. These effects were mainly observed at the dose of 375 mg/kg. These observations confirm the traditional use in the treatment of hypertension and support the importance of the conservation of local knowledge as well as the conservation of Cameroonian biodiversity.

  19. Characterization of a plasminogen activator produced by Acanthamoeba castellanii.

    Science.gov (United States)

    Mitra, M M; Alizadeh, H; Gerard, R D; Niederkorn, J Y

    1995-07-01

    Serine proteases play an important role in a diverse array of biological processes, including embryogenesis, metastasis, angiogenesis, thrombolysis and tissue invasion by certain parasites. The latter observation prompted us to explore the possibility that the tissue-invasive ocular parasite Acanthamoeba castellanii elaborates one or more serine proteases. Acanthamoeba sp. are pathogenic free-living amoebae that can produce an invasive, blinding inflammatory disease of the cornea, termed Acanthamoeba keratitis. The present study reports the preliminary purification and characterization of a novel plasminogen activator from an ocular isolate of A. castellanii. The parasite-derived enzyme has a molecular mass of approx. 40 kDa and produces a single band of lysis on fibrinogen-agarose zymographs. Activity of the enzyme is completely inhibited by treatment with diisopropylfluorophosphate, indicating that it is a serine protease. The parasite-derived serine protease is not inhibited by amiloride which is a strong inhibitor of urokinase-type plasminogen activator. Additionally, the enzyme is not inhibited by plasminogen activator inhibitor-1 which is the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator. It does not cross-react with antibodies specific for human urokinase or tissue-type plasminogen activator. The parasite-derived enzyme activates plasminogen from several mammalian species, including human, cow and pig. Thus, it is possible that this parasite-derived serine protease contributes to the pathogenesis of Acanthamoeba keratitis.

  20. Tubular transport and metabolism of cimetidine in chicken kidneys

    Energy Technology Data Exchange (ETDEWEB)

    Rennick, B.; Ziemniak, J.; Smith, I.; Taylor, M.; Acara, M.

    1984-02-01

    Renal tubular transport and renal metabolism of (/sup 14/C)cimetidine (CIM) were investigated by unilateral infusion into the renal portal circulation in chickens (Sperber technique). (/sup 14/C)CIM was actively transported at a rate 88% that of simultaneously infused p-aminohippuric acid, and its transport was saturable. The following organic cations competitively inhibited the tubular transport of (/sup 14/C)CIM with decreasing potency: CIM, ranitidine, thiamine, procainamide, guanidine and choline. CIM inhibited the transport of (/sup 14/C)thiamine, (/sup 14/C)amiloride and (/sup 14/C)tetraethylammonium. During CIM infusion, two renal metabolites, CIM sulfoxide and hydroxymethylcimetidine, were found in urine. When CIM sulfoxide was infused, its transport efficiency was 32% and not saturable. CIM sulfoxide did ot inhibit the simultaneous renal tubular transport of p-aminohippuric acid or tetraethylammonium. CIM is transported by the organic cation transport system and the kidney metabolizes CIM. Transport of CIM and other cationic drugs could produce a drug interaction to alter drug excretion.

  1. Embryonic epithelial membrane transporters.

    Science.gov (United States)

    Horster, M

    2000-12-01

    Embryonic epithelial membrane transporters are organized into transporter families that are functional in several epithelial organs, namely, in kidney, lung, pancreas, intestine, and salivary gland. Family members (subtypes) are developmentally expressed in plasma membranes in temporospatial patterns that are 1) similar for one subtype within different organs, like aquaporin-1 (AQP1) in lung and kidney; 2) different between subtypes within the same organ, like the amiloride-sensitive epithelial sodium channel (ENaC) in lung; and 3) apparently matched among members of different transporter families, as alpha-ENaC with AQP1 and -4 in lung and with AQP2 in kidney. Finally, comparison of temporal expression patterns in early embryonic development of transporters from different families [e.g., cystic fibrosis transmembrane conductance regulator (CFTR), ENaC, and outer medullary potassium channel] suggests regulatory activating or inactivating interactions in defined morphogenic periods. This review focuses on embryonic patterns, at the mRNA and immunoprotein level, of the following transporter entities expressed in epithelial cell plasma membranes: ENaC; the chloride transporters CFTR, ClC-2, bumetanide-sensitive Na-K-Cl cotransporter, Cl/OH, and Cl/HCO(3); the sodium glucose transporter-glucose transporter; the sodium/hydrogen exchanger; the sodium-phosphate cotransporter; the ATPases; and AQP. The purpose of this article is to relate temporal and spatial expression patterns in embryonic and in early postnatal epithelia to developmental changes in organ structure and function.

  2. Evidence that membrane transduction of oligoarginine does not require vesicle formation

    International Nuclear Information System (INIS)

    Zaro, Jennica L.; Shen Weichiang

    2005-01-01

    The involvement of vesicular formation processes in the membrane transduction and nuclear transport of oligoarginine is currently a subject of controversy. In this report, a novel quantitative method which allows for the selective measurement of membrane transduction excluding concurrent endocytosis was used to determine the effects of temperature, endosomal acidification, endosomolysis, and several known inhibitors of endocytic pathways on the internalization of oligoarginine. The results show that, unlike endocytosis, transduction of oligoarginine was not affected by incubation at 16 deg. C as compared to the 37 deg. C control, and was only partially inhibited at 4 deg. C incubation. Additionally, membrane transduction was not inhibited to the same extent as endocytosis following treatment with ammonium chloride, hypertonic medium, amiloride, or filipin. The endosomolytic activity of oligoarginine was investigated by examining the leakage of FITC-dextran into the cytosolic compartment, which was not higher in the presence of oligoarginine. Furthermore, ammonium chloride showed no effect on the nuclear transport of oligoarginine. The data presented in this report indicate that membrane transduction is likely to occur at the plasma membrane without the formation of membrane vesicles, and the nuclear localization involves membrane transduction, rather than endocytosis of oligoarginine

  3. Acid-Sensing Ion Channels Expression, Identity and Role in the Excitability of the Cochlear Afferent Neurons

    Science.gov (United States)

    González-Garrido, Antonia; Vega, Rosario; Mercado, Francisco; López, Iván A.; Soto, Enrique

    2015-01-01

    Acid-sensing ion channels (ASICs) are activated by an increase in the extracellular proton concentration. There are four genes (ASIC1-4) that encode six subunits, and they are involved in diverse neuronal functions, such as mechanosensation, learning and memory, nociception, and modulation of retinal function. In this study, we characterize the ASIC currents of spiral ganglion neurons (SGNs). These ASIC currents are primarily carried by Na+, exhibit fast activation and desensitization, display a pH50 of 6.2 and are blocked by amiloride, indicating that these are ASIC currents. The ASIC currents were further characterized using several pharmacological tools. Gadolinium and acetylsalicylic acid reduced these currents, and FMRFamide, zinc (at high concentrations) and N,N,N’,N’–tetrakis-(2-piridilmetil)-ethylenediamine increased them, indicating that functional ASICs are composed of the subunits ASIC1, ASIC2, and ASIC3. Neomycin and streptomycin reduced the desensitization rate of the ASIC current in SGNs, indicating that ASICs may contribute to the ototoxic action of aminoglycosides. RT-PCR of the spiral ganglion revealed significant expression of all ASIC subunits. By immunohistochemistry the expression of the ASIC1a, ASIC2a, ASIC2b, and ASIC3 subunits was detected in SGNs. Although only a few SGNs exhibited action potential firing in response to an acidic stimulus, protons in the extracellular solution modulated SGN activity during sinusoidal stimulation. Our results show that protons modulate the excitability of SGNs via ASICs. PMID:26733809

  4. Ba2+-inhibitable /sup 86/Rb+ fluxes across membranes of vesicles from toad urinary bladder

    Energy Technology Data Exchange (ETDEWEB)

    Garty, H.; Civan, M.M.

    1987-01-01

    /sup 86/Rb+ fluxes have been measured in suspensions of vesicles prepared from the epithelium of toad urinary bladder. A readily measurable barium-sensitive, ouabain-insensitive component has been identified; the concentration of external Ba2+ required for half-maximal inhibition was 0.6 mM. The effects of externally added cations on /sup 86/Rb+ influx and efflux have established that this pathway is conductive, with a selectivity for K+, Rb+ and Cs+ over Na+ and Li+. The Rb+ uptake is inversely dependent on external pH, but not significantly affected by internal Ca2+ or external amiloride, quinine, quinidine or lidocaine. It is likely, albeit not yet certain, that the conductive Rb+ pathway is incorporated in basolateral vesicles oriented right-side-out. It is also not yet clear whether this pathway comprises the principle basolateral K+ channel in vivo, and that its properties have been unchanged during the preparative procedures. Subject to these caveats, the data suggest that the inhibition by quinidine of Na+ transport across toad bladder does not arise primarily from membrane depolarization produced by a direct blockage of the basolateral channels. It now seems more likely that the quinidine-induced elevation of intracellular Ca2+ activity directly blocks apical Na+ entry.

  5. Functional hair cell mechanotransducer channels are required for aminoglycoside ototoxicity.

    Directory of Open Access Journals (Sweden)

    Abdelrahman Alharazneh

    Full Text Available Aminoglycosides (AG are commonly prescribed antibiotics with potent bactericidal activities. One main side effect is permanent sensorineural hearing loss, induced by selective inner ear sensory hair cell death. Much work has focused on AG's initiating cell death processes, however, fewer studies exist defining mechanisms of AG uptake by hair cells. The current study investigated two proposed mechanisms of AG transport in mammalian hair cells: mechanotransducer (MET channels and endocytosis. To study these two mechanisms, rat cochlear explants were cultured as whole organs in gentamicin-containing media. Two-photon imaging of Texas Red conjugated gentamicin (GTTR uptake into live hair cells was rapid and selective. Hypocalcemia, which increases the open probability of MET channels, increased AG entry into hair cells. Three blockers of MET channels (curare, quinine, and amiloride significantly reduced GTTR uptake, whereas the endocytosis inhibitor concanavalin A did not. Dynosore quenched the fluorescence of GTTR and could not be tested. Pharmacologic blockade of MET channels with curare or quinine, but not concanavalin A or dynosore, prevented hair cell loss when challenged with gentamicin for up to 96 hours. Taken together, data indicate that the patency of MET channels mediated AG entry into hair cells and its toxicity. Results suggest that limiting permeation of AGs through MET channel or preventing their entry into endolymph are potential therapeutic targets for preventing hair cell death and hearing loss.

  6. The effects of environmental deuterium on normal and neoplastic cultured cell development

    International Nuclear Information System (INIS)

    Bild, W.; Schuller, T.; Zhihai, Qin; Blankenstein, T.; Nastasa, V.; Haulica, I.

    2000-01-01

    The powdered culture media (RPMI - 1640) were reconstituted either with normal distilled water (150 ppm deuterium) either with deuterium - depleted water (DDW) in various concentrations (30, 60, 90 ppm) and sterilized by filtration with 0.2 μm filters. The cell lines used were NIH (normal mouse fibroblasts), RAG (mouse renal carcinoma) and TS/A (mouse mammary adenocarcinoma). In auxiliary tests, BAIBC mouse splenocytes in direct culture were used, stimulated for growth with concanavalin A or LPS (bacterial lipopolysaccharide). The estimation of the growth was made using the MTT assay or direct counting with trypan blue exclusion. The following results were obtained: Deuterium - depleted water had a stimulating effect on cell growth, the most important stimulating action being from the 90 ppm deuterium-water. The growth curves show, in a first phase, a stimulation of the rapid -growing neoplastic cells, followed by a slower growth of the normal cells. Amiloride 100 mM blocking of the Na + /K + membrane pump did not affect the cell growth curves, while the lansoprazole 100 mM blocking of the K + /H + ATP-ase brought the growth curves at the level of those with normal water. This might show an eventual involvement of the K + /H + antiport in the stimulating effects of the DDW. (authors)

  7. Na(+) /H(+) exchanger 1 (NHE1) function is necessary for maintaining mammary tissue architecture.

    Science.gov (United States)

    Jenkins, Edmund C; Debnath, Shawon; Varriano, Sophia; Gundry, Stephen; Fata, Jimmie E

    2014-02-01

    The mammary gland is an ideal model to study the link between form and function in normal tissue. Perhaps as interesting as the cues necessary to generate this structure are the signals required to maintain its branched architecture over the lifetime of the organism, since likely these pathways are de-regulated in malignancies. Previously, we have shown that the Na(+) /H(+) exchanger 1 (NHE1), a critical regulator of intracellular pH, was necessary for mammary branching morphogenesis. Here we provide strong evidence that NHE1 function is also necessary for maintaining mammary branched architecture. Inhibition of NHE1 with 5-N-Methy-N-isobutyl amiloride (MIA) on branched structures resulted in a rapid (within 24 hr) and reversible loss of branched architecture that was not accompanied by any overt changes in cell proliferation or cell death. NHE1 inhibition led to a significant acidification of intracellular pH in the branched end buds that preceded a number of events, including altered tissue polarity of myoepithelial cells, loss of NHE1 basal polarity, F-actin rearrangements, and decreased E-cadherin expression. Our results implicate NHE1 function and intracellular pH homeostasis as key factors that maintain mammary tissue architecture, thus, indirectly allowing for mammary function as a milk-providing (form) and -producing (function) gland. Copyright © 2013 Wiley Periodicals, Inc.

  8. Characterization of the Sweet Taste Receptor Tas1r2 from an Old World Monkey Species Rhesus Monkey and Species-Dependent Activation of the Monomeric Receptor by an Intense Sweetener Perillartine.

    Directory of Open Access Journals (Sweden)

    Chenggu Cai

    Full Text Available Sweet state is a basic physiological sensation of humans and other mammals which is mediated by the broadly acting sweet taste receptor-the heterodimer of Tas1r2 (taste receptor type 1 member 2 and Tas1r3 (taste receptor type 1 member 3. Various sweeteners interact with either Tas1r2 or Tas1r3 and then activate the receptor. In this study, we cloned, expressed and functionally characterized the taste receptor Tas1r2 from a species of Old World monkeys, the rhesus monkey. Paired with the human TAS1R3, it was shown that the rhesus monkey Tas1r2 could respond to natural sugars, amino acids and their derivates. Furthermore, similar to human TAS1R2, rhesus monkey Tas1r2 could respond to artificial sweeteners and sweet-tasting proteins. However, the responses induced by rhesus monkey Tas1r2 could not be inhibited by the sweet inhibitor amiloride. Moreover, we found a species-dependent activation of the Tas1r2 monomeric receptors of human, rhesus monkey and squirrel monkey but not mouse by an intense sweetener perillartine. Molecular modeling and sequence analysis indicate that the receptor has the conserved domains and ligand-specific interactive residues, which have been identified in the characterized sweet taste receptors up to now. This is the first report of the functional characterization of sweet taste receptors from an Old World monkey species.

  9. The Na(+)/H(+) exchanger SM-20220 attenuates ischemic injury in in vitro and in vivo models.

    Science.gov (United States)

    Horikawa, N; Nishioka, M; Itoh, N; Kuribayashi, Y; Matsui, K; Ohashi, N

    2001-01-01

    The aim of this study is to clarify whether the activation of a Na(+)/H(+) exchanger (NHE) is tightly concerned with neuronal and glial cell injury induced by ischemia using a selective NHE inhibitor, SM-20220 (N-(aminoiminomethyl)-1-methyl-1H-indole-2-carboxamide methanesulfonate). Two hours of hypoxia followed by 24 h of reoxygenation induced lactate dehydrogenase (LDH) release, a marker of cell membrane damage, in cultured neurons and glia derived from rats. SM-20220 significantly reduced LDH release in both cells in a concentration-dependent manner, and this effect was statistically significant at concentrations of more than 10(-8) mol/l for neurons and 10(-7) mol/l for glia. A standard NHE inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride, also reduced LDH release in neurons at concentrations of more than 10(-7) mol/l. In a rat transient middle cerebral artery occlusion model, intravenous infusion of SM-20220 reduced cerebral infarction when the serum concentration of SM- 20220 was maintained at about 10(-7) mol/l. These results suggest that the activation of the NHE plays an important role in ischemic neuronal and glial cell injury, and NHE inhibitor may have good therapeutic value for the treatment of ischemic stroke. Copyright 2001 S. Karger AG, Basel.

  10. Preparation and characterization of viable epithelial cells from rabbit distal colon

    International Nuclear Information System (INIS)

    Kaunitz, J.D.

    1988-01-01

    A preparation of viable epithelial cells, suitable for transport studies, was prepared from rabbit distal colon. Enzymatic digestion of scraped mucosa liberated a population of intact colonic glands that were dissociated into cells by gentle agitation in culture medium. Metabolism of [ 14 C]glucose was constant over 2 h and was inhibited 70% by 1 mM ouabain. Cells were loaded with the trapped, pH-sensitive fluorescent dye 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxy methyl ester and leaked 0.1% of the dye per minute. Cell pH was 7.21 ± 0.03 in pH 7.4. Ringer medium. Intracellular potassium concentration, as measured by a nigericin null-point technique, was 128 ± 8 mM. Plasma membrane potential measured with the potential-sensitive fluorescent dye 3,3-dipropylthiadicarboxycanine iodide was -52 ± 2 mV. Recovery of intracellular pH in acid-loaded cells occurred after exposure to sodium-containing medium and was inhibited by 5 x 10 -4 M amiloride. It is concluded that viable epithelial cells can be prepared from rabbit distal colon with relative simplicity and in high yield. These cells are suitable for measurement of intracellular pH and membrane potential and are thus a convenient model system for study of colonic cell physiology

  11. Lithium absorption by the rabbit gall-bladder

    DEFF Research Database (Denmark)

    Hansen, C P; Holstein-Rathlou, N H; Skøtt, O

    1991-01-01

    was elicited from the mucosal side and was not accounted for by compensatory Li+ absorption; water and Na+ absorption rates decreased nearly in parallel. The effects of 0.4 mM amiloride and of substitution with 20 mM Li+ were only partly additive. It is concluded that Li+ absorption in the rabbit gall......Lithium (Li+) absorption across the low-resistance epithelium of the rabbit gall-bladder was studied in order to elucidate possible routes and mechanisms of Li+ transfer. Li+ at a concentration of 0.4 mM in both mucosal and serosal media did not affect isosmotic mucosa-to-serosa fluid absorption....... At this low concentration net mucosa-to-serosa Li+ absorption was insignificant when the ambient Na+ concentration was 115 mM, although the gall-bladder had a significant Li+ permeability (2.7 X 10(-5) cm s-1) and a significant mucosa-to-serosa Li+ gradient developed as a result of fluid absorption. Net Li...

  12. Calcium reduces the sodium permeability of luminal membrane vesicles from toad bladder. Studies using a fast-reaction apparatus

    International Nuclear Information System (INIS)

    Chase, H.S. Jr.; Al-Awqati, Q.

    1983-01-01

    Regulation of the sodium permeability of the luminal membrane is the major mechanism by which the net rate of sodium transport across tight epithelia is varied. Previous evidence has suggested that the permeability of the luminal membrane might be regulated by changes in intracellular sodium or calcium activities. To test this directly, we isolated a fraction of the plasma membrane from the toad urinary bladder, which contains a fast, amiloride-sensitive sodium flux with characteristics similar to those of the native luminal membrane. Using a flow-quench apparatus to measure the initial rate of sodium efflux from these vesicles in the millisecond time range, we have demonstrated that the isotope exchange permeability of these vesicles is very sensitive to calcium. Calcium reduces the sodium permeability, and the half-maximal inhibitory concentration is 0.5 microM, well within the range of calcium activity found in cells. Also, the permeability of the luminal membrane vesicles is little affected by the ambient sodium concentration. These results, when taken together with studies on whole tissue, suggest that cell calcium may be an important regulator of transepithelial sodium transport by its effect on luminal sodium permeability. The effect of cell sodium on permeability may be mediated by calcium rather than by sodium itself

  13. Pre- and post-treatment urinary tract findings in children with nephrogenic diabetes insipidus.

    Science.gov (United States)

    Caletti, María Gracia; Balestracci, Alejandro; Di Pinto, Diana

    2014-03-01

    Nephrogenic diabetes insipidus (NDI) is characterized by the kidney's inability to concentrate urine, which causes intense polyuria that may lead to urinary tract dilation. We report the morphological findings of the urinary tract in ten boys with NDI specifically addressing the presence and changes of urinary tract dilation during treatment. Patients were diagnosed at a median age of 1.6 years (range, 0.16-6.33 years) and treated with a low osmotic diet, hydrochlorothiazide-amiloride and indomethacin, which decreased the diuresis from a median of 10.5 ml/kg/h to 4.4 ml/kg/h (p < 0.001). Three patients showed normal renal ultrasound before treatment until last control, while the remaining seven showed urinary tract dilation. In this second group, dilation was reduced with treatment in four patients and disappeared in the remaining three. Children without dilation or in whom the dilation disappeared were diagnosed and treated earlier than those with persistent dilation (median 1.66 versus 4.45 years, respectively). After a median of 10.4 (range, 2.3-20.3) years of follow-up, no patients showed urological complications. Medical treatment of the disease improved the dilation in all cases, preventing its potential complications. Regardless of the good outcome of our patients, periodic urologic follow-up is recommended in NDI patients.

  14. Diuretics prevent thiazolidinedione-induced cardiac hypertrophy without compromising insulin-sensitizing effects in mice.

    Science.gov (United States)

    Chang, Cherng-Shyang; Tsai, Pei-Jane; Sung, Junne-Ming; Chen, Ju-Yi; Ho, Li-Chun; Pandya, Kumar; Maeda, Nobuyo; Tsai, Yau-Sheng

    2014-02-01

    Much concern has arisen regarding critical adverse effects of thiazolidinediones (TZDs), including rosiglitazone and pioglitazone, on cardiac tissue. Although TZD-induced cardiac hypertrophy (CH) has been attributed to an increase in plasma volume or a change in cardiac nutrient preference, causative roles have not been established. To test the hypothesis that volume expansion directly mediates rosiglitazone-induced CH, mice were fed a high-fat diet with rosiglitazone, and cardiac and metabolic consequences were examined. Rosiglitazone treatment induced volume expansion and CH in wild-type and PPARγ heterozygous knockout (Pparg(+/-)) mice, but not in mice defective for ligand binding (Pparg(P465L/+)). Cotreatment with the diuretic furosemide in wild-type mice attenuated rosiglitazone-induced CH, hypertrophic gene reprogramming, cardiomyocyte apoptosis, hypertrophy-related signal activation, and left ventricular dysfunction. Similar changes were observed in mice treated with pioglitazone. The diuretics spironolactone and trichlormethiazide, but not amiloride, attenuated rosiglitazone effects on volume expansion and CH. Interestingly, expression of glucose and lipid metabolism genes in the heart was altered by rosiglitazone, but these changes were not attenuated by furosemide cotreatment. Importantly, rosiglitazone-mediated whole-body metabolic improvements were not affected by furosemide cotreatment. We conclude that releasing plasma volume reduces adverse effects of TZD-induced volume expansion and cardiac events without compromising TZD actions in metabolic switch in the heart and whole-body insulin sensitivity. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  15. [Diuretics in monotherapy and in combination with other diuretics and nondiuretics in the treatment of hypertension].

    Science.gov (United States)

    Spinar, J; Spinarová, L; Vítovec, J

    2013-06-01

    Diuretics belong to the basic group of medicines for the treatment of hypertension and heart failure. In the case of hypertension treatment, their main indication is higher age and isolated systolic hypertension. In the case of heart failure they are used for the treatment of swellings and shortness of breath. The most frequently prescribed group of diuretics is thiazides and similar products. In patients with renal insufficiency, loop diuretics are administered. In the case of hypertension, diuretics are mainly used in the combination treatment. The most frequently used diuretic in combination is again hydrochlorothiazide, which is combined with reninangiotensin system blockers. It is mainly the combination of an ACE inhibitor + indapamide that seems to be modern and promising, and it is, on the basis of large clinical trials, recommended also for diabetics (ADVANCE) or for secondary prevention following a cerebrovascular accident (PROGRESS) or for the elderly (HYVET). Also a combination of two diuretics is popular -  mainly hydrochlorothiazide + amiloride. A combination of a betablocker and diuretic is less suitable.

  16. A new HDAC inhibitor cinnamoylphenazine shows antitumor activity in association with intensive macropinocytosis.

    Science.gov (United States)

    Zhu, Bing-Yan; Shang, Bo-Yang; Du, Yue; Li, Yi; Li, Liang; Xu, Xian-Dong; Zhen, Yong-Su

    2017-02-28

    Previous studies have shown that intensive macropinocytosis occurs in cancer cells and neutral red (NR) is noted for its capability to enter into the cell massively through a process mimetic to macropinocytosis. In addition, trans-cinnamic acid (tCA) has been found to be an inhibitor of histone deacetylase (HDAC). In the present study, cinnamoylphenazine (CA-PZ) that consists of NR and tCA moieties was synthesized and evaluated. As shown, CA-PZ massively entered into colon carcinoma HT-29 cells and pancreatic carcinoma MIA PaCa-2 cells and this entry was blocked by 5-(N-ethyl-N-isopropyl) amiloride (EIPA, an inhibitor of macropinocytosis), indicating a macropinocytosis-mediated uptake. Furthermore, CA-PZ markedly increased the protein expression levels of acetyl-H3, acetyl-H4 and p21 in HT-29 cells and MIA PaCa-2 cells. CA-PZ significantly inhibited the growth of colon carcinoma HT-29 and pancreatic carcinoma MIA PaCa-2 xenografts. By in vivo imaging, CA-PZ displayed prominent accumulation in the tumor xenografts. The study indicates that the newly synthesized CA-PZ acts as an HDAC inhibitor in association with intensive macropinocytosis-mediated intracellular delivery in cancer cells. The use of neutral red for preparation of chimeric molecules with the attribute of macropinocytosis-mediated intracellular delivery might open an alternative way for development of HDAC inhibitors.

  17. Dissecting Bacterial Cell Wall Entry and Signaling in Eukaryotic Cells: an Actin-Dependent Pathway Parallels Platelet-Activating Factor Receptor-Mediated Endocytosis

    Directory of Open Access Journals (Sweden)

    Lip Nam Loh

    2017-01-01

    Full Text Available The Gram-positive bacterial cell wall (CW peptidoglycan-teichoic acid complex is released into the host environment during bacterial metabolism or death. It is a highly inflammatory Toll-like receptor 2 (TLR2 ligand, and previous in vivo studies have demonstrated its ability to recapitulate pathological features of pneumonia and meningitis. We report that an actin-dependent pathway is involved in the internalization of the CW by epithelial and endothelial cells, in addition to the previously described platelet-activating factor receptor (PAFr-dependent uptake pathway. Unlike the PAFr-dependent pathway, which is mediated by clathrin and dynamin and does not lead to signaling, the alternative pathway is sensitive to 5-(N-ethyl-N-isopropyl amiloride (EIPA and engenders Rac1, Cdc42, and phosphatidylinositol 3-kinase (PI3K signaling. Upon internalization by this macropinocytosis-like pathway, CW is trafficked to lysosomes. Intracellular CW trafficking is more complex than previously recognized and suggests multiple points of interaction with and without innate immune signaling.

  18. Uptake of advanced glycation end products by proximal tubule epithelial cells via macropinocytosis.

    Science.gov (United States)

    Gallicchio, Marisa A; Bach, Leon A

    2013-12-01

    Chronic hyperglycaemia during diabetes leads to non-enzymatic glycation of proteins to form advanced glycation end products (AGEs) that contribute to nephropathy. We describe AGE uptake in LLC-PK1 and HK2 proximal tubule cell lines by macropinocytosis, a non-specific, endocytic mechanism. AGE-BSA induced dorsal circular actin ruffles and amiloride-sensitive dextran-TRITC uptake, significantly increased AGE-BSA-FITC uptake (167±20% vs BSA control, pmacropinocytosis. PAK1 and PIP5Kγ siRNA significantly decreased AGE-BSA-FITC uptake (81±6% and 64±7%, respectively, pmacropinocytosis inhibitors and a neutralising TGF-β antibody, reversed the AGE-induced decrease in surface Na(+)K(+)ATPase, suggesting AGE uptake by macropinocytosis may contribute to diabetic kidney fibrosis and/or EMT by modulating this pump. Understanding methods of cellular uptake and signalling by AGEs may lead to novel therapies for diabetic nephropathy. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Duffy antigen receptor for chemokines mediates chemokine endocytosis through a macropinocytosis-like process in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Yani Zhao

    Full Text Available The Duffy antigen receptor for chemokines (DARC shows high affinity binding to multiple inflammatory CC and CXC chemokines and is expressed by erythrocytes and endothelial cells. Recent evidence suggests that endothelial DARC facilitates chemokine transcytosis to promote neutrophil recruitment. However, the mechanism of chemokine endocytosis by DARC remains unclear.We investigated the role of several endocytic pathways in DARC-mediated ligand internalization. Here we report that, although DARC co-localizes with caveolin-1 in endothelial cells, caveolin-1 is dispensable for DARC-mediated (125I-CXCL1 endocytosis as knockdown of caveolin-1 failed to inhibit ligand internalization. (125I-CXCL1 endocytosis by DARC was also independent of clathrin and flotillin-1 but required cholesterol and was, in part, inhibited by silencing Dynamin II expression.(125I-CXCL1 endocytosis was inhibited by amiloride, cytochalasin D, and the PKC inhibitor Gö6976 whereas Platelet Derived Growth Factor (PDGF enhanced ligand internalization through DARC. The majority of DARC-ligand interactions occurred on the endothelial surface, with DARC identified along plasma membrane extensions with the appearance of ruffles, supporting the concept that DARC provides a high affinity scaffolding function for surface retention of chemokines on endothelial cells.These results show DARC-mediated chemokine endocytosis occurs through a macropinocytosis-like process in endothelial cells and caveolin-1 is dispensable for CXCL1 internalization.

  20. A Novel Role of Dickkopf-Related Protein 3 in Macropinocytosis in Human Bladder Cancer T24 Cells

    Directory of Open Access Journals (Sweden)

    Nonoka Tsujimura

    2016-11-01

    Full Text Available Dickkopf-related protein 3 (Dkk-3 is a potential tumor suppressor reported in various cancer entities. However, we found that Dkk-3 was exceptionally upregulated in bladder cancer T24 cells. To validate the biological role of Dkk-3 other than a tumor suppressor, we examined the function of Dkk-3 in T24 cells. Gene silencing of Dkk-3 inhibited cell growth through inducing G0/G1 cell-cycle arrest. Furthermore, Dkk-3 knock-down caused macropinocytosis accompanied by autophagy, which were canceled in part by their inhibitors 5-(N-ethyl-N-isopropyl amiloride (EIPA and 3-methyladenine (3-MA. The macropinocytosis was induced by the Dkk-3 knock-down when there were sufficient extracellular nutrients. On the other hand, when the nutritional condition was poor, the autophagy was mainly induced by the Dkk-3 knock-down. These data indicated that Dkk-3 has a role in modulating macropinocytotic and autophagic pathways, a distinct function other than a Wnt antagonist.

  1. A Novel Role of Dickkopf-Related Protein 3 in Macropinocytosis in Human Bladder Cancer T24 Cells.

    Science.gov (United States)

    Tsujimura, Nonoka; Yamada, Nami O; Kuranaga, Yuki; Kumazaki, Minami; Shinohara, Haruka; Taniguchi, Kohei; Akao, Yukihiro

    2016-11-05

    Dickkopf-related protein 3 (Dkk-3) is a potential tumor suppressor reported in various cancer entities. However, we found that Dkk-3 was exceptionally upregulated in bladder cancer T24 cells. To validate the biological role of Dkk-3 other than a tumor suppressor, we examined the function of Dkk-3 in T24 cells. Gene silencing of Dkk-3 inhibited cell growth through inducing G₀/G₁ cell-cycle arrest. Furthermore, Dkk-3 knock-down caused macropinocytosis accompanied by autophagy, which were canceled in part by their inhibitors 5-( N -ethyl- N -isopropyl) amiloride (EIPA) and 3-methyladenine (3-MA). The macropinocytosis was induced by the Dkk-3 knock-down when there were sufficient extracellular nutrients. On the other hand, when the nutritional condition was poor, the autophagy was mainly induced by the Dkk-3 knock-down. These data indicated that Dkk-3 has a role in modulating macropinocytotic and autophagic pathways, a distinct function other than a Wnt antagonist.

  2. Infectious pancreatic necrosis virus enters CHSE-214 cells via macropinocytosis.

    Science.gov (United States)

    Levican, Jorge; Miranda-Cárdenas, Camila; Soto-Rifo, Ricardo; Aguayo, Francisco; Gaggero, Aldo; León, Oscar

    2017-06-08

    Infectious pancreatic necrosis virus (IPNV) is a non-enveloped virus belonging to the Birnaviridae family. IPNV produces an acute disease in salmon fingerlings, with high mortality rates and persistent infection in survivors. Although there are reports of IPNV binding to various cells, the viral receptor and entry pathways remain unknown. The aim of this study was to determine the endocytic pathway that allows for IPNV entry. We observed that IPNV stimulated fluid uptake and virus particles co-localysed with the uptake marker dextran in intracellular compartments, suggesting a role for macropinocytosis in viral entry. Consistent with this idea, viral infection was significantly reduced when the Na+/H+ exchanger NHE1 was inhibited with 5-(N-Ethyl-N-isopropyl) amiloride (EIPA). Neither chlorpromazine nor filipin complex I affected IPNV infection. To examine the role of macropinocytosis regulators, additional inhibitors were tested. Inhibitors of the EGFR pathway and the effectors Pak1, Rac1 and PKC reduced viral infection. Together, our results indicate that IPNV is mainly internalized into CHSE-214 cells by macropinocytosis.

  3. A review on the use of bulk specimen X-ray microanalysis in cancer research

    International Nuclear Information System (INIS)

    Zs-Nagy, I.

    1989-01-01

    The freeze-fracture, freeze-drying (FFFD) method of biological bulk specimen preparation combined with quantitative X-ray microanalysis is suitable for the measurement of intracellular concentrations of biologically relevant elements in human biopsy or experimental animal materials. Especially useful information can be obtained regarding the intracellular Na+/K+ ratios being independent of the actual (and unknown) water content of the cytoplasm. The sustained increase of this ratio indicates a sustained depolarization of the cell membrane. These data are of importance from the point of view of the membrane hypothesis of mitogenesis (MHM). It has been revealed that the distribution histograms of the intracellular Na+/K+ ratio display a very significant broadening and an increase of the average values in human urogenital, thyroid and laryngeal tumors, as well as in experimentally induced cell proliferation models. Although MHM has been claimed to be invalid on the basis of some atomic absorption measurements of the intracellular monovalent ion concentrations as well as of some in vitro results obtained with amiloride, this review paper demonstrates that MHM may still be a valid hypothesis for the explanation of mitotic regulation.97 references

  4. p-aminohippurate transport in the airways: Role of Na sup + and HCO sub 3 -

    Energy Technology Data Exchange (ETDEWEB)

    Cloutier, M.M. (Univ. of Connecticut Health Center, Farmington (USA))

    1989-12-01

    The role of Na{sup +} and HCO{sub 3}- in the transport of p-aminohippurate (PAH) across the canine tracheal epithelium was investigated using Ussing chamber techniques and radiolabeled PAH. Under control conditions, net PAH absorption or a tendency toward net PAH absorption was observed. Neither amiloride (10(-4) M), furosemide (10(-3) M), ouabain (2 x 10(-4) M), nor Na+ substitution of the Ringer solution with choline had any effect on unidirectional PAH fluxes. When the Ringer solution was replaced with a HCO{sub 3}(-)-free solution, net PAH absorption was consistently observed. In HCO{sub 3}(-)-free experiments, unidirectional PAH absorptive fluxes were inhibited by mucosal addition of either of the stilbene derivatives, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 10(-4) M) or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS, 10(-4) M). DIDS was more effective than SITS and was also effective in inhibiting PAH absorption in tissues bathed in Ringer solution. Submucosal DIDS or SITS had no effect on PAH fluxes either in HCO{sub 3}(-)-free or Ringer experiments. We conclude that PAH transport in canine tracheal epithelium occurs by a HCO{sub 3}(-)-PAH exchange process located on the luminal membrane. PAH transport is not Na{sup +} dependent but is inhibited by both DIDS and SITS.

  5. Three homologous subunits form a high affinity peptide-gated ion channel in Hydra

    DEFF Research Database (Denmark)

    Dürrnagel, Stefan; Kuhn, Anne; Tsiairis, Charisios D

    2010-01-01

    Recently, three ion channel subunits of the degenerin (DEG)/epithelial Na(+) channel (ENaC) gene family have been cloned from the freshwater polyp Hydra magnipapillata, the Hydra Na(+) channels (HyNaCs) 2-4. Two of them, HyNaC2 and HyNaC3, co-assemble to form an ion channel that is gated by the n......Recently, three ion channel subunits of the degenerin (DEG)/epithelial Na(+) channel (ENaC) gene family have been cloned from the freshwater polyp Hydra magnipapillata, the Hydra Na(+) channels (HyNaCs) 2-4. Two of them, HyNaC2 and HyNaC3, co-assemble to form an ion channel that is gated......NaC2/3/5 channel has altered pore properties and amiloride affinity, more similarly to other DEG/ENaC channels. Collectively, our results suggest that the three homologous subunits HyNaC2, -3, and -5 form a peptide-gated ion channel in Hydra that could contribute to fast synaptic transmission....

  6. Ion transport by mitochondria-rich cells in toad skin

    DEFF Research Database (Denmark)

    Larsen, Erik Hviid; Ussing, H H; Spring, K R

    1987-01-01

    The optical sectioning video imaging technique was used for measurements of the volume of mitochondria-rich (m.r.) cells of the isolated epithelium of toad skin. Under short-circuit conditions, cell volume decreased by about 14% in response to bilateral exposure to Cl-free (gluconate substitution....... Unilateral exposure to a Cl-free solution did not prevent ouabain-induced cell swelling. It is concluded that m.r. cells have an amiloride-blockable Na conductance in the apical membrane, a ouabain-sensitive Na pump in the basolateral membrane, and a passive Cl permeability in both membranes. From...... the initial rate of ouabain-induced cell volume increase the active Na current carried by a single m.r. cell was estimated to be 9.9 +/- 1.3 pA. Voltage clamping of the preparation in the physiological range of potentials (0 to -100 mV, serosa grounded) resulted in a cell volume increase with a time course...

  7. Stretch-activated cation channel from larval bullfrog skin

    DEFF Research Database (Denmark)

    Hillyard, Stanley D; Willumsen, Niels J; Marrero, Mario B

    2010-01-01

    Cell-attached patches from isolated epithelial cells from larval bullfrog skin revealed a cation channel that was activated by applying suction (-1 kPa to -4.5 kPa) to the pipette. Activation was characterized by an initial large current spike that rapidly attenuated to a stable value and showed...... was markedly reduced with N-methyl-D-glucamide (NMDG)-Cl Ringer's solution in the pipette. Neither amiloride nor ATP, which are known to stimulate an apical cation channel in Ussing chamber preparations of larval frog skin, produced channel activation nor did these compounds affect the response to suction....... Stretch activation was not affected by varying the pipette concentrations of Ca(2+) between 0 mmol l(-1) and 4 mmol l(-1) or by varying pH between 6.8 and 8.0. However, conductance was reduced with 4 mmol l(-1) Ca(2+). Western blot analysis of membrane homogenates from larval bullfrog and larval toad skin...

  8. Na sup + -H sup + exchanger in proximal cells isolated from kidney. II. Short-term regulation by glucocorticoids

    Energy Technology Data Exchange (ETDEWEB)

    Bidet, M.; Merot, J.; Tauc, M.; Poujeol, P. (Institut National de la Sante et de la Recherche Medicale (France))

    1987-11-01

    The purpose of this study was to investigate the acute regulation by glucocorticoid of the Na{sup +}-H{sup +} exchanger in isolated renal proximal cells of the rabbit. The changes of intracellular pH (pH{sub i}) were determined in a bicarbonate-free buffer by the use of a fluorescent pH probe that may be trapped intracellularly, 2,7-biscarboxyethyl- 5(6)- carboxyfluorescein (BCECF). The activity of the Na{sup +}-H{sup +} exchanger was estimated by measuring the Na{sup +}-induced H{sup +} efflux in BCECF-loaded cells acid loaded with nigericin in choline medium. The uptake of 1.5 mM {sup 22}Na was also studied in Na{sup +}-depleted cells. Acute application of dexamethasone increased the activity of the Na{sup +}-H{sup +} exchanger. The effect on the kinetics of amiloride-sensitive Na{sup +}-H{sup +} exchange indicated that dexamethasone (DEX) increased the activity by increasing the V{sub max} of the carrier for external sodium and for external H{sup +}. The apparent affinity was not modified either for Na{sup +} or for H{sup +}. The glucocorticoid action was undetectable after pretreatment of cells with actinomycin D or cycloheximide. Acute glucocorticoid activation of the Na{sup +}-H{sup +} exchanger in isolated proximal cells required RNA and protein synthesis and was consistent with an increase in the number of carriers in the membrane.

  9. Candidate pheromone receptors of codling moth Cydia pomonella respond to pheromones and kairomones.

    Science.gov (United States)

    Cattaneo, Alberto Maria; Gonzalez, Francisco; Bengtsson, Jonas M; Corey, Elizabeth A; Jacquin-Joly, Emmanuelle; Montagné, Nicolas; Salvagnin, Umberto; Walker, William B; Witzgall, Peter; Anfora, Gianfranco; Bobkov, Yuriy V

    2017-01-24

    Olfaction plays a dominant role in the mate-finding and host selection behaviours of the codling moth (Cydia pomonella), an important pest of apple, pear and walnut orchards worldwide. Antennal transcriptome analysis revealed a number of abundantly expressed genes related to the moth olfactory system, including those encoding the olfactory receptors (ORs) CpomOR1, CpomOR3 and CpomOR6a, which belong to the pheromone receptor (PR) lineage, and the co-receptor (CpomOrco). Using heterologous expression, in both Drosophila olfactory sensory neurones and in human embryonic kidney cells, together with electrophysiological recordings and calcium imaging, we characterize the basic physiological and pharmacological properties of these receptors and demonstrate that they form functional ionotropic receptor channels. Both the homomeric CpomOrco and heteromeric CpomOrco + OR complexes can be activated by the common Orco agonists VUAA1 and VUAA3, as well as inhibited by the common Orco antagonists amiloride derivatives. CpomOR3 responds to the plant volatile compound pear ester ethyl-(E,Z)-2,4-decadienoate, while CpomOR6a responds to the strong pheromone antagonist codlemone acetate (E,E)-8,10-dodecadien-1-yl acetate. These findings represent important breakthroughs in the deorphanization of codling moth pheromone receptors, as well as more broadly into insect ecology and evolution and, consequently, for the development of sustainable pest control strategies based on manipulating chemosensory communication.

  10. Jet dispensing of multi-layered films for the co-delivery of three antihypertensive agents.

    Science.gov (United States)

    Scoutaris, Nicolaos; Malamatari, Maria; Letellier, Adrien; Douroumis, Dennis

    2018-02-01

    Three-layer thin films comprising of two polymers as substrate (ethyl cellulose and, copovidone K28) and three antihypertensive agents (hydrochlorothiazide, amiloride HCl, and carvedilol) were printed using jet dispensing technology. Two film formulations with different ethyl cellulose to copovidone K28 ratio (i.e., 90/10 and 50/50 w/w) were prepared using a three-course dispensing. The films were characterized regarding surface morphology, solid-state properties, polymer-drug interactions, drug distribution in each layer, and in vitro drug release. All the components of the films were found to be in the amorphous state apart from hydrochlorothiazide which retained its crystallinity. FT-IR spectroscopy revealed hydrogen bond interactions between carvedilol and copovidone K28. Combinations of ethyl cellulose and copovidone K28 provide suitable polymeric film substrates with the ability to modify drug release. Particularly, decreased ethyl cellulose to copovidone K28 weight ratio was found to suppress the crystallization of hydrochlorothiazide and to increase the release rate of the dispensed drugs. Jet dispensing was found to be a rapid technology for the preparation of multi-layered films that can be used as personalized formulations for the delivery of combinations of drugs.

  11. Acid-sensing ion channels expression, identity and role in the excitability of the cochlear afferent neurons

    Directory of Open Access Journals (Sweden)

    Antonia eGonzález-Garrido

    2015-12-01

    Full Text Available Acid-sensing ion channels (ASICs are activated by an increase in the extracellular proton concentration. There are four genes (ASIC1-4 that encode six subunits, and they are involved in diverse neuronal functions, such as mechanosensation, learning and memory, nociception, and modulation of retinal function. In this study, we characterize the ASIC currents of spiral ganglion neurons (SGNs. These ASIC currents are primarily carried by Na+, exhibit fast activation and desensitization, display a pH50 of 6.2 and are blocked by amiloride, indicating that these are ASIC currents. The ASIC currents were further characterized using several pharmacological tools. Gadolinium and acetylsalicylic acid reduced these currents, and FMRFamide, zinc (at high concentrations and N,N,N’,N’–tetrakis-(2-piridilmetil-etilendiamina (TPEN increased them, indicating that functional ASICs are composed of the subunits ASIC1, ASIC2 and ASIC3. Neomycin and streptomycin reduced the desensitization rate of the ASIC current in SGNs, indicating that ASICs may contribute to the ototoxic action of aminoglycosides. RT-PCR of the spiral ganglion revealed significant expression of all ASIC subunits. By immunohistochemistry the expression of the ASIC1a, ASIC2a, ASIC2b and ASIC3 subunits was detected in SGNs. Although only a few SGNs exhibited action potential firing in response to an acidic stimulus, protons in the extracellular solution modulated SGN activity during sinusoidal stimulation. Our results show that protons modulate the excitability of SGNs via ASICs.

  12. Acid-sensing ion channel (ASIC) 4 predominantly localizes to an early endosome-related organelle upon heterologous expression.

    Science.gov (United States)

    Schwartz, Verena; Friedrich, Katharina; Polleichtner, Georg; Gründer, Stefan

    2015-12-15

    Acid-sensing ion channels (ASICs) are voltage-independent proton-gated amiloride sensitive sodium channels, belonging to the DEG/ENaC gene family. Six different ASICs have been identified (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4) that are activated by a drop in extracellular pH, either as homo- or heteromers. An exception is ASIC4, which is not activated by protons as a homomer and which does not contribute to functional heteromeric ASICs. Insensitivity of ASIC4 to protons and its comparatively low sequence identity to other ASICs (45%) raises the question whether ASIC4 may have different functions than other ASICs. In this study, we therefore investigated the subcellular localization of ASIC4 in heterologous cell lines, which revealed a surprising accumulation of the channel in early endosome-related vacuoles. Moreover, we identified an unique amino-terminal motif as important for forward-trafficking from the ER/Golgi to the early endosome-related compartment. Collectively, our results show that heterologously expressed ASIC4 predominantly resides in an intracellular endosomal compartment.

  13. Protein kinase D1 modulates aldosterone-induced ENaC activity in a renal cortical collecting duct cell line.

    LENUS (Irish Health Repository)

    McEneaney, Victoria

    2010-08-30

    Aldosterone treatment of M1-CCD cells stimulated an increase in epithelial Na(+) channel (ENaC) alpha-subunit expression that was mainly localized to the apical membrane. PKD1-suppressed cells constitutively expressed ENaCalpha at low abundance, with no increase after aldosterone treatment. In the PKD1-suppressed cells, ENaCalpha was mainly localized proximal to the basolateral surface of the epithelium both before and after aldosterone treatment. Apical membrane insertion of ENaCbeta in response to aldosterone treatment was also sensitive to PKD1 suppression as was the aldosterone-induced rise in the amiloride-sensitive, trans-epithelial current (I(TE)). The interaction of the mineralocorticoid receptor (MR) with specific elements in the promoters of aldosterone responsive genes is stabilized by ligand interaction and phosphorylation. PKD1 suppression inhibited aldosterone-induced SGK-1 expression. The nuclear localization of MR was also blocked by PKD1 suppression and MEK antagonism implicating both these kinases in MR nuclear stabilization. PKD1 thus modulates aldosterone-induced ENaC activity through the modulation of sub-cellular trafficking and the stabilization of MR nuclear localization.

  14. Phorbol esters inhibit ammoniagenesis and gluconeogenesis in proximal tubular segments

    Energy Technology Data Exchange (ETDEWEB)

    Chobanian, M.C.; Hammerman, M.R.

    1987-06-01

    To characterize the regulation of ammoniagenesis and gluconeogenesis in renal proximal tubule, ammonia and glucose productions were measured in suspension of canine proximal tubular segments incubated with 10 mM L-glutamine. Productions were linear functions of time for 120 min and were decreased as extracellular pH was increased from 7.0 to 7.5. To ascertain whether activation of protein kinase c affects either process, the authors incubated segments with tumor-promoting phorbol esters, 12-O-tetradecanoylphorbol-13-acetate (TPA), or phorbol 12,13-dibutyrate, or with the inactive phorbol ester 4..cap alpha..-phorbol. Ammoniagenesis and gluconeogenesis were inhibited by incubation with 10/sup /minus/6/M of the two former compounds but not the latter compound. Phorbol ester-induced inhibition was observed under conditions such that extracellular (Na/sup +/) was greater than intracellular (Na/sup +/), but not when extracellular (Na/sup +/) equaled intracellular (Na/sup +/), and was not observed in the presence of amiloride. These findings are consistent with a role for protein kinase c in the control of ammoniagenesis and gluconeogenesis in proximal tubule. Such control could be mediated via stimulation of Na/sup 1/-H/sup +/ exchange.

  15. Tumor-promoting phorbol esters effect alkalinization of canine renal proximal tubular cells

    Energy Technology Data Exchange (ETDEWEB)

    Mellas, J.; Hammerman, M.R.

    1986-03-01

    We have demonstrated the presence of specific receptors for tumor-promoting phorbol esters in the plasma membrane of the canine renal proximal tubular cell. These compounds affect proximal tubular metabolism in vitro. For example, we have shown that they inhibit gluconeogenesis in canine renal proximal tubular segments. Tumor-promoting phorbol esters have been shown to effect alkalinization of non-renal cells, by enhancing Na/sup +/-H/sup +/ exchange across the plasma membrane. To determine whether the actions of tumor-promoting phorbol esters in proximal tubular segments might be mediated by a similar process, we incubated suspensions of segments from dog kidney with these compounds and measured changes in intracellular pH using (/sup 14/C)-5,5-dimethoxazoladine-2-4-dione (DMO) and flow dialysis. Incubation of segments with phorbol 12,13 dibutyrate, but not inactive phorbol ester, 4 ..gamma.. phorbol, effected alkalinization of cells within the segments in a concentration-dependent manner. Alkalinization was dependent upon the presence of extracellular (Na/sup +/) > intracellular (Na/sup +/), was prevented by amiloride and was demonstrable in the presence of SITS. Our findings suggest that tumor-promoting esters stimulate the Na/sup +/-H/sup +/ exchanger known to be present in the brush border membrane of the renal proximal tubular cell. It is possible that the stimulation reflects a mechanism by which phorbol esters affect metabolic processes in these cells.

  16. Crude venom from nematocysts of Pelagia noctiluca (Cnidaria: Scyphozoa) elicits a sodium conductance in the plasma membrane of mammalian cells

    Science.gov (United States)

    Morabito, Rossana; Costa, Roberta; Rizzo, Valentina; Remigante, Alessia; Nofziger, Charity; La Spada, Giuseppa; Marino, Angela; Paulmichl, Markus; Dossena, Silvia

    2017-01-01

    Cnidarians may negatively impact human activities and public health but concomitantly their venom represents a rich source of bioactive substances. Pelagia noctiluca is the most venomous and abundant jellyfish of the Mediterranean Sea and possesses a venom with hemolytic and cytolytic activity for which the mechanism is largely unknown. Here we show that exposure of mammalian cells to crude venom from the nematocysts of P. noctiluca profoundly alters the ion conductance of the plasma membrane, therefore affecting homeostatic functions such as the regulation and maintenance of cellular volume. Venom-treated cells exhibited a large, inwardly rectifying current mainly due to permeation of Na+ and Cl-, sensitive to amiloride and completely abrogated following harsh thermal treatment of crude venom extract. Curiously, the plasma membrane conductance of Ca2+ and K+ was not affected. Current-inducing activity was also observed following delivery of venom to the cytosolic side of the plasma membrane, consistent with a pore-forming mechanism. Venom-induced NaCl influx followed by water and consequent cell swelling most likely underlie the hemolytic and cytolytic activity of P. noctiluca venom. The present study underscores unique properties of P. noctiluca venom and provides essential information for a possible use of its active compounds and treatment of envenomation.

  17. Chloride secretion by cultures of pig tracheal gland cells

    Science.gov (United States)

    Borthwell, Rachel M.; Hajighasemi-Ossareh, Mohammad; Lachowicz-Scroggins, Marrah E.; Finkbeiner, W. E.; Stevens, Jeremy E.; Modlin, Sara

    2012-01-01

    Malfunction of airway submucosal glands contributes to the pathology of cystic fibrosis (CF), and cell cultures of CF human airway glands show defects in Cl− and water transport. Recently, a transgenic pig model of CF (the CF pig) has been developed. Accordingly, we have developed cell cultures of pig airway gland epithelium for use in investigating alterations in gland function in CF. Our cultures form tight junctions (as evidenced by high transepithelial electrical resistance) and show high levels of active anion secretion (measured as amiloride-insensitive short-circuit current). In agreement with recent results on human airway glands, neurohumoral agents that elevate intracellular Ca2+ potently stimulated anion secretion, while elevation of cAMP was comparatively ineffective. Our cultures express lactoferrin and lysozyme (serous gland cell markers) and MUC5B (the main mucin of airway glands). They are, therefore, potentially useful in determining if CF-related alterations in anion transport result in altered secretion of serous cell antimicrobial agents or mucus. PMID:22367783

  18. Severe respiratory phenotype caused by a de novo Arg528Gly mutation in the CACNA1S gene in a patient with hypokalemic periodic paralysis.

    Science.gov (United States)

    Kil, Tae-Hwan; Kim, June-Bum

    2010-05-01

    Hypokalemic periodic paralysis (HOKPP) is a rare disorder characterized by episodic muscle weakness with hypokalemia. Mutations in the CACNA1S gene, which encodes the alpha 1-subunit of the skeletal muscle L-type voltage-dependent calcium channel, have been reported to be mainly responsible for HOKPP. The paralytic attacks generally spare the respiratory muscles and the heart. Here, we report the case of a 16-year-old boy who presented with frequent respiratory insufficiency during the severe attacks. Mutational analysis revealed a heterozygous c.1582C>G substitution in the CACNA1S gene, leading to an Arg528Gly mutation in the protein sequence. The parents were clinically unaffected and did not show a mutation in the CACNA1S gene. A de novo Arg528Gly mutation has not previously been reported. The patient described here presents the unique clinical characteristics, including a severe respiratory phenotype and a reduced susceptibility to cold exposure. The patient did not respond to acetazolamide and showed a marked improvement of the paralytic symptoms on treatment with a combination of spironolactone, amiloride, and potassium supplements. Copyright 2009 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

  19. Regulation of renal peripheral benzodiazepine receptors by anion transport inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Basile, A.S.; Lueddens, W.M.; Skolnick, P.

    1988-01-01

    The in vitro and in vivo regulation of (/sup 3/H)Ro 5-4864 binding to peripheral benzodiazepine receptors (PBR) by ion transport/exchange inhibitors was studied in the kidney. The potencies of 9-anthroic acid, furosemide, bumetanide, hydrochlorothiazide and SITS as inhibitors of (/sup 3/H)Ro 5-4864 binding to renal membranes were consistent with their actions as anion transport inhibitors (Ki approx. = 30 - 130 ..mu..M). In contrast, spironolactone, amiloride, acetazolamide, and ouabain were less potent (Ki=100-1000 ..mu..M). Administration of furosemide to rats for five days resulted in a profound diuresis accompanied by a significant increase in PBR density (43%) that was apparent by the fifth day of treatment. Administration of hydrochlorothiazide or Ro 5-4864 for five days also caused diuresis and increased renal PBR density. Both the diuresis and increased density of PBR produced by Ro 5-4864 were blocked by coadministration of PK 11195, which alone had no effect on either PBR density or urine volume. The equilibrium binding constants of (/sup 3/H)Ro 5-4864 to cardiac membranes were unaffected by administration of any of these drugs. These findings suggest that renal PBR may be selectively modulated in vivo and in vitro by administration of ion transport/exchange inhibitors. 36 references, 4 tables.

  20. The Thiazide-Sensitive Co-Transporter Promotes the Development of Sodium Retention in Mice with Diet-Induced Obesity

    Directory of Open Access Journals (Sweden)

    Matthew R P Davies

    2015-09-01

    Full Text Available Background/Aims: Intravascular volume expansion due to sodium retention is involved in the pathogenesis of obesity-related hypertension. Institution of high fat diet (HFD feeding leads to an initial state of positive sodium balance due to enhanced tubular reabsorption of sodium, but which tubular sodium transporters are responsible for this remains undefined. Methods: C57/Bl6 mice were fed control or HFD for 3 weeks. Blood pressures were recorded by tail cuff method. Sodium transporter expression and phosphorylation were determined by Western blotting. In vivo activity of NCC was determined using natriuretic responses to hydrochlorothiazide. Expression of NCC mRNA was determined using qPCR. Results: At 3 weeks HFD mice had significant weight gains compared to control mice, but blood pressures were not yet elevated. There were no changes in expression or phosphorylation of the bumetanide-sensitive cotransporter, NKCC2, or in expression of subunits of the amiloride-sensitive ion channel, ENaC. However, there were significant increases in mRNA and protein expression of the thiazide-sensitive co-transporter, NCC, in kidneys from HFD mice. Consistent with this, HFD mice had increased in vivo activity of NCC. Conclusions: Increased expression of NCC promotes the sodium loading response to institution of HFD feeding before onset of hypertension.

  1. Insulin regulation of Na/K pump activity in rat hepatoma cells

    International Nuclear Information System (INIS)

    Gelehrter, T.D.; Shreve, P.D.; Dilworth, V.M.

    1984-01-01

    Insulin rapidly increases Na/K pump activity in HTC rat hepatoma cells in tissue culture, as measured by the ouabain-sensitive influx of the potassium analogue 86Rb+. Increased influx is observed within minutes and is maximal (70% above control) within 1-2 h. The effect appears to be mediated by the insulin receptors, as: the concentration dependence on insulin is identical to that for insulin induction of tyrosine aminotransferase and stimulation of 2-aminoisobutyric acid transport, proinsulin is 6% as potent as insulin, and the effect is blocked by anti-receptor antibodies. The early stimulation of potassium influx is not blocked by cycloheximide and is not associated with an increased number of pump sites as measured by 3 H-ouabain binding. The insulin effect is blocked by amiloride, which blocks sodium influx, and is mimicked by the sodium ionophore monensin, which increases sodium influx and intracellular accumulation. Insulin also rapidly increases the initial rate of 22 Na+ influx, suggesting that insulin may enhance Na/K pump activity, in part, by increasing intracellular sodium concentration. Incubation of HTC cells with insulin for 24 h causes complete unresponsiveness to the insulin induction of transaminase and stimulation of amino acid transport, a phenomenon mediated by postbinding mechanisms. In contrast, similar incubation with insulin does not cause unresponsiveness to the insulin stimulation of Na/K pump activity. Therefore, the site of regulation of responsiveness to insulin must be distal to, or separate from, those events causing stimulation of ion fluxes

  2. Role of diuretics and lipid formulations in the prevention of amphotericin B-induced nephrotoxicity.

    Science.gov (United States)

    Karimzadeh, Iman; Khalili, Hossein; Farsaei, Shadi; Dashti-Khavidaki, Simin; Sagheb, Mohammad Mahdi

    2013-07-01

    To collect available clinical data to define the role of diuretics and lipid formulations in the prevention of amphotericin B (AmB)-induced nephrotoxicity (AIN) in human populations. A literature search was performed in the following databases: Scopus, Medline, Embase, Cochrane Central Register of Controlled Trials, and Cochrane Database of Systematic Reviews. Co-administration of mannitol failed to show any clinically significant benefit in preventing AIN. Potassium-sparing diuretics, such as amiloride and spironolactone, have been shown to have beneficial effects as an alternative or adjunct to oral/parenteral potassium supplements in preventing hypokalemia due to AmB. Lipid-based formulations of AmB are clinically effective and safe in preventing AIN. However, due to their high cost and limited accessibility, these formulations are generally used as second-line antifungal therapy in cases of conventional AmB refractoriness and/or intolerance or pre-existing renal dysfunction. The potential effects of other nephroprotective agents, such as N-acetylcysteine, AIN merit further considerations and investigations.

  3. Exercise reduces airway sodium ion reabsorption in cystic fibrosis but not in exercise asthma.

    Science.gov (United States)

    Schmitt, L; Wiebel, M; Frese, F; Dehnert, C; Zugck, C; Bärtsch, P; Mairbäurl, H

    2011-02-01

    When ventilating large volumes of air during exercise, airway fluid secretion is essential for airway function. Since these are impaired in cystic fibrosis and exercise-induced asthma, it was the aim of this study to determine how exercise affects airway Na(+) and Cl(-) transport and whether changes depend on exercise intensity. Nasal potential was measured in Ringer's solution, with amiloride to block Na(+) transport, and in low chloride-containing isoproterenol to assess Cl(-) channels. Nasal potential was measured at rest and during submaximal and maximal bicycle ergometer exercise in individuals with cystic fibrosis, exercise-induced asthma and controls. At rest, nasal potential was significantly higher in cystic fibroses than in the others. Maximal exercise decreased nasal potentials in cystic fibrosis and controls but not in exercise asthma. Submaximal exercise decreased nasal potentials only in cystic fibrosis. Cl(-) transport was not affected. Our results indicate that nasal potentials and Na(+) transport were decreased by maximal exercise in healthy and cystic fibrosis, whereas submaximal exercise decreased potentials in cystic fibrosis only. Exercise did not affect nasal potentials in asthmatics. Decreased reabsorption during exercise might favour airway fluid secretion during hyperpnoea. This protective effect appears blunted in patients with exercise-induced asthma.

  4. Interleukin-17A induces bicarbonate secretion in normal human bronchial epithelial cells

    Science.gov (United States)

    Kreindler, James L.; Bertrand, Carol A.; Lee, Robert J.; Karasic, Thomas; Aujla, Shean; Pilewski, Joseph M.; Frizzell, Raymond A.; Kolls, Jay K.

    2009-01-01

    The innate immune functions of human airways include mucociliary clearance and antimicrobial peptide activity. Both functions may be affected by changes in epithelial ion transport. Interleukin-17A (IL-17A), which has a receptor at the basolateral membrane of airway epithelia, is a T cell cytokine that has been shown to increase mucus secretion and antimicrobial peptide production by human bronchial epithelial (HBE) cells. Furthermore, IL-17A levels are increased in sputum from patients during pulmonary exacerbations of cystic fibrosis. Therefore, we investigated the effects of IL-17A on basal, amiloride-sensitive, and forskolin-stimulated ion transport in mature, well-differentiated HBE cells. Exposure of HBE monolayers to IL-17A for 48 h induced a novel forskolin-stimulated bicarbonate secretion in addition to forskolin-stimulated chloride secretion and resulted in alkalinization of liquid on the mucosal surface of polarized cells. IL-17A-induced bicarbonate secretion was cystic fibrosis transmembrane conductance regulator (CFTR)-dependent, mucosal chloride-dependent, partially Na+-dependent, and sensitive to serosal, but not mucosal, stilbene inhibition. These data suggest that IL-17A modulates epithelial bicarbonate secretion and implicate a mechanism by which airway surface liquid pH changes may be abnormal in cystic fibrosis. PMID:19074559

  5. Eplerenone-Resistant Salt-Sensitive Hypertension in Nedd4-2 C2 KO Mice.

    Science.gov (United States)

    Kino, Tabito; Ishigami, Tomoaki; Murata, Tsumugi; Doi, Hiroshi; Nakashima-Sasaki, Rie; Chen, Lin; Sugiyama, Michiko; Azushima, Kengo; Wakui, Hiromichi; Minegishi, Shintaro; Tamura, Kouichi

    2017-06-11

    The epithelial sodium channel (ENaC) plays critical roles in maintaining fluid and electrolyte homeostasis and is located in the aldosterone-sensitive distal nephron (ASDN). We previously found that Nedd4-2 C2 knockout (KO) mice showed salt-sensitive hypertension with paradoxically enhanced ENaC gene expression in ASDN under high oral salt intake. Eplerenone (EPL), a selective aldosterone blocker, is a promising therapeutic option for resistant or/and salt-sensitive hypertension. We examined the effect of EPL on Nedd4-2 C2 KO mice with respect to blood pressure, metabolic parameters, and molecular level changes in ASDN under high oral salt intake. We found that EPL failed to reduce blood pressure in KO mice with high oral salt intake and upregulated ENaC expression in ASDN. Thus, salt-sensitive hypertension in Nedd4-2 C2 KO was EPL-resistant. Gene expression analyses of laser-captured specimens in ASDN suggested the presence of non-aldosterone-dependent activation of ENaC transcription in ASDN of Nedd4-2 C2 KO mice, which was abolished by amiloride treatment. Our results from Nedd4-2 C2 KO mice suggest that enhanced ENaC gene expression is critically involved in salt-sensitive hypertension under certain conditions of specific enzyme isoforms for their ubiquitination.

  6. Acute stress enhances learning and memory by activating acid-sensing ion channels in rats.

    Science.gov (United States)

    Ye, Shunjie; Yang, Rong; Xiong, Qiuju; Yang, Youhua; Zhou, Lianying; Gong, Yeli; Li, Changlei; Ding, Zhenhan; Ye, Guohai; Xiong, Zhe

    2018-04-15

    Acute stress has been shown to enhance learning and memory ability, predominantly through the action of corticosteroid stress hormones. However, the valuable targets for promoting learning and memory induced by acute stress and the underlying molecular mechanisms remain unclear. Acid-sensing ion channels (ASICs) play an important role in central neuronal systems and involves in depression, synaptic plasticity and learning and memory. In the current study, we used a combination of electrophysiological and behavioral approaches in an effort to explore the effects of acute stress on ASICs. We found that corticosterone (CORT) induced by acute stress caused a potentiation of ASICs current via glucocorticoid receptors (GRs) not mineralocorticoid receptors (MRs). Meanwhile, CORT did not produce an increase of ASICs current by pretreated with GF109203X, an antagonist of protein kinase C (PKC), whereas CORT did result in a markedly enhancement of ASICs current by bryostatin 1, an agonist of PKC, suggesting that potentiation of ASICs function may be depended on PKC activating. More importantly, an antagonist of ASICs, amiloride (10 μM) reduced the performance of learning and memory induced by acute stress, which is further suggesting that ASICs as the key components involves in cognitive processes induced by acute stress. These results indicate that acute stress causes the enhancement of ASICs function by activating PKC signaling pathway, which leads to potentiated learning and memory. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. The mechanism of fluid secretion in the rabbit pancreas studied by means of various inhibitors.

    Science.gov (United States)

    Kuijpers, G A; Van Nooy, I G; De Pont, J J; Bonting, S L

    1984-12-05

    In order to increase our understanding of the mechanism of pancreatic fluid secretion we have studied the effects of various transport inhibitors on this process in the isolated rabbit pancreas. In this preparation, a high rate of unstimulated fluid secretion occurs, which probably originates from the ductular cells. Inhibitory are ouabain, furosemide, bumetanide, piretanide, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and acetazolamide, with their half-inhibitory concentrations: 2 X 10(-6) M (ouabain), 1.3 X 10(-3) M (furosemide), 2.2 X 10(-3) M (bumetanide and piretanide) and 1.4 X 10(-4) M (SITS). With acetazolamide a maximal inhibition of only 20% is found at 10(-3) M. Amiloride (10(-3) M) has no effect on pancreatic fluid secretion. The inhibitory effects on HCO-3 output are always larger and those on Cl- output lower than those on fluid secretion. The results suggest that the ouabain-sensitive (Na+ + K+)-ATPase system provides the energy for a Na+-gradient-driven Cl--HCO-3-exchange transport system, sensitive to the loop diuretics furosemide, bumetanide and piretanide and to SITS. This system would drive the transcellular transport of HCO-3 and secondarily that of cations, Cl- and water.

  8. Veneno de Latrodectus mactans de Chile (Araneae, Theridiidae: su efecto sobre músculo liso

    Directory of Open Access Journals (Sweden)

    Fernando Romero M.

    2003-06-01

    Full Text Available El veneno de arañas del género Latrodectus induce contracción de músculo liso de mamíferos, postulándose como posible mecanismo la liberación de mediadores adrenérgicos y colinérgicos. El veneno de Latrodectus mactans de Chile contiene neurotoxinas que inducen actividad contráctil en músculo liso en forma parcialmente independiente de actividades adrenérgicas y colinérgicas, lo que deja abiertas interrogantes sobre el mecanismo de acción del veneno. La respuesta tónica en músculo liso depende de la movilidad de los iones sodio (Na+ y calcio (Ca2+ y, más es-pecíficamente, de la concentración de Ca2+ citoplasmático. Una de las vías de ingreso del calcio al músculo liso, además de los canales tipo L de Ca2+ , es el intercambiador Na+/Ca2+ . En el presente trabajo se estudia la posible participación de este intercambiador en la respuesta tónica inducida por el veneno sobre músculo liso del conducto deferente de rata, en un modelo de órgano aislado. Utilizando bloqueadores de canales de Na+ (amiloride y Ca2+ (nifedipina y una solución estimuladora del intercambiador (Tyrode pobre en sodio, se realizaron registros de tensión isométrica inducida por el veneno. Simultáneamente al uso de nifedipina, se registraron las variaciones de la [Ca2+] citoplasmática mediante microfluorimetría. Se observó que la inhibición de la contracción en presencia de amiloride depende de su concentración, mostrando una participación de los canales de Ca2+ dependientes de voltaje en la contracción. En presencia de nifedipina, la contracción inducida por el veneno sólo fue parcialmente inhibida, y la microfluorimetría mostró un aumento de la concentración de Ca2+ citoplasmático en presencia del bloqueador, lo que indica una participación de otros mecanismos para el ingreso de Ca2+ a la célula. Por último, al disminuir la concentración de Na+ extracelular se estimuló la contracción tónica en un 30.7%, atribuible, al menos en

  9. Synthetic ion transporters can induce apoptosis by facilitating chloride anion transport into cells.

    Science.gov (United States)

    Ko, Sung-Kyun; Kim, Sung Kuk; Share, Andrew; Lynch, Vincent M; Park, Jinhong; Namkung, Wan; Van Rossom, Wim; Busschaert, Nathalie; Gale, Philip A; Sessler, Jonathan L; Shin, Injae

    2014-10-01

    Anion transporters based on small molecules have received attention as therapeutic agents because of their potential to disrupt cellular ion homeostasis. However, a direct correlation between a change in cellular chloride anion concentration and cytotoxicity has not been established for synthetic ion carriers. Here we show that two pyridine diamide-strapped calix[4]pyrroles induce coupled chloride anion and sodium cation transport in both liposomal models and cells, and promote cell death by increasing intracellular chloride and sodium ion concentrations. Removing either ion from the extracellular media or blocking natural sodium channels with amiloride prevents this effect. Cell experiments show that the ion transporters induce the sodium chloride influx, which leads to an increased concentration of reactive oxygen species, release of cytochrome c from the mitochondria and apoptosis via caspase activation. However, they do not activate the caspase-independent apoptotic pathway associated with the apoptosis-inducing factor. Ion transporters, therefore, represent an attractive approach for regulating cellular processes that are normally controlled tightly by homeostasis.

  10. Liddle Syndrome: Review of the Literature and Description of a New Case

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    Martina Tetti

    2018-03-01

    Full Text Available Liddle syndrome is an inherited form of low-renin hypertension, transmitted with an autosomal dominant pattern. The molecular basis of Liddle syndrome resides in germline mutations of the SCNN1A, SCNN1B and SCNN1G genes, encoding the α, β, and γ-subunits of the epithelial Na+ channel (ENaC, respectively. To date, 31 different causative mutations have been reported in 72 families from four continents. The majority of the substitutions cause an increased expression of the channel at the distal nephron apical membrane, with subsequent enhanced renal sodium reabsorption. The most common clinical presentation of the disease is early onset hypertension, hypokalemia, metabolic alkalosis, suppressed plasma renin activity and low plasma aldosterone. Consequently, treatment of Liddle syndrome is based on the administration of ENaC blockers, amiloride and triamterene. Herein, we discuss the genetic basis, clinical presentation, diagnosis and treatment of Liddle syndrome. Finally, we report a new case in an Italian family, caused by a SCNN1B p.Pro618Leu substitution.

  11. Apparent mineralocorticoid excess: time of manifestation and complications despite treatment.

    Science.gov (United States)

    Knops, Noël B B; Monnens, Leo A; Lenders, Jacques W; Levtchenko, Elena N

    2011-06-01

    Here we describe the case of a patient followed from birth because of a positive family history for apparent mineralocorticoid excess (AME) in an older brother. The patient, a girl, had normal serum electrolyte and blood pressure measurements in the first months after birth. Not until the age of 11 months did she develop anorexia and failure to thrive in combination with hypertension, hypokalemia, and metabolic alkalosis, which are consistent with the diagnosis of AME. This diagnosis was confirmed by mutation analysis of the HSD11B2 gene (C1228T). Treatment with amiloride and furosemide electrolyte disturbances normalized her blood pressure. At the age of 19 years she unexpectedly suffered a stroke. Additional investigations revealed no accepted risk factor for stroke. We discuss the possible underlying mechanisms for the delayed manifestation of hypertension and electrolyte disturbances in AME, propose an additional explanation for the stroke in this patient, and advise treatment with a mineralocorticoid receptor antagonist to reduce stroke risk in patients with AME.

  12. Retinoic acid treatment of fibroblasts causes a rapid Decrease in [3H]inositol uptake

    International Nuclear Information System (INIS)

    Sinha, R.; Creek, K.E.; Silverman-Jones, C.; de Luca, L.M.

    1989-01-01

    NIH 3T3 fibroblasts treated with all-trans-retinoic acid (RA) showed a dramatic decrease in the uptake of [ 3 H]inositol compared to solvent-treated controls. The onset of RA-induced inhibition of [ 3 H]inositol uptake was rapid with a 10-15% decrease occurring after 2-3 h of RA exposure and 60-70% reduction after 16 h of RA treatment. A progressive dose-dependent decrease in inositol uptake was found as the concentration of RA increased from 10 -8 to 10 -5 M and the effect was fully reversible within 48 h after RA removal. RA inhibition of inositol uptake was also observed in 3T3-Swiss and Balb/3T3 cells but not in two virally transformed 3T3 cell lines. Phlorizin, amiloride, and monensin inhibited inositol uptake by 66, 74, and 58%, respectively, and this inhibition was additive when the cells were treated with RA as well as these inhibitors. A decreased incorporation of [ 3 H]inositol into polyphosphoinositides was also observed in RA-treated cells but not to the same extent as for [ 3 H]inositol uptake. In conclusion, RA treatment of 3T3 fibroblasts decreases the uptake of [ 3 H]inositol by up to 70% within 8 to 10 h at near physiological concentrations in a reversible and specific manner

  13. Theoretical study of structure, pKa, lipophilicity, solubility, absorption, and polar surface area of some centrally acting antihypertensives.

    Science.gov (United States)

    Remko, Milan; Swart, Marcel; Bickelhaupt, F Matthias

    2006-03-15

    The methods of theoretical chemistry have been used to elucidate the molecular properties of the substituted imidazoline and oxazoline structures, a class of potent agonists and antagonists of imidazoline receptors. The geometries of various tautomers and isomers of 2-[2,6-dichlorophenylimino]imidazolidine (clonidine), 1-(N-dicyclopropylmethyl)amino-2-oxazoline (rilmenidine), 4-chloro-N-(4,5-dihydro-1H-imidazol-2yl)-6-methoxy-2-methyl-5-pyrimidinamine (moxonidine), N-(dicyclopropylmethyl)-4,5-dihydro-1H-pyrrol-2-amine (aminopyrroline), N-dicyclopropylmethyl-4,5-dihydrothiazol-2-amine (aminothiazoline), 4,5-dihydro-2-(2-methoxyphenyl)-1H-imidazole (compound_6), 4,5-dihydro-2-(3-methylthiophen-2-yl)-1H-imidazole (compound_7), N-(2-chloro-4-iodophenyl)-4,5-dihydro-5-methyl-3H-pyrrol-2-amine (LNP_911), N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride), 2-(1,4-benzodioxan-2-yl)-2-imidazoline (idazoxan), (+/-)-2-(2-ethyl-2,3-dihydro-2-benzofuranyl)-2-imidazoline (efaroxan), (4-aminobutyl)guaninine (agmatine), and 1-methyl-9H-pyrido[3,4-b]indole (harmane) have been studied using Becke3LYP/6-31+G(d,p) and BP86/TZ2P DFT methods. The optimized geometries indicate that these molecules show a distinctly nonplanar configuration of the imidazoline and oxazoline moieties. In the gas-phase, rilmenidine and aminothiazoline exist in two forms (amino and imino), the amino tautomers being more stable by about 6 kJ/mol. The calculations showed, in agreement with experiments, that clonidine, moxonidine, and LNP_911 exist in a more stable imino tautomer. The tautomer containing the amino group is by about 30 kJ/mol less stable. Computations that include the effect of solvation indicated that also in water the relative stability order of individual tautomers (amino and imino forms) is preserved. The computed pKa values varied between 6.7 and 9.0, and correlate well with the available experimental pKa's found in the literature. Among the clinically useful antihypertensives

  14. TNF Lectin-Like Domain Restores Epithelial Sodium Channel Function in Frameshift Mutants Associated with Pseudohypoaldosteronism Type 1B

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    Anita Willam

    2017-05-01

    Full Text Available Previous in vitro studies have indicated that tumor necrosis factor (TNF activates amiloride-sensitive epithelial sodium channel (ENaC current through its lectin-like (TIP domain, since cyclic peptides mimicking the TIP domain (e.g., solnatide, showed ENaC-activating properties. In the current study, the effects of TNF and solnatide on individual ENaC subunits or ENaC carrying mutated glycosylation sites in the α-ENaC subunit were compared, revealing a similar mode of action for TNF and solnatide and corroborating the previous assumption that the lectin-like domain of TNF is the relevant molecular structure for ENaC activation. Accordingly, TNF enhanced ENaC current by increasing open probability of the glycosylated channel, position N511 in the α-ENaC subunit being identified as the most important glycosylation site. TNF significantly increased Na+ current through ENaC comprising only the pore forming subunits α or δ, was less active in ENaC comprising only β-subunits, and showed no effect on ENaC comprising γ-subunits. TNF did not increase the membrane abundance of ENaC subunits to the extent observed with solnatide. Since the α-subunit is believed to play a prominent role in the ENaC current activating effect of TNF and TIP, we investigated whether TNF and solnatide can enhance αβγ-ENaC current in α-ENaC loss-of-function frameshift mutants. The efficacy of solnatide has been already proven in pathological conditions involving ENaC in phase II clinical trials. The frameshift mutations αI68fs, αT169fs, αP197fs, αE272fs, αF435fs, αR438fs, αY447fs, αR448fs, αS452fs, and αT482fs have been reported to cause pseudohypoaldosteronism type 1B (PHA1B, a rare, life-threatening, salt-wasting disease, which hitherto has been treated only symptomatically. In a heterologous expression system, all frameshift mutants showed significantly reduced amiloride-sensitive whole-cell current compared to wild type αβγ-ENaC, whereas membrane

  15. Optimizing nasal potential difference analysis for CFTR modulator development: assessment of ivacaftor in CF subjects with the G551D-CFTR mutation.

    Directory of Open Access Journals (Sweden)

    Steven M Rowe

    Full Text Available Nasal potential difference (NPD is used as a biomarker of the cystic fibrosis transmembrane conductance regulator (CFTR and epithelial sodium channel (ENaC activity. We evaluated methods to detect changes in chloride and sodium transport by NPD based on a secondary analysis of a Phase II CFTR-modulator study. Thirty-nine subjects with CF who also had the G551D-CFTR mutation were randomized to receive ivacaftor (Kalydeco™; also known as VX-770 in four doses or placebo twice daily for at least 14 days. All data were analyzed by a single investigator who was blinded to treatment assignment. We compared three analysis methods to determine the best approach to quantify changes in chloride and sodium transport: (1 the average of both nostrils; (2 the most-polarized nostril at each visit; and (3 the most-polarized nostril at screening carried forward. Parameters of ion transport included the PD change with zero chloride plus isoproterenol (CFTR activity, the basal PD, Ringer's PD, and change in PD with amiloride (measurements of ENaC activity, and the delta NPD (measuring CFTR and ENaC activity. The average and most-polarized nostril at each visit were most sensitive to changes in chloride and sodium transport, whereas the most-polarized nostril at screening carried forward was less discriminatory. Based on our findings, NPD studies should assess both nostrils rather than a single nostril. We also found that changes in CFTR activity were more readily detected than changes in ENaC activity, and that rigorous standardization was associated with relatively good within-subject reproducibility in placebo-treated subjects (± 2.8 mV. Therefore, we have confirmed an assay of reasonable reproducibility for detecting chloride-transport improvements in response to CFTR modulation.

  16. Pharmacological characterization of the diuretic effect of Hibiscus sabdariffa Linn (Malvaceae) extract.

    Science.gov (United States)

    Alarcón-Alonso, Javier; Zamilpa, Alejandro; Aguilar, Francisco Alarcón; Herrera-Ruiz, Maribel; Tortoriello, Jaime; Jimenez-Ferrer, Enrique

    2012-02-15

    Hibiscus sabdariffa L. (Malvaceae) populary known in Mexico as "Jamaica", "flor de Jamaica", has widely used in Mexican Traditional Medicine as antihypertensive and diuretic, although the latter activity has been reported the present work show evidence about the diuretic, natriuretic and potassium-sparing effects. To evaluate the diuretic activity of Hibiscus sabdariffa aqueous extract on in vivo and in situ models. The Hibiscus sabdariffa aqueous extract was administrated in increasing doses and evaluated the diuresis produced and disposal of electrolytes. Moreover, in isolated kidney was determined the renal filtration rate with plant extract, furosemide and amiloride. The yield of Hibiscus sabdariffa aqueous extraction was 28.3% and the chemical standardization from 1 g of extract was: 56.5 mg delphinidin-3-O-sambubioside, 20.8 mg/g cyanidin-3-O-sambubioside, 3.2 mg/g quercetin, 2.1 mg/g rutin and 2.7 mg/g chlorogenic acid. The diuretic and natriuretic effect of Hibiscus sabdariffa aqueous extract showed a dose-dependent behavior. The pharmacological constants of natriuretic effect was ED50=86 mg/kg and Emax=0.9 mEq/100 g/5 h. In the model of kidney in situ was observed that renal filtration increased 48% with the aqueous extract of Hibiscus sabdariffa and an additive effect when was perfuse with furosemide. The compound presents in Hibiscus sabdariffa as quercetin had effect on the vascular endothelium causing oxide nitric release, increasing renal vasorelaxation by increasing kidney filtration. Therefore, the diuretic effect of Hibiscus sabdariffa may be mediated by nitric oxide release. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  17. [Glucose-6-phosphatase from nuclear envelope in rat liver].

    Science.gov (United States)

    González-Mujica, Freddy

    2008-06-01

    Nuclear envelope (NE) and microsomal glucosa-6-phosphatase (G-6-Pase) activities were compared. Intact microsomes were unable to hydrolyze mannose-6-phosphate (M-6-P), on the other hand, intact NE hydrolyzes this substrate. Galactose-6-phosphate showed to be a good substrate for both NE and microsomal enzymes, with similar latency to that obtained with M-6-P using microsomes. In consequence, this substrate was used to measure the NE integrity. The kinetic parameters (Kii and Kis) of the intact NE G-6-Pase for the phlorizin inhibition using glucose-6-phosphate (G-6-P) and M-6-P as substrates, were very similar. The NE T1 transporter was more sensitive to amiloride than the microsomal T1. The microsomal system was more sensitive to N-ethylmalemide (NEM) than the NE and the latter was insensitive to anion transport inhibitors DIDS and SITS, which strongly affect the microsomal enzyme. The above results allowed to postulate the presence of a hexose-6-phosphate transporter in the NE which is able to carry G-6-P and M-6-P, and perhaps other hexose-6-phosphate which could be different from that present in microsomes or, if it is the same, its activity could by modified by the membrane system where it is included. The higher PPi hydrolysis activity of the intact NE G-6-Pase in comparison to the intact microsomal, suggests differences between the Pi/PPi transport (T2) of both systems. The lower sensitivity of the NE G-6-Pase to NEM suggests that the catalytic subunit of this system has some differences with the microsomal isoform.

  18. Identification of genes regulating migration and invasion using a new model of metastatic prostate cancer

    International Nuclear Information System (INIS)

    Banyard, Jacqueline; Chung, Ivy; Migliozzi, Matthew; Phan, Derek T; Wilson, Arianne M; Zetter, Bruce R; Bielenberg, Diane R

    2014-01-01

    Understanding the complex, multistep process of metastasis remains a major challenge in cancer research. Metastasis models can reveal insights in tumor development and progression and provide tools to test new intervention strategies. To develop a new cancer metastasis model, we used DU145 human prostate cancer cells and performed repeated rounds of orthotopic prostate injection and selection of subsequent lymph node metastases. Tumor growth, metastasis, cell migration and invasion were analyzed. Microarray analysis was used to identify cell migration- and cancer-related genes correlating with metastasis. Selected genes were silenced using siRNA, and their roles in cell migration and invasion were determined in transwell migration and Matrigel invasion assays. Our in vivo cycling strategy created cell lines with dramatically increased tumorigenesis and increased ability to colonize lymph nodes (DU145LN1-LN4). Prostate tumor xenografts displayed increased vascularization, enlarged podoplanin-positive lymphatic vessels and invasive margins. Microarray analysis revealed gene expression profiles that correlated with metastatic potential. Using gene network analysis we selected 3 significantly upregulated cell movement and cancer related genes for further analysis: EPCAM (epithelial cell adhesion molecule), ITGB4 (integrin β4) and PLAU (urokinase-type plasminogen activator (uPA)). These genes all showed increased protein expression in the more metastatic DU145-LN4 cells compared to the parental DU145. SiRNA knockdown of EpCAM, integrin-β4 or uPA all significantly reduced cell migration in DU145-LN4 cells. In contrast, only uPA siRNA inhibited cell invasion into Matrigel. This role of uPA in cell invasion was confirmed using the uPA inhibitors, amiloride and UK122. Our approach has identified genes required for the migration and invasion of metastatic tumor cells, and we propose that our new in vivo model system will be a powerful tool to interrogate the metastatic

  19. Kinetics, stoichiometry, and anion selectivity of cAMP-stimulated Cl-HCO3 exchange in rabbit cortical collecting tubule (CCT)

    International Nuclear Information System (INIS)

    Schuster, V.L.

    1986-01-01

    Cyclic AMP stimulates net HCO 3 secretion in rabbit CCT (Schuster, JCI 75:2056). Because cAMP induces Cl-independent (electrogenic) HCO 3 secretion in several epithelia, I studied the anion dependence of the CCT cAMP effect. Tubules were perfused in vitro with lumen amiloride; bath cAMP was continuously present to stimulate HCO 3 secretion. First, the dependence of HCO 3 secretion on lumen [Cl] was determined. With bath [Cl]=O mM, perfusate [Cl] was varied (2-150 mM, gluconate substitution). Mean lumen [Cl] was determined either by a silver electrode in the collected fluid (2-12 mM perfusate) or by 36 Cl (12-150 mM). Total bath-to-lumen HCO 3 flux, J/sup Ib//sub HCO3/, was measured (bath HCO 3 =25 mM, perfusate HCO 3 =O), Passive J/sub HCO3/ was estimated from the GHK equation using a previously-determined HCO 3 permeability = 1.9 x 10 -6 cm/s. Mediated J/sub HCO3/ vs. mean lumen [Cl] showed saturation kinetics, apparent K/sub m/ = 5.8 mM and V/sub max/ = 8.7 pmol/mm/min. Second, the stoichiometry was estimated. When bath HCO 3 was replaced by HEPES at various perfusate [Cl] (12-150 mM), ΔJ/sub HCO3/ varied linearly with ΔJ/sup Ib/sub Cl/ (slope = .85 +- .27). Third, in anion selectivity studies Br supported HCO 3 secretion (89% rate with Cl) but I - and SO 4 /sup =/ did not. In rabbit CCT, as opposed to several other HCO 3 -secreting epithelia, cAMP stimulates 1:1 Cl-HCO 3 exchange

  20. Role of Aquaporin Water Channels in Airway Fluid Transport, Humidification, and Surface Liquid Hydration

    Science.gov (United States)

    Song, Yuanlin; Jayaraman, Sujatha; Yang, Baoxue; Matthay, Michael A.; Verkman, A.S.

    2001-01-01

    Several aquaporin-type water channels are expressed in mammalian airways and lung: AQP1 in microvascular endothelia, AQP3 in upper airway epithelia, AQP4 in upper and lower airway epithelia, and AQP5 in alveolar epithelia. Novel quantitative methods were developed to compare airway fluid transport–related functions in wild-type mice and knockout mice deficient in these aquaporins. Lower airway humidification, measured from the moisture content of expired air during mechanical ventilation with dry air through a tracheotomy, was 54–56% efficient in wild-type mice, and reduced by only 3–4% in AQP1/AQP5 or AQP3/AQP4 double knockout mice. Upper airway humidification, measured from the moisture gained by dry air passed through the upper airways in mice breathing through a tracheotomy, decreased from 91 to 50% with increasing ventilation from 20 to 220 ml/min, and reduced by 3–5% in AQP3/AQP4 knockout mice. The depth and salt concentration of the airway surface liquid in trachea was measured in vivo using fluorescent probes and confocal and ratio imaging microscopy. Airway surface liquid depth was 45 ± 5 μm and [Na+] was 115 ± 4 mM in wild-type mice, and not significantly different in AQP3/AQP4 knockout mice. Osmotic water permeability in upper airways, measured by an in vivo instillation/sample method, was reduced by ∼40% by AQP3/AQP4 deletion. In doing these measurements, we discovered a novel amiloride-sensitive isosmolar fluid absorption process in upper airways (13% in 5 min) that was not affected by aquaporin deletion. These results establish the fluid transporting properties of mouse airways, and indicate that aquaporins play at most a minor role in airway humidification, ASL hydration, and isosmolar fluid absorption. PMID:11382807

  1. Sodium hydrogen exchangers contribute to arenavirus cell entry.

    Science.gov (United States)

    Iwasaki, Masaharu; Ngo, Nhi; de la Torre, Juan C

    2014-01-01

    Several arenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever (HF) disease in humans and pose a great public health concern in the regions in which they are endemic. Moreover, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. The limited existing armamentarium to combat human-pathogenic arenaviruses underscores the importance of developing novel antiarenaviral drugs, a task that would be facilitated by the identification and characterization of virus-host cell factor interactions that contribute to the arenavirus life cycle. A genome-wide small interfering RNA (siRNA) screen identified sodium hydrogen exchanger 3 (NHE3) as required for efficient multiplication of LCMV in HeLa cells, but the mechanisms by which NHE activity contributed to the life cycle of LCMV remain unknown. Here we show that treatment with the NHE inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) resulted in a robust inhibition of LCMV multiplication in both rodent (BHK-21) and human (A549) cells. EIPA-mediated inhibition was due not to interference with virus RNA replication, gene expression, or budding but rather to a blockade of virus cell entry. EIPA also inhibited cell entry mediated by the glycoproteins of the HF arenaviruses LASV and Junin virus (JUNV). Pharmacological and genetic studies revealed that cell entry of LCMV in A549 cells depended on actin remodeling and Pak1, suggesting a macropinocytosis-like cell entry pathway. Finally, zoniporide, an NHE inhibitor being explored as a therapeutic agent to treat myocardial infarction, inhibited LCMV propagation in culture cells. Our findings indicate that targeting NHEs could be a novel strategy to combat human-pathogenic arenaviruses.

  2. Bacterial endotoxin enhances colorectal cancer cell adhesion and invasion through TLR-4 and NF-kappaB-dependent activation of the urokinase plasminogen activator system.

    LENUS (Irish Health Repository)

    Killeen, S D

    2009-05-19

    Perioperative exposure to lipopolysaccharide (LPS) is associated with accelerated metastatic colorectal tumour growth. LPS directly affects cells through Toll-like receptor 4 (TLR-4) and the transcription factor NF-kappaB. The urokinase plasminogen activator (u-PA) system is intimately implicated in tumour cell extracellular matrix (ECM) interactions fundamental to tumour progression. Thus we sought to determine if LPS directly induces accelerated tumour cell ECM adhesion and invasion through activation of the u-PA system and to elucidate the cellular pathways involved. Human colorectal tumour cell lines were stimulated with LPS. u-PA concentration, u-PA activity, active u-PA, surface urokinase plasminogen activator receptor (u-PAR) and TLR-4 expression were assessed by ELISA, colorimetric assay, western blot analysis and flow cytometry respectively. In vitro tumour cell vitronectin adhesion and ECM invasion were analysed by vitronectin adhesion assay and ECM invasion chambers. u-PA and u-PAR function was inhibited with anti u-PA antibodies or the selective u-PA inhibitors amiloride or WXC-340, TLR-4 by TLR-4-blocking antibodies and NF-kappaB by the selective NF-kappaB inhibitor SN-50. LPS upregulates u-PA and u-PAR in a dose-dependent manner, enhancing in vitro tumour cell vitronectin adhesion and ECM invasion by >40% (P<0.01). These effects were ameliorated by u-PA and u-PAR inhibition. LPS activates NF-kappaB through TLR-4. TLR-4 and NF-kappaB inhibition ameliorated LPS-enhanced u-PA and u-PAR expression, tumour cell vitronectin adhesion and ECM invasion. LPS promotes tumour cell ECM adhesion and invasion through activation of the u-PA system in a TLR-4- and NF-kappaB-dependent manner.

  3. Hyperviscous airway periciliary and mucous liquid layers in cystic fibrosis measured by confocal fluorescence photobleaching

    Science.gov (United States)

    Derichs, Nico; Jin, Byung-Ju; Song, Yuanlin; Finkbeiner, Walter E.; Verkman, A. S.

    2011-01-01

    Airway surface liquid (ASL) volume depletion and mucus accumulation occur in cystic fibrosis (CF). The ASL comprises a superficial mucus layer (ML) overlying a periciliary fluid layer (PCL) that contacts surface epithelial cells. We measured viscosity of the ML and PCL from the diffusion of FITC-dextran dissolved in the ASL of unperturbed, well-differentiated primary cultures of human bronchial epithelia grown at an air-liquid interface. Diffusion was measured by fluorescence recovery after photobleaching, using a perfluorocarbon immersion lens and confocal fluorescence detection. Bleaching of an in-plane 6-μm-wide region was done in which diffusion coefficients were computed using solution standards of specified viscosity and finite-element computations of 2-layer dye diffusion in 3 dimensions. We found remarkably elevated viscosity in both ML and PCL of CF vs. non-CF bronchial epithelial cell cultures. Relative viscosities (with saline=1) were in the range 7–10 in the non-CF ML and PCL, and 25–30 in both ML and PCL in CF, and greatly reduced by amiloride treatment or mucin washout. These data indicate that the CF airway surface epithelium, even without hyperviscous secretions from submucosal glands, produces an intrinsically hyperviscous PCL and ML, which likely contributes to CF lung disease by impairment of mucociliary clearance. Our results challenge the view that the PCL is a relatively watery, nonviscous fluid layer in contact with a more viscous ML, and offer an explanation for CF lung disease in the gland-free lower airways.—Derichs, N., Jin, B. -J., Song, Y., Finkbeiner, W. E., Verkman, A. S. Hyperviscous airway periciliary and mucous liquid layers in cystic fibrosis measured by confocal fluorescence photobleaching. PMID:21427214

  4. Na+/H+ Exchanger Regulates Amino Acid-Mediated Autophagy in Intestinal Epithelial Cells

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    Huiying Shi

    2017-08-01

    Full Text Available Background/Aims: Dysfunctional autophagy has been reported to be associated with aberrant intestinal metabolism. Amino acids can regulate autophagic activity in intestinal epithelial cells (IECs. Na+/H+-exchanger 3 (NHE3 has been found to participate in the absorption of amino acids in the intestine, but whether NHE3 is involved in the regulation of autophagy in IECs is unclear. Methods: In the present study, an amino acid starvation-induced autophagic model was established. Then, the effects of alanine and proline with or without the NHE inhibitor 5-(N-ethyl-N-isopropyl amiloride (EIPA were evaluated. Autophagy was examined based on the microtubule-associated light chain 3 (LC3 levels, transmission electron microscopy (TEM, tandem GFP-mCherry-LC3 construct, sequestosome-1 (SQSTM1, P62 mRNA and protein levels, and autophagy-related gene (ATG 5, 7, and 12 expression levels. The autophagic flux was evaluated as the ratio of yellow (autophagosomes to red (autolysosomes LC3 puncta. Results: Following amino acid starvation, we found the LC3-II and ATG expression levels were enhanced in the IEC-18 cells. An increase in the number of autophagic vacuoles was concomitantly observed by TEM and confocal microscopy. Based on the results, supplementation with either alanine or proline depressed autophagy in the IEC-18 cells. Consistent with the elevated LC3-II levels, ATG expression increased upon NHE3 inhibition. Moreover, the mCherry-GFP-LC3 autophagic puncta representing both autophagosomes and autolysosomes per cell increased after EIPA treatment. Conclusions: These results demonstrate that NHE (most likely NHE3 may participate in the amino acid regulation of autophagy in IECs, which would aid in the design of better treatments for intestinal inflammation.

  5. Inhibitors of the influenza A virus M2 proton channel discovered using a high-throughput yeast growth restoration assay.

    Directory of Open Access Journals (Sweden)

    Aruna D Balgi

    Full Text Available The M2 proton channel of the influenza A virus is the target of the anti-influenza drugs amantadine and rimantadine. The effectiveness of these drugs has been dramatically limited by the rapid spread of drug resistant mutations, mainly at sites S31N, V27A and L26F in the pore of the channel. Despite progress in designing inhibitors of V27A and L26F M2, there are currently no drugs targeting these mutated channels in clinical trials. Progress in developing new drugs has been hampered by the lack of a robust assay with sufficient throughput for discovery of new active chemotypes among chemical libraries and sufficient sensitivity to provide the SAR data essential for their improvement and development as drugs. In this study we adapted a yeast growth restoration assay, in which expression of the M2 channel inhibits yeast growth and exposure to an M2 channel inhibitor restores growth, into a robust and sensitive high-throughput screen for M2 channel inhibitors. A screen of over 250,000 pure chemicals and semi-purified fractions from natural extracts identified 21 active compounds comprising amantadine, rimantadine, 13 related adamantanes and 6 non-adamantanes. Of the non-adamantanes, hexamethylene amiloride and a triazine derivative represented new M2 inhibitory chemotypes that also showed antiviral activity in a plaque reduction assay. Of particular interest is the fact that the triazine derivative was not sufficiently potent for detection as an inhibitor in the traditional two electrode voltage clamp assay for M2 channel activity, but its discovery in the yeast assay led to testing of analogues of which one was as potent as amantadine.

  6. Medium dependent dual turn on/turn off fluorescence sensing for Cu2+ions using AMI/SDS assemblies.

    Science.gov (United States)

    Gujar, Varsha B; Ottoor, Divya

    2017-02-15

    Behavior of Amiloride (AMI) as a metal ion sensor in anionic surfactant assemblies of varying concentrations at different pH is depicted in this work. From a non-sensor fluorophore, AMI has been transformed in to a tunable fluorosensor for Cu 2+ ions in various SDS concentrations. At premicellar concentration of SDS, ion-pair complex is expected to be formed between AMI and SDS due to electrostatic interactions between them. However at CMC concentrations of SDS, fluorescence intensity of AMI is greatly enhanced with red shift in emission, due to the incorporation of AMI molecule in the hydrophobic micellar interface. The behavior of metal sensing by AMI-SDS assemblies gives rise to several interesting observations. Micellation of SDS has been greatly enhanced by increasing copper ion concentrations, as these counter ions screens the charge on monomers of SDS which lead to the aggregation at premicellar concentrations only. Concentrations and pH dependent discrete trends of interactions between SDS-AMI and SDS-Cu 2+ ions, have given tunable fluorescence responses (fluorescence turn on/turn off) of AMI for added Cu 2+ ions. The electrostatic interaction between the metal cations and the anionic surfactants is the driving force for bringing the metal ions near to the vicinity of micelle where AMI resides. Thus, a comprehensive understanding of the mechanism related to the 'turn on-turn off' fluorescence response of AMI with respect to pH and SDS concentration for effective Cu 2+ ion sensing is illustrated in this work. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Correction of chloride transport and mislocalization of CFTR protein by vardenafil in the gastrointestinal tract of cystic fibrosis mice.

    Directory of Open Access Journals (Sweden)

    Barbara Dhooghe

    Full Text Available Although lung disease is the major cause of mortality in cystic fibrosis (CF, gastrointestinal (GI manifestations are the first hallmarks in 15-20% of affected newborns presenting with meconium ileus, and remain major causes of morbidity throughout life. We have previously shown that cGMP-dependent phosphodiesterase type 5 (PDE5 inhibitors rescue defective CF Transmembrane conductance Regulator (CFTR-dependent chloride transport across the mouse CF nasal mucosa. Using F508del-CF mice, we examined the transrectal potential difference 1 hour after intraperitoneal injection of the PDE5 inhibitor vardenafil or saline to assess the amiloride-sensitive sodium transport and the chloride gradient and forskolin-dependent chloride transport across the GI tract. In the same conditions, we performed immunohistostaining studies in distal colon to investigate CFTR expression and localization. F508del-CF mice displayed increased sodium transport and reduced chloride transport compared to their wild-type littermates. Vardenafil, applied at a human therapeutic dose (0.14 mg/kg used to treat erectile dysfunction, increased chloride transport in F508del-CF mice. No effect on sodium transport was detected. In crypt colonocytes of wild-type mice, the immunofluorescence CFTR signal was mostly detected in the apical cell compartment. In F508del-CF mice, a 25% reduced signal was observed, located mostly in the subapical region. Vardenafil increased the peak of intensity of the fluorescence CFTR signal in F508del-CF mice and displaced it towards the apical cell compartment. Our findings point out the intestinal mucosa as a valuable tissue to study CFTR transport function and localization and to evaluate efficacy of therapeutic strategies in CF. From our data we conclude that vardenafil mediates potentiation of the CFTR chloride channel and corrects mislocalization of the mutant protein. The study provides compelling support for targeting the cGMP signaling pathway in CF

  8. Choline transport via choline transporter-like protein 1 in conditionally immortalized rat syncytiotrophoblast cell lines TR-TBT.

    Science.gov (United States)

    Lee, N-Y; Choi, H-M; Kang, Y-S

    2009-04-01

    Choline is an essential nutrient for phospholipids and acetylcholine biosynthesis in normal development of fetus. In the present study, we investigated the functional characteristics of choline transport system and inhibitory effect of cationic drugs on choline transport in rat conditionally immortalized syncytiotrophoblast cell line (TR-TBT). Choline transport was weakly Na(+) dependent and significantly influenced by extracellular pH and by membrane depolarization. The transport process of choline is saturable with Michaelis-Menten constants (K(m)) of 68microM and 130microM in TR-TBT 18d-1 and TR-TBT 18d-2 respectively. Choline uptake in the cells was inhibited by unlabeled choline and hemicholinium-3 as well as various organic cations including guanidine, amiloride and acetylcholine. However, the prototypical organic cation tetraethylammonium and cimetidine showed very little inhibitory effect of choline uptake in TR-TBT cells. RT-PCR revealed that choline transporter-like protein 1 (CTL1) and organic cation transporter 2 (OCT2) are expressed in TR-TBT cells. The transport properties of choline in TR-TBT cells were similar or identical to that of CTL1 but not OCT2. CTL1 was also detected in human placenta. In addition, several cationic drugs such as diphenhydramine and verapamil competitively inhibited choline uptake in TR-TBT 18d-1 with K(i) of 115microM and 55microM, respectively. Our results suggest that choline transport system, which has intermediate affinity and weakly Na(+) dependent, in TR-TBT seems to occur through a CTL1 and this system may have relevance with the uptake of pharmacologically important organic cation drugs.

  9. Gene expression of the concentration-sensitive sodium channel is suppressed in lipopolysaccharide-induced acute lung injury in mice.

    Science.gov (United States)

    Hagiwara, Teruki; Yoshida, Shigeru; Hidaka, Yuji

    2017-04-01

    The concentration-sensitive sodium channel (Na C ) is expressed in alveolar type II epithelial cells and pulmonary microvascular endothelial cells in mouse lungs. We recently reported that Na C contributes to amiloride-insensitive sodium transport in mouse lungs (Respiratory Physiology & Neurobiology, 2016). However, details regarding its physiological role in the lung remain unknown. To examine whether Na C is involved in alveolar fluid clearance during an acute lung injury (ALI), we analyzed the relationship between Na C gene expression in the lung and the development of pulmonary edema in lipopolysaccharide (LPS)-induced ALI mice. LPS-induced ALI mice were prepared by the intratracheal administration of LPS. Bronchoalveolar lavage (BAL) neutrophils and lung water content (LWCs) were used as a marker of ALI and pulmonary edema, respectively. Na C protein production in the lung was detected by immunoblotting and immunofluorescence. The gene expressions of Na C and the epithelial sodium channel (ENaC) of LPS-induced ALI mice were examined by quantitative RT-PCR over a time course of 14 days. The BAL neutrophil count increased until day 2 after LPS administration and had nearly recovered by day 6. LWCs in LPS-induced mice gradually increased until day 8 and had recovered by day 14. The expression of the Na C protein in the lungs of LPS-induced mice dramatically decreased from day 2 to day 6, but recovered by day 8. The mRNA expression of Na C decreased in the lung, as well as those for α-, β-, and γ-ENaC during ALI. Thus, Na C expression is suppressed during the development stage of pulmonary edema and then recovers in the convalescent phase. Our results suggest that suppression of the gene expression of Na C is involved in the development of pulmonary edema in ALI.

  10. Error signals as powerful stimuli for the operant conditioning-like process of the fictive respiratory output in a brainstem-spinal cord preparation from rats.

    Science.gov (United States)

    Formenti, Alessandro; Zocchi, Luciano

    2014-10-01

    Respiratory neuromuscular activity needs to adapt to physiologic and pathologic conditions. We studied the conditioning effects of sensory fiber (putative Ia and II type from neuromuscular spindles) stimulation on the fictive respiratory output to the diaphragm, recorded from C4 phrenic ventral root, of in-vitro brainstem-spinal cord preparations from rats. The respiratory burst frequency in these preparations decreased gradually (from 0.26±0.02 to 0.09±0.003 bursts(-1)±SEM) as the age of the donor rats increased from zero to 4 days. The frequency greatly increased when the pH of the bath was lowered, and was significantly reduced by amiloride. C4 low threshold, sensory fiber stimulation, mimicking a stretched muscle, induced a short-term facilitation of the phrenic output increasing burst amplitude and frequency. When the same stimulus was applied contingently on the motor bursts, in an operant conditioning paradigm (a 500ms pulse train with a delay of 700ms from the beginning of the burst) a strong and persistent (>1h) increase in burst frequency was observed (from 0.10±0.007 to 0.20±0.018 bursts(-1)). Conversely, with random stimulation burst frequency increased only slightly and declined again within minutes to control levels after stopping stimulation. A forward model is assumed to interpret the data, and the notion of error signal, i.e. the sensory fiber activation indicating an unexpected stretched muscle, is re-considered in terms of the reward/punishment value. The signal, gaining hedonic value, is reviewed as a powerful unconditioned stimulus suitable in establishing a long-term operant conditioning-like process. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Action of cholera toxin in the intestinal epithelial cells

    International Nuclear Information System (INIS)

    Hyun, C.S.

    1982-01-01

    The primary event in the action of cholera toxin on the isolated chick intestinal epithelial cell is its interaction with a large number of high affinity binding sites in the cell membrane. Binding of 125 I-labeled toxin is rapid, temperature-dependent, reversible, and saturable over a wide range of concentrations and includes only a small contribution from nonspecific sites. A characteristic lag phase of 10 min occurs following the complete binding of toxin before any increase in cellular cAMP levels can be detected. The response (elevation of cellular cAMP) is linear with time for 40 to 50 min and causes a six- to eight-fold increase over control levels (10 to 15 picomole cAMP/mg cellular protein) at steady state. cAMP and agents that increase cAMP production inhibit Cl - -independent Na + influx into the isolated enterocytes whereas chlorpromazine (CPZ) which completely abolishes toxin-induced elevation of cAMP both reverses and prevents the cAMP-mediated inhibition of Na + entry. Correlation between cellular cAMP levels and the magnitude of Na + influx provides evidence for a cAMP-mediated control of intestinal Na + uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT on Na + during induction of intestinal secretion. The effect of cAMP on Na + but not Cl - influx preparations can be partially explained in terms of a cAMP-regulated Na + /H + neutral exchange system. Data on the coupling relationship between Na + transport and the intra- and extracellular pH in the enterocytes show that an amiloride-sensitive electroneutral Na + /H + exchange process occurs. This coupling between Na + and H + is partially inhibited by CT and dbcAMP, suggesting that the Na + /H + exchange may be a cAMP-regulated process. 31 references, 32 figures, 5 tables

  12. Reduced Sodium Transport With Nasal Administration of the Prostasin Inhibitor Camostat in Subjects With Cystic Fibrosis

    Science.gov (United States)

    Reeves, Ginger; Hathorne, Heather; Solomon, G. Martin; Abbi, Smita; Renard, Didier; Lock, Ruth; Zhou, Ping; Danahay, Henry; Clancy, John P.; Waltz, David A.

    2013-01-01

    Background: Prostasin, a trypsin-like serine protease, is a channel-activating protease and major regulator of epithelial sodium channel-mediated sodium absorption. Its direct inhibition by camostat represents a potential approach to inhibiting sodium transport in cystic fibrosis (CF). Methods: To determine whether a topical formulation of camostat represents an efficacious and tolerable approach to reducing Na+ transport in the CF airway, we conducted a two-part randomized, double-blind, placebo-controlled, crossover, ascending single-dose study to evaluate the pharmacodynamics, safety, and pharmacokinetics of camostat administered through a nasal spray pump in subjects with CF. Nasal potential difference (PD) was measured before and after treatment, and safety and pharmacokinetics were assessed by a standardized approach. Results: In part 1, nine subjects were enrolled, and six completed crossover dosing at the maximally tolerated dose. The change in maximal (most polarizing) basal PD 2 h following administration of camostat was +13.1 mV (1.6-mg dose group) compared with −8.6 mV following placebo (P < .005). Intrasubject change in Ringer and amiloride-sensitive PDs exhibited similar and consistent responses. Bayesian analysis in an additional six subjects in part 2 estimated a dose of 18 μg/mL to provide 50% of the maximum effect. There was no significant change in chloride transport or total nasal symptom score, nasal examination rating, and laboratory parameters. Conclusions: This study establishes the proof of concept that a reduction in sodium transport in the human CF airway can be achieved through inhibition of prostasin activity, identifying a potential therapeutic target in the disease. Trial registration: ClinicalTrials.gov; No.: NCT00506792; URL: www.clinicaltrials.gov PMID:23412700

  13. Potentially severe drug-drug interactions among older people and associations in assisted living facilities in Finland: a cross-sectional study.

    Science.gov (United States)

    Teramura-Grönblad, Mariko; Raivio, Minna; Savikko, Niina; Muurinen, Seija; Soini, Helena; Suominen, Merja; Pitkälä, Kaisu

    2016-09-01

    This study aims to assess potentially severe class D drug-drug interactions (DDDIs) in residents 65 years or older in assisted living facilities with the use of a Swedish and Finnish drug-drug interaction database (SFINX). A cross-sectional study of residents in assisted living facilities in Helsinki, Finland. A total of 1327 residents were assessed in this study. Drugs were classified according to the Anatomical Therapeutic Chemical (ATC) classification system and DDDIs were coded according to the SFINX. Prevalence of DDDIs, associated factors and 3-year mortality among residents. Of the participants (mean age was 82.7 years, 78.3% were females), 5.9% (N = 78) are at risk for DDDIs, with a total of 86 interactions. Participants with DDDIs had been prescribed a higher number of drugs (10.8 (SD 3.8) vs. 7.9 (SD 3.7), p assisted living, 5.9% were exposed to DDDIs associated with the use of a higher number of drugs. Physicians should be trained to find safer alternatives to drugs associated with DDDIs. KEY POINTS   Potentially severe, class D drug-drug interactions (DDDIs) have been defined in the SFINX database as clinically relevant drug interactions that should be avoided.  • Of the residents in assisted living, 5.9% were exposed to DDDIs that were associated with the use of a higher number of drugs.  • The most frequent DDDIs were related to the concomitant use of potassium with amiloride or spironolactone. Carbamazepine and methotrexate were also linked to DDDIs.  • No difference in mortality was observed between residents exposed to DDDIs and residents not exposed to DDDIs.

  14. Comparison of the responses of the chorda tympani and glossopharyngeal nerves to taste stimuli in C57BL/6J mice

    Directory of Open Access Journals (Sweden)

    Hellekant Göran

    2003-03-01

    Full Text Available Abstract Background Recent progress in discernment of molecular pathways of taste transduction underscores the need for comprehensive phenotypic information for the understanding of the influence of genetic factors in taste. To obtain information that can be used as a base line for assessment of effects of genetic manipulations in mice taste, we have recorded the whole-nerve integrated responses to a wide array of taste stimuli in the chorda tympani (CT and glossopharyngeal (NG nerves, the two major taste nerves from the tongue. Results In C57BL/6J mice the responses in the two nerves were not the same. In general sweeteners gave larger responses in the CT than in the NG, while responses to bitter taste in the NG were larger. Thus the CT responses to cyanosuosan, fructose, NC00174, D-phenylalanline and sucrose at all concentrations were significantly larger than in the NG, whereas for acesulfame-K, L-proline, saccharin and SC45647 the differences were not significant. Among bitter compounds amiloride, atropine, cycloheximide, denatonium benzoate, L-phenylalanine, 6-n-propyl-2-thiouracil (PROP and tetraethyl ammonium chloride (TEA gave larger responses in the NG, while the responses to brucine, chloroquine, quinacrine, quinine hydrochloride (QHCl, sparteine and strychnine, known to be very bitter to humans, were not significantly larger in the NG than in the CT. Conclusion These data provide a comprehensive survey and comparison of the taste sensitivity of the normal C57BL/6J mouse against which the effects of manipulations of its gustatory system can be better assessed.

  15. Decrease of intracellular pH as possible mechanism of embryotoxicity of glycol ether alkoxyacetic acid metabolites

    International Nuclear Information System (INIS)

    Louisse, Jochem; Bai Yanqing; Verwei, Miriam; Sandt, Johannes J.M. van de; Blaauboer, Bas J.; Rietjens, Ivonne M.C.M.

    2010-01-01

    Embryotoxicity of glycol ethers is caused by their alkoxyacetic acid metabolites, but the mechanism underlying the embryotoxicity of these acid metabolites is so far not known. The present study investigates a possible mechanism underlying the embryotoxicity of glycol ether alkoxyacetic acid metabolites using the methoxyacetic acid (MAA) metabolite of ethylene glycol monomethyl ether as the model compound. The results obtained demonstrate an MAA-induced decrease of the intracellular pH (pH i ) of embryonic BALB/c-3T3 cells as well as of embryonic stem (ES)-D3 cells, at concentrations that affect ES-D3 cell differentiation. These results suggest a mechanism for MAA-mediated embryotoxicity similar to the mechanism of embryotoxicity of the drugs valproic acid and acetazolamide (ACZ), known to decrease the pH i in vivo, and therefore used as positive controls. The embryotoxic alkoxyacetic acid metabolites ethoxyacetic acid, butoxyacetic acid and phenoxyacetic acid also caused an intracellular acidification of BALB/c-3T3 cells at concentrations that are known to inhibit ES-D3 cell differentiation. Two other embryotoxic compounds, all-trans-retinoic acid and 5-fluorouracil, did not decrease the pH i of embryonic cells at concentrations that affect ES-D3 cell differentiation, pointing at a different mechanism of embryotoxicity of these compounds. MAA and ACZ induced a concentration-dependent inhibition of ES-D3 cell differentiation, which was enhanced by amiloride, an inhibitor of the Na + /H + -antiporter, corroborating an important role of the pH i in the embryotoxic mechanism of both compounds. Together, the results presented indicate that a decrease of the pH i may be the mechanism of embryotoxicity of the alkoxyacetic acid metabolites of the glycol ethers.

  16. NHE- and diffusion-dependent proton fluxes across the tubular system membranes of fast-twitch muscle fibers of the rat.

    Science.gov (United States)

    Launikonis, Bradley S; Cully, Tanya R; Csernoch, Laszlo; Stephenson, D George

    2018-01-02

    The complex membrane structure of the tubular system (t-system) in skeletal muscle fibers is open to the extracellular environment, which prevents measurements of H + movement across its interface with the cytoplasm by conventional methods. Consequently, little is known about the t-system's role in the regulation of cytoplasmic pH, which is different from extracellular pH. Here we describe a novel approach to measure H + -flux measurements across the t-system of fast-twitch fibers under different conditions. The approach involves loading the t-system of intact rat fast-twitch fibers with a strong pH buffer (20 mM HEPES) and pH-sensitive fluorescent probe (10 mM HPTS) before the t-system is sealed off. The pH changes in the t-system are then tracked by confocal microscopy after rapid changes in cytoplasmic ionic conditions. T-system sealing is achieved by removing the sarcolemma by microdissection (mechanical skinning), which causes the tubules to pinch off and seal tight. After this procedure, the t-system repolarizes to physiological levels and can be electrically stimulated when placed in K + -based solutions of cytosolic-like ionic composition. Using this approach, we show that the t-system of fast-twitch skeletal fibers displays amiloride-sensitive Na + /H + exchange (NHE), which decreases markedly at alkaline cytosolic pH and has properties similar to that in mammalian cardiac myocytes. We observed mean values for NHE density and proton permeability coefficient of 339 pmol/m 2 of t-system membrane and 158 µm/s, respectively. We conclude that the cytosolic pH in intact resting muscle can be quantitatively explained with respect to extracellular pH by assuming that these values apply to the t-system membrane and the sarcolemma. © 2018 Launikonis et al.

  17. ACh-induced depolarization in inner ear artery is generated by activation of a TRP-like non-selective cation conductance and inactivation of a potassium conductance.

    Science.gov (United States)

    Ma, Ke-Tao; Guan, Bing-Cai; Yang, Yu-Qin; Zhao, Hui; Jiang, Zhi-Gen

    2008-05-01

    Adequate cochlear blood supply by the spiral modiolar artery (SMA) is critical for normal hearing. ACh may play a role in neuroregulation of the SMA but several key issues including its membrane action mechanisms remain poorly understood. Besides its well-known endothelium-dependent hyperpolarizing action, ACh can induce a depolarization in vascular cells. Using intracellular and whole-cell recording techniques on cells in guinea pig in vitro SMA, we studied the ionic mechanism underlying the ACh-depolarization and found that: (1) ACh induced a DAMP-sensitive depolarization when intermediate conductance KCa channels were blocked by charybdotoxin or nitrendipine. The ACh-depolarization was associated with a decrease in input resistance (R(input)) in high membrane potential (V(m)) ( approximately -40 mV) cells but with no change or an increase in R input in low Vm ( approximately -75 mV) cells. ACh-depolarization was attenuated by background membrane depolarization from approximately -70 mV in the majority of cells; (2) ACh-induced inward current in smooth muscle cells embedded in a SMA segment often showed a U-shaped I/V curve, the reversal potential of its two arms being near EK and 0 mV, respectively; (3) ACh-depolarization was reduced by low Na+, zero K+ or 20mM K+ bath solutions; (4) ACh-depolarization was inhibited by La3+ in all cells tested, by 4-AP and flufenamic acid in low Vm cells, but was not sensitive to Cd2+, Ni2+, nifedipine, niflumic acid, DIDS, IAA94, linopirdine or amiloride. We conclude that ACh-induced vascular depolarization was generated mainly by activation of a TRP-like non-selective cation channel and by inactivation of an inward rectifier K+ channel.

  18. A first screening and risk assessment of pharmaceuticals and additives in personal care products in waste water, sludge, recipient water and sediment from Faroe Islands, Iceland and Greenland.

    Science.gov (United States)

    Huber, Sandra; Remberger, Mikael; Kaj, Lennart; Schlabach, Martin; Jörundsdóttir, Hrönn Ó; Vester, Jette; Arnórsson, Mímir; Mortensen, Inge; Schwartson, Richard; Dam, Maria

    2016-08-15

    A screening of a broad range of pharmaceuticals and additives in personal care products (PPCPs) in sub-arctic locations of the Faroe Islands (FO), Iceland (IS) and Greenland (GL) was conducted. In total 36 pharmaceuticals including some metabolites, and seven additives in personal care products were investigated in influent and effluent waters as well as sludge of waste water treatment plants (WWTPs) and in water and sediment of recipients. Concentrations and distribution patterns for PPCPs discharged via sewage lines (SLs) to the marine environment were assessed. Of the 36 pharmaceuticals or metabolites analysed 33 were found close to or above the limit of detection (LOD) in all or a part of the samples. All of the seven investigated additives in personal care products were detected above the LOD. Some of the analysed PPCPs occurred in every or almost every sample. Among these were diclofenac, ibuprofen, lidocaine, naproxen, metformin, citalopram, venlafaxine, amiloride, furosemide, metoprolol, sodium dodecyl sulphate (SDS) and cetrimonium salt (ATAC-C16). Additionally, the study encompasses ecotoxicological risk assessment of 2/3 of the analysed PPCPs in recipient and diluted effluent waters. For candesartan only a small margin to levels with inacceptable risks was observed in diluted effluent waters at two locations (FO). Chronical risks for aquatic organisms staying and/or living around WWTP effluent pipe-outlets were indicated for 17β-estradiol and estriol in the three countries. Additives in PCPs were found to pose the largest risk to the aquatic environment. The surfactants CAPB and ATAC-C16 were found in concentrations resulting in risk factors up to 375 for CAPB and 165 for ATAC-C16 in recipients for diluted effluents from Iggia, Nuuk (GL) and Torshavn (FO) respectively. These results demonstrates a potentially high ecological risk stemming from discharge of surfactants as used in household and industrial detergents as well as additives in personal care

  19. Intracellular acidification by inhibition of the Na+/H+-exchanger leads to caspase-independent death of cerebellar granule neurons resembling paraptosis.

    Science.gov (United States)

    Schneider, D; Gerhardt, E; Bock, J; Müller, M M; Wolburg, H; Lang, F; Schulz, J B

    2004-07-01

    Potassium withdrawal is commonly used to induce caspase-mediated apoptosis in cerebellar granule neurons in vitro. However, the underlying and cell death-initiating mechanisms are unknown. We firstly investigated potassium efflux through the outward delayed rectifier K+ current (Ik) as a potential mediator. However, tetraethylammoniumchloride, an inhibitor of Ik, was ineffective to block apoptosis after potassium withdrawal. Since potassium withdrawal reduced intracellular pH (pHi) from 7.4 to 7.2, we secondly investigated the effects of intracellular acidosis. To study intracellular acidosis in cerebellar granule neurons, we inhibited the Na+/H+ exchanger (NHE) with 4-isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate (HOE 642) and 5-(N-ethyl-N-isopropyl)-amiloride. Both inhibitors concentration-dependently induced cell death and potentiated cell death after potassium withdrawal. Although inhibition of the NHE induced cell death with morphological criteria of apoptosis in light and electron microscopy including chromatin condensation, positive TUNEL staining and cell shrinkage, no internucleosomal DNA cleavage or activation of caspases was detected. In contrast to potassium withdrawal-induced apoptosis, cell death induced by intracellular acidification was not prevented by insulin-like growth factor-1, cyclo-adenosine-monophosphate, caspase inhibitors and transfection with an adenovirus expressing Bcl-XL. However, cycloheximide protected cerebellar granule neurons from death induced by potassium withdrawal as well as from death after treatment with HOE 642. Therefore, the molecular mechanisms leading to cell death after acidification appear to be different from the mechanisms after potassium withdrawal and resemble the biochemical but not the morphological characteristics of paraptosis.

  20. High salt loading induces urinary storage dysfunction via upregulation of epithelial sodium channel alpha in the bladder epithelium in Dahl salt-sensitive rats

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    Seiji Yamamoto

    2017-11-01

    Full Text Available We aimed to investigate whether high salt intake affects bladder function via epithelial sodium channel (ENaC by using Dahl salt-resistant (DR and salt-sensitive (DS rats. Bladder weight of DR + high-salt diet (HS, 8% NaCl and DS + HS groups were significantly higher than those of DR + normal-salt diet (NS, 0.3% NaCl and DS + NS groups after one week treatment. We thereafter used only DR + HS and DS + HS group. Systolic and diastolic blood pressures were significantly higher in DS + HS group than in DR + HS group after the treatment period. Cystometrogram showed the intercontraction intervals (ICI were significantly shorter in DS + HS group than in DR + HS group during infusion of saline. Subsequent infusion of amiloride significantly prolonged ICI in DS + HS group, while no intra-group difference in ICI was observed in DR + HS group. No intra- or inter-group differences in maximum intravesical pressure were observed. Protein expression levels of ENaCα in the bladder were significantly higher in DS + HS group than in DR + HS group. ENaCα protein was localized at bladder epithelium in both groups. In conclusion, high salt intake is considered to cause urinary storage dysfunction via upregulation of ENaC in the bladder epithelium with salt-sensitive hypertension, suggesting that ENaC might be a candidate for therapeutic target for urinary storage dysfunction.

  1. Differential Contribution of TRPA1, TRPV4 and TRPM8 to Colonic Nociception in Mice.

    Directory of Open Access Journals (Sweden)

    Sonja M Mueller-Tribbensee

    Full Text Available Various transient receptor potential (TRP channels in sensory neurons contribute to the transduction of mechanical stimuli in the colon. Recently, even the cold-sensing menthol receptor TRPM(melastatin8 was suggested to be involved in murine colonic mechano-nociception.To analyze the roles of TRPM8, TRPA1 and TRPV4 in distension-induced colonic nociception and pain, TRP-deficient mice and selective pharmacological blockers in wild-type mice (WT were used. Visceromotor responses (VMR to colorectal distension (CRD in vivo were recorded and distension/pressure-induced CGRP release from the isolated murine colon ex vivo was measured by EIA.Distension-induced colonic CGRP release was markedly reduced in TRPA1-/- and TRPV4-/- mice at 90/150 mmHg compared to WT. In TRPM8-deficient mice the reduction was only distinct at 150 mmHg. Exposure to selective pharmacological antagonists (HC030031, 100 μM; RN1734, 10 μM; AMTB, 10 μM showed corresponding effects. The unselective TRP blocker ruthenium red (RR, 10 μM was as efficient in inhibiting distension-induced CGRP release as the unselective antagonists of mechanogated DEG/ENaC (amiloride, 100 μM and stretch-activated channels (gadolinium, 50 μM. VMR to CRD revealed prominent deficits over the whole pressure range (up to 90 mmHg in TRPA1-/- and TRPV4-/- but not TRPM8-/- mice; the drug effects of the TRP antagonists were again highly consistent with the results from mice lacking the respective TRP receptor gene.TRPA1 and TRPV4 mediate colonic distension pain and CGRP release and appear to govern a wide and congruent dynamic range of distensions. The role of TRPM8 seems to be confined to signaling extreme noxious distension, at least in the healthy colon.

  2. Dynamics of intracellular ionic concentrations in single living cells using videomicrofluorometry: application to pHi variations

    Science.gov (United States)

    Viallet, Pierre M.; Yassine, Mohamed; Salmon, Jean-Marie; Vigo, Jean

    1996-01-01

    The intracellular concentration of ions such as H+, Mg2+, Ca2+, is known to monitor the activity of many fundamental enzymes. Furthermore these ions are generally considered as intracellular messengers involved in the transduction of extracellular signals. Recent technological progress, occulting the physicochemical properties of the probe, led to the feeling that accurate data on microvolumes are instantly accessible. Unfortunately fluorescent probes are supposed to fill up conflicting requirements for ionic affinity, absence of fading and intracellular calibration. Such a situation generally precludes the use of the simplest methods of data acquisition and treatment. This paper is based on the use of microspectrofluorometry, resolution of single cell complex fluorescence spectrum, and videomicrofluorometry. The methods of data handling allow us to demonstrate that most of the problems met in intracellular calibration come from the fighting of cells against the modification of the extracellular pH. Using these techniques allows us to restrict the need of comparison between results in cuvettes and intracellular results to the physiological pH range. A consequence of such an approach is that the effect with time of known concentrations of amiloride and nigericin on pHi became accessible. Data is presented allowing us to get information on the behavior of the ionic channels and/or cation/H+ exchangers involved in the pHi regulation. Such a method leads the way to direct investigations and monitoring of the different processes of regulation of the intracellular ionic concentrations in different cell lines at the level of single cells. Using different specific modifiers (activators or blocking agents) and convenient specific fluorescent probes, the efficiency of such pathways is expected to be checked at will. Compared to the patch clamp techniques, the method can be extended to the study of pathways located on the inner cell membranes.

  3. Effects of thyroid hormone on Na sup + -K sup + transport in resting and stimulated rat skeletal muscle

    Energy Technology Data Exchange (ETDEWEB)

    Everts, M.E.; Clausen, T. (Aarhus Univ. (Denmark))

    1988-11-01

    The effects of hypothyroidism and 3,5,3{prime}-triiodothyronine (T{sub 3}) treatment on passive Na{sup +}-K{sup +} fluxes and Na{sup +}-K{sup +} pump concentration were investigated in isolated rat muscle. Within 12 h after a single dose of T{sub 3} (20 {mu}g/100 g body wt), K{sup +} efflux had increased by 21% in soleus and by 20% in extensor digitorum longus muscle. In the presence of ouabain, even larger effects were observed. These changes were associated with a 12% rise in amiloride-suppressible Na{sup +} influx but no significant increase in ({sup 3}H)ouabain binding site concentration. After 3 days of T{sub 3} treatment, the stimulating effect on K{sup +} efflux and Na{sup +} influx in soleus reached a plateau {approximately}80 and 40% above control levels, respectively, whereas the maximum increase in ({sup 3}H)ouabain binding site concentration (103%) was only fully developed after 8 days. Hypothyroidism decreased {sup 86}Rb efflux by 30%. The efflux of K{sup +} and the influx of Na{sup +} per contraction (both {approximately}7 nmol/g wet wt) as well as the net loss of K{sup +} induced by electrical stimulation were unaffected by T{sub 3} treatment. The rise in resting K{sup +} efflux after 12-24 h of T{sub 3} treatment could be partly blocked by dantrolene or trifluoroperazine, indicating that an increase in the cytoplasmic Ca{sup 2+} concentration may contribute to the early rise in K{sup +} efflux. It is concluded that the early rise in the resting passive leaks of Na{sup +} and K{sup +} induced by T{sub 3} is a major driving force for Na{sup +}-K{sup +} pump synthesis.

  4. Regulation of ENaC in mice lacking renal insulin receptors in the collecting duct

    Science.gov (United States)

    Pavlov, Tengis S.; Ilatovskaya, Daria V.; Levchenko, Vladislav; Li, Lijun; Ecelbarger, Carolyn M.; Staruschenko, Alexander

    2013-01-01

    The epithelial sodium channel (ENaC) is one of the central effectors involved in regulation of salt and water homeostasis in the kidney. To study mechanisms of ENaC regulation, we generated knockout mice lacking the insulin receptor (InsR KO) specifically in the collecting duct principal cells. Single-channel analysis in freshly isolated split-open tubules demonstrated that the InsR-KO mice have significantly lower ENaC activity compared to their wild-type (C57BL/6J) littermates when animals were fed either normal or sodium-deficient diets. Immunohistochemical and Western blot assays demonstrated no significant changes in expression of ENaC subunits in InsR-KO mice compared to wild-type littermates. Insulin treatment caused greater ENaC activity in split-open tubules isolated from wild-type mice but did not have this effect in the InsR-KO mice. Thus, these results suggest that insulin increases ENaC activity via its own receptor affecting the channel open probability. To further determine the mechanism of the action of insulin on ENaC, we used mouse mpkCCDc14 principal cells. Insulin significantly augmented amiloride-sensitive transepithelial flux in these cells. Pretreatment of the mpkCCDc14 cells with phosphatidylinositol 3-kinase (LY294002; 10 μM) or mTOR (PP242; 100 nM) inhibitors precluded this effect. This study provides new information about the importance of insulin receptors expressed in collecting duct principal cells for ENaC activity.—Pavlov, T. S., Ilatovskaya, D. V., Levchenko, V., Li, L., Ecelbarger, C. M., Staruschenko, A. Regulation of ENaC in mice lacking renal insulin receptors in the collecting duct. PMID:23558339

  5. Diuretic exposure in premature infants from 1997–2011

    Science.gov (United States)

    Laughon, Matthew M.; Chantala, Kim; Aliaga, Sofia; Herring, Amy H.; Hornik, Christoph P.; Hughes, Rachel; Clark, Reese H.; Smith, P. Brian

    2014-01-01

    Objective Diuretics are often prescribed off-label to premature infants, particularly to prevent or treat bronchopulmonary dysplasia (BPD). We examined their use and safety in this group. Study Design Retrospective cohort study of infants diuretics in 333 neonatal intensive care units from 1997–2011. We examined use of acetazolamide, amiloride, bumetanide, chlorothiazide, diazoxide, ethacrynic acid, furosemide, hydrochlorothiazide, mannitol, metolazone, or spironolactone combination. Respiratory support and FiO2 on the first day of each course of diuretic use were identified. Results Thirty-seven percent (39,357/107,542) of infants were exposed to at least 1 diuretic; furosemide was the most commonly used (93% with ≥1 recorded dose), followed by spironolactone, chlorothiazide, hydrochlorothiazide, bumetanide, and acetazolamide. Seventy-four percent were exposed to 1 diuretic at a time, 19% to 2 diuretics simultaneously, and 6% to 3 diuretics simultaneously. The most common combination was furosemide/spironolactone, followed by furosemide/chlorothiazide and chlorothiazide/spironolactone. Many infants were not receiving mechanical ventilation on the first day of each new course of furosemide (47%), spironolactone (69%), chlorothiazide (61%), and hydrochlorothiazide (68%). Any adverse event occurred on 42 per 1000 infant-days for any diuretic and 35 per 1000 infant-days for furosemide. Any serious adverse event occurred in 3.8 for any diuretic and 3.2 per 1000 infant-days for furosemide. The most common laboratory abnormality associated with diuretic exposure was thrombocytopenia. Conclusion Despite no FDA indication and little safety data, over one third of premature infants in our population were exposed to a diuretic, many with minimal respiratory support. PMID:24801161

  6. Clathrin and LRP-1-independent constitutive endocytosis and recycling of uPAR.

    Directory of Open Access Journals (Sweden)

    Katia Cortese

    Full Text Available BACKGROUND: The urokinase receptor (uPAR/CD87 is highly expressed in malignant tumours. uPAR, as a GPI anchored protein, is preferentially located at the cell surface, where it interacts with its ligands urokinase (uPA and the extracellular matrix protein vitronectin, thus promoting plasmin generation, cell-matrix interactions and intracellular signalling events. Interaction with a complex formed by uPA and its inhibitor PAI-1 induces cell surface down regulation and recycling of the receptor via the clathrin-coated pathway, a process dependent on the association to LRP-1. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have found that along with the ligand-induced down-regulation, uPAR also internalizes and recycles constitutively through a second pathway that is independent of LRP-1 and clathrin but shares some properties with macropinocytosis. The ligand-independent route is amiloride-sensitive, does not require uPAR partitioning into lipid rafts, is independent of the activity of small GTPases RhoA, Rac1 and Cdc42, and does not require PI3K activity. Constitutively endocytosed uPAR is found in EEA1 positive early/recycling endosomes but does not reach lysosomes in the absence of ligands. Electron microscopy analysis reveals the presence of uPAR in ruffling domains at the cell surface, in macropinosome-like vesicles and in endosomal compartments. CONCLUSIONS/SIGNIFICANCE: These results indicate that, in addition to the ligand-induced endocytosis of uPAR, efficient surface expression and membrane trafficking might also be driven by an uncommon macropinocytic mechanism coupled with rapid recycling to the cell surface.

  7. Dissection of the Influenza A Virus Endocytic Routes Reveals Macropinocytosis as an Alternative Entry Pathway

    Science.gov (United States)

    de Vries, Erik; Tscherne, Donna M.; Wienholts, Marleen J.; Cobos-Jiménez, Viviana; Scholte, Florine; García-Sastre, Adolfo; Rottier, Peter J. M.; de Haan, Cornelis A. M.

    2011-01-01

    Influenza A virus (IAV) enters host cells upon binding of its hemagglutinin glycoprotein to sialylated host cell receptors. Whereas dynamin-dependent, clathrin-mediated endocytosis (CME) is generally considered as the IAV infection pathway, some observations suggest the occurrence of an as yet uncharacterized alternative entry route. By manipulating entry parameters we established experimental conditions that allow the separate analysis of dynamin-dependent and -independent entry of IAV. Whereas entry of IAV in phosphate-buffered saline could be completely inhibited by dynasore, a specific inhibitor of dynamin, a dynasore-insensitive entry pathway became functional in the presence of fetal calf serum. This finding was confirmed with the use of small interfering RNAs targeting dynamin-2. In the presence of serum, both IAV entry pathways were operational. Under these conditions entry could be fully blocked by combined treatment with dynasore and the amiloride derivative EIPA, the hallmark inhibitor of macropinocytosis, whereas either drug alone had no effect. The sensitivity of the dynamin-independent entry pathway to inhibitors or dominant-negative mutants affecting actomyosin dynamics as well as to a number of specific inhibitors of growth factor receptor tyrosine kinases and downstream effectors thereof all point to the involvement of macropinocytosis in IAV entry. Consistently, IAV particles and soluble FITC-dextran were shown to co-localize in cells in the same vesicles. Thus, in addition to the classical dynamin-dependent, clathrin-mediated endocytosis pathway, IAV enters host cells by a dynamin-independent route that has all the characteristics of macropinocytosis. PMID:21483486

  8. Enzymatically Modified Low-Density Lipoprotein Promotes Foam Cell Formation in Smooth Muscle Cells via Macropinocytosis and Enhances Receptor-Mediated Uptake of Oxidized Low-Density Lipoprotein.

    Science.gov (United States)

    Chellan, Bijoy; Reardon, Catherine A; Getz, Godfrey S; Hofmann Bowman, Marion A

    2016-06-01

    Enzyme-modified nonoxidized low-density lipoprotein (ELDL) is present in human atherosclerotic lesions. Our objective is to understand the mechanisms of ELDL uptake and its effects on vascular smooth muscle cells (SMC). Transformation of murine aortic SMCs into foam cells in response to ELDL was analyzed. ELDL, but not acetylated or oxidized LDL, was potent in inducing SMC foam cell formation. Inhibitors of macropinocytosis (LY294002, wortmannin, amiloride) attenuated ELDL uptake. In contrast, inhibitors of receptor-mediated endocytosis (dynasore, sucrose) and inhibitor of caveolae-/lipid raft-mediated endocytosis (filipin) had no effect on ELDL uptake in SMC, suggesting that macropinocytosis is the main mechanism of ELDL uptake by SMC. Receptor for advanced glycation end products (RAGE) is not obligatory for ELDL-induced SMC foam cell formation, but primes SMC for the uptake of oxidized LDL in a RAGE-dependent manner. ELDL increased intracellular reactive oxygen species, cytosolic calcium, and expression of lectin-like oxidized LDL receptor-1 in wild-type SMC but not in RAGE(-/-) SMC. The macropinocytotic uptake of ELDL is regulated predominantly by intracellular calcium because ELDL uptake was completely inhibited by pretreatment with the calcium channel inhibitor lacidipine in wild-type and RAGE(-/-) SMC. This is in contrast to pretreatment with PI3 kinase inhibitors which completely prevented ELDL uptake in RAGE(-/-) SMC, but only partially in wild-type SMC. ELDL is highly potent in inducing foam cells in murine SMC. ELDL endocytosis is mediated by calcium-dependent macropinocytosis. Priming SMC with ELDL enhances the uptake of oxidized LDL. © 2016 American Heart Association, Inc.

  9. Uptake of ricinB-quantum dot nanoparticles by a macropinocytosis-like mechanism

    Directory of Open Access Journals (Sweden)

    Iversen Tore

    2012-07-01

    Full Text Available Abstract Background There is a huge effort in developing ligand-mediated targeting of nanoparticles to diseased cells and tissue. The plant toxin ricin has been shown to enter cells by utilizing both dynamin-dependent and -independent endocytic pathways. Thus, it is a representative ligand for addressing the important issue of whether even a relatively small ligand-nanoparticle conjugate can gain access to the same endocytic pathways as the free ligand. Results Here we present a systematic study concerning the internalization mechanism of ricinB:Quantum dot (QD nanoparticle conjugates in HeLa cells. Contrary to uptake of ricin itself, we found that internalization of ricinB:QDs was inhibited in HeLa cells expressing dominant-negative dynamin. Both clathrin-, Rho-dependent uptake as well as a specific form of macropinocytosis involve dynamin. However, the ricinB:QD uptake was not affected by siRNA-mediated knockdown of clathrin or inhibition of Rho-dependent uptake caused by treating cells with the Clostridium C3 transferase. RicinB:QD uptake was significantly reduced by cholesterol depletion with methyl-β-cyclodextrin and by inhibitors of actin polymerization such as cytochalasin D. Finally, we found that uptake of ricinB:QDs was blocked by the amiloride analog EIPA, an inhibitor of macropinocytosis. Upon entry, the ricinB:QDs co-localized with dextran, a marker for fluid-phase uptake. Thus, internalization of ricinB:QDs in HeLa cells critically relies on a dynamin-dependent macropinocytosis-like mechanism. Conclusions Our results demonstrate that internalization of a ligand-nanoparticle conjugate can be dependent on other endocytic mechanisms than those used by the free ligand, highlighting the challenges of using ligand-mediated targeting of nanoparticles-based drug delivery vehicles to cells of diseased tissues.

  10. Response of copper deficient rats to inhibitors of renal sodium reabsorption

    Energy Technology Data Exchange (ETDEWEB)

    Noordewier, B.; Saari, J.T. (Northwestern College, Orange City, IA (United States) USDA/ARS, Grand Forks, ND (United States))

    1991-03-11

    This study examined the effects of furosemide (Furo), a Loop diuretic, and amiloride (Am), a potassium (K)-sparing diuretic, on the excretion of sodium (Na) and K in copper-adequate (CuAdeq) and copper-deficient (CuDef) rats. Weanling male Sprague Dawley rats were fed a CuDef or CuAdeq diet ad libitum and given deionized water to drink. After 5 weeks on the diets, rats were assigned to one of four treatment regimens: Furo, Am or Furo + Am. Rats were anesthetized and electrolyte excretion was measured in 2 {times} 15 min control periods followed by 3 {times} 15 min treatment periods. Furo increased Na excretion in a dose dependent manner in both the CuAdeq and the CuDef rats. The response of the CuAdeq rats was slightly greater than that of the CuDef rats in each of the 3 treatment groups in which Furo was given. K excretion following Furo increased to the same extent in the CuAdeq and CuDef rats. The natriuretic response to Am alone was slightly greater in the CuDef than the CuAdeq rats. The antikaliuretic response of the CuDef rats was similar to that of the CuAdeq rats whether Am was given alone or in combination with Furo. These data show that CuDef rats respond to Furo and Am in a manner which is similar to that of CuAdeq rats, this indicates that the sensitivity of the Na reabsorption mechanisms to inhibition by diuretics is not markedly affected by copper deficiency.

  11. Role of diacylglycerol in adrenergic-stimulated sup 86 Rb uptake by proximal tubules

    Energy Technology Data Exchange (ETDEWEB)

    Baines, A.D.; Drangova, R.; Ho, P. (Univ. of Toronto, Ontario (Canada))

    1990-05-01

    We used rat proximal tubule fragments purified by Percoll centrifugation to examine the role of diacylglycerol (DAG) in noradrenergic-stimulated Na+ reabsorption. Tubular DAG concentration and ouabain-inhibitable 86Rb uptake increased within 30 s after adding norepinephrine (NE) and remained elevated for at least 5 min. NE (1 microM) increased DAG content 17% and ouabain-inhibitable 86Rb uptake 23%. Cirazoline-stimulated 86Rb uptake was not inhibited by BaCl, quinidine, or bumetanide (1-10 microM) or by the omission of HCO3- or Cl- from the medium, but it was completely inhibited by ouabain and furosemide. Oleoyl-acetyl glycerol, L-alpha-1,2-dioctanoylglycerol, and L-alpha-1,2-dioleoylglycerol (DOG) increased total 86Rb uptake 8-11%. 12-O-tetradecanoylphorbol-13-acetate (TPA) (5 nM) increased uptake by only 4%. Staurosporine at 5 nM inhibited DOG stimulation completely, whereas 50 nM staurosporine was required to inhibit NE stimulation completely. Sphingosine inhibited DOG stimulation by 66% but did not inhibit NE stimulation. Amiloride (1 mM) completely blocked DOG stimulation. Monensin increased 86Rb uptake 31% and completely blocked the DOG effect but reduced the NE effect by only 26% (P = 0.08). In tubules from salt-loaded rats, NE did not increase DAG concentration, but NE-stimulated 86Rb uptake was reduced by only 23% (P = 0.15). Thus DAG released by NE may stimulate Na+ entry through Na(+)-H+ exchange. NE predominantly stimulates Na(+)-K(+)-adenosinetriphosphatase (ATPase) by activating a protein kinase that is insensitive to DAG and TPA and is inhibited by staurosporine but not by sphingosine. NE may also stimulate K+ efflux through a BaCl-insensitive K+ channel that is inhibited by millimolar furosemide.

  12. Atrial natriuretic peptide and cGMP activate sodium transport through PKA-dependent pathway in the urinary bladder of the Japanese tree frog.

    Science.gov (United States)

    Yamada, Toshiki; Matsuda, Kouhei; Uchiyama, Minoru

    2006-03-01

    The effects of atrial natriuretic peptide (ANP) and cGMP on transepithelial ion transport were examined in the urinary bladder of the Japanese tree frog, Hyla japonica, using Ussing chamber voltage-clamp and whole-cell patch-clamp techniques. When the bladders were exposed to 4.4 x 10(-11) to 10(-6) M ANP or 10(-7) to 3 x 10(-4) M 8-Br-cGMP, both the transepithelial potential difference (PD) and the short-circuit current (Isc) were significantly increased in a concentration-response manner. The cGMP-dependent responses were inhibited in a Na+-free bath solution and in the presence of amiloride. The cGMP-dependent increases in Isc were significantly inhibited by specific PKA inhibitors (5 x 10(-7) M KT-5720 and >10(-5) M H-89), but not by a specific PKG inhibitor (5 x 10(-7) M KT-5823). ANP-dependent increases in Isc were also significantly inhibited by KT-5720. In the patch-clamp study, ANP and cGMP significantly increased in inward currents involving Na+ uptake. These results suggest that a cross-talk mechanism exists between cAMP and cGMP signaling pathways, which leads to Na+ transport in the frog urinary bladder. In addition, the cGMP-dependent increases in Isc were partially inhibited by 10(-4) M l-cis-diltiazem, a specific inhibitor of cyclic nucleotide-gated (CNG) channels. These results also suggest a relation between CNG channels and the cGMP-dependent increases in Na+ absorption of the frog urinary bladder.

  13. Effect of ventilation pressure on alveolar fluid clearance and beta-agonist responses in mice.

    Science.gov (United States)

    Yu, Erin N Z; Traylor, Zachary P; Davis, Ian C

    2009-10-01

    High tidal volume ventilation is detrimental to alveolar fluid clearance (AFC), but effects of ventilation pressure (P) on AFC are unknown. In anesthetized BALB/c mice ventilated at constant tidal volume (8 ml/kg), mean AFC rate was 12.8% at 6 cmH(2)O P, but increased to 37.3% at 18 cmH(2)O P. AFC rate declined at 22 cmH(2)O P, which also induced lung damage. Increased AFC at 18 cmH(2)O P did not result from elevated plasma catecholamines, hypercapnia, or hypocapnia, but was due to augmented Na(+) and Cl(-) absorption. PKA agonists and beta-agonists stimulated AFC at 10 cmH(2)O P by upregulating amiloride-sensitive Na(+) transport. However, at 18 cmH(2)O P, PKA agonists and beta-agonists reduced AFC. At 15 cmH(2)O P, the AFC rate was intermediate (mean 26.6%), and forskolin and beta-agonists had no effect. Comparable P dependency of AFC and beta-agonist responsiveness was found in C57BL/6 mice. The effect on AFC of increasing P to 18 cmH(2)O was blocked by adenosine deaminase or an A(2b)-adenosine receptor antagonist, and could be mimicked by adenosine in mice ventilated at 10 cmH(2)O P. Modulation of adenosine signaling also resulted in altered responsiveness to beta-agonists. These findings indicate that, in the normal mouse lung, basal AFC rates and responses to beta-agonists are impacted by ventilation pressure in an adenosine-dependent manner.

  14. Scanning ion-selective electrode technique and X-ray microanalysis provide direct evidence of contrasting Na+ transport ability from root to shoot in salt-sensitive cucumber and salt-tolerant pumpkin under NaCl stress.

    Science.gov (United States)

    Lei, Bo; Huang, Yuan; Sun, Jingyu; Xie, Junjun; Niu, Mengliang; Liu, Zhixiong; Fan, Molin; Bie, Zhilong

    2014-12-01

    Grafting onto salt-tolerant pumpkin rootstock can increase cucumber salt tolerance. Previous studies have suggested that this can be attributed to pumpkin roots with higher capacity to limit the transport of Na(+) to the shoot than cucumber roots. However, the mechanism remains unclear. This study investigated the transport of Na(+) in salt-tolerant pumpkin and salt-sensitive cucumber plants under high (200 mM) or moderate (90 mM) NaCl stress. Scanning ion-selective electrode technique showed that pumpkin roots exhibited a higher capacity to extrude Na(+), and a correspondingly increased H(+) influx under 200 or 90 mM NaCl stress. The 200 mM NaCl induced Na(+)/H(+) exchange in the root was inhibited by amiloride (a Na(+)/H(+) antiporter inhibitor) or vanadate [a plasma membrane (PM) H(+) -ATPase inhibitor], indicating that Na(+) exclusion in salt stressed pumpkin and cucumber roots was the result of an active Na(+)/H(+) antiporter across the PM, and the Na(+)/H(+) antiporter system in salt stressed pumpkin roots was sufficient to exclude Na(+) X-ray microanalysis showed higher Na(+) in the cortex, but lower Na(+) in the stele of pumpkin roots than that in cucumber roots under 90 mM NaCl stress, suggesting that the highly vacuolated root cortical cells of pumpkin roots could sequester more Na(+), limit the radial transport of Na(+) to the stele and thus restrict the transport of Na(+) to the shoot. These results provide direct evidence for pumpkin roots with higher capacity to limit the transport of Na(+) to the shoot than cucumber roots. © 2014 Scandinavian Plant Physiology Society.

  15. Induced thyme product prevents VEGF-induced migration in human umbilical vein endothelial cells.

    Science.gov (United States)

    Krill, Diane; Madden, John; Huncik, Kevin; Moeller, Peter D

    2010-12-17

    Compounds with anti-angiogenic properties are useful in combating cancer by preventing new blood vessel formation to support the tumor. In this report we introduce a rapid method for screening potential anti-angiogenic compounds in a model system that stimulates the production of secondary defense chemicals in plants. This methodology identified an inducible vascular factor (IVF3), which was found to be inhibitory in all of the model systems tested. Thyme plants were exposed to highly vascular mint plants and the methanol extracts were analyzed by reverse phase HPLC. The thyme compounds induced by the invading mint tissue, and not present in the thyme plants grown alone, were tested in a vertical plate assay measuring root length as a quantitative assay for drug sensitivity. The HPLC-purified extract, referred to as IVF3, reduced the growth of root vascular tissue compared to the control and vehicle control, and 50% as well as known angiogenesis inhibitors, VEGF receptor tyrosine kinase inhibitor and amiloride hydrochloride. Extracted compounds that were effective inhibitors of plant roots were assayed in Madin Darby canine kidney epithelial cells (MDCK) for toxicity, and in human umbilical vein endothelial cells (HUVEC) for their effect on migration. IVF3 was effective at limiting HUVEC migration in VEGF-stimulated cultures. In vivo video capture of intersegmental vessel circulation between 48 and 72 h post fertilization in the developing vasculature of zebrafish embryos showed IVF3 also significantly reduced ISV functional circulation. This report demonstrates the anti-angiogenic effects of IVF3 extract in endothelial cells and in an intact vertebrate model for angiogenesis. Copyright © 2010 Elsevier Inc. All rights reserved.

  16. Extracellular acidosis activates ASIC-like channels in freshly isolated cerebral artery smooth muscle cells.

    Science.gov (United States)

    Chung, Wen-Shuo; Farley, Jerry M; Swenson, Alyssa; Barnard, John M; Hamilton, Gina; Chiposi, Rumbidzayi; Drummond, Heather A

    2010-05-01

    Recent studies suggest that certain acid-sensing ion channels (ASIC) are expressed in vascular smooth muscle cells (VSMCs) and are required for VSMC functions. However, electrophysiological evidence of ASIC channels in VSMCs is lacking. The purpose of this study was to test the hypothesis that isolated cerebral artery VSMCs express ASIC-like channels. To address this hypothesis, we used RT-PCR, Western blotting, immunolabeling, and conventional whole cell patch-clamp technique. We found extracellular H(+)-induced inward currents in 46% of cells tested (n = 58 of 126 VSMCs, pH 6.5-5.0). The percentage of responsive cells and the current amplitude increased as the external H(+) concentration increased (pH(6.0), n = 28/65 VSMCs responsive, mean current density = 8.1 +/- 1.2 pA/pF). Extracellular acidosis (pH(6.0)) shifted the whole cell reversal potential toward the Nernst potential of Na(+) (n = 6) and substitution of extracellular Na(+) by N-methyl-d-glucamine abolished the inward current (n = 6), indicating that Na(+) is a major charge carrier. The broad-spectrum ASIC blocker amiloride (20 microM) inhibited proton-induced currents to 16.5 +/- 8.7% of control (n = 6, pH(6.0)). Psalmotoxin 1 (PcTx1), an ASIC1a inhibitor and ASIC1b activator, had mixed effects: PcTx1 either 1) abolished H(+)-induced currents (11% of VSMCs, 5/45), 2) enhanced or promoted activation of H(+)-induced currents (76%, 34/45), or 3) failed to promote H(+) activation in nonresponsive VSMCs (13%, 6/45). These findings suggest that freshly dissociated cerebral artery VSMCs express ASIC-like channels, which are predominantly formed by ASIC1b.

  17. Acid and stretch, but not capsaicin, are effective stimuli for ATP release in the porcine bladder mucosa: Are ASIC and TRPV1 receptors involved?

    Science.gov (United States)

    Sadananda, Prajni; Kao, Felicity C L; Liu, Lu; Mansfield, Kylie J; Burcher, Elizabeth

    2012-05-15

    Stretch-evoked ATP release from the bladder mucosa is a key event in signaling bladder fullness. Our aim was to examine whether acid and capsaicin can also release ATP and to determine the receptors involved, using agonists and antagonists at TRPV1 and acid-sensing ion channels (ASICs). Strips of porcine bladder mucosa were exposed to acid, capsaicin or stretch. Strip tension was monitored. Bath fluid was collected for ATP measurement. Gene expression of ASICs and TRPV1 in porcine bladders was quantified using quantitative real-time PCR (qRT-PCR). Stretch stimulus (150% of original length) repeatedly and significantly increased ATP release to approximately 45 times basal release. Acid (pH 6.5, 6.0, 5.6) contracted mucosal strips and also increased ATP release up to 30-fold, without evidence of desensitization. Amiloride (0.3 μM) reduced the acid-evoked ATP release by approximately 70%, while capsazepine (10 μM) reduced acid-evoked ATP release at pH 6.0 and pH 5.6 (by 68% and 61%, respectively). Capsaicin (0.1-10 μM) was ineffective in causing ATP release, and also failed to contract porcine mucosal or detrusor strips. Gene expression for ASIC1, ASIC2, ASIC3 and TRPV1 was seen in the lateral wall, dome, trigone and neck of both detrusor and mucosa. In conclusion, stretch and acid induce ATP release in the porcine bladder mucosa, but capsaicin is ineffective. The pig bladder is a well-known model for the human bladder, however these data suggest that it should be used with caution, particularly for TRPV1 related studies. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Modulation of ASIC channels in rat cerebellar purkinje neurons by ischaemia-related signals

    Science.gov (United States)

    Allen, Nicola J; Attwell, David

    2002-01-01

    Acid-sensing ion channels (ASICs), activated by a decrease of extracellular pH, are found in neurons throughout the nervous system. They have an amino acid sequence similar to that of ion channels activated by membrane stretch, and have been implicated in touch sensation. Here we characterize the pH-dependent activation of ASICs in cerebellar Purkinje cells and investigate how they are modulated by factors released in ischaemia. Lowering the external pH from 7.4 activated an inward current at −66 mV, carried largely by Na+ ions, which was half-maximal for a step to pH 6.4 and was blocked by amiloride and gadolinium. The H+-gated current desensitized within a few seconds, but approximately 30% of cells showed a sustained inward current (11% of the peak current) in response to the maintained presence of pH 6 solution. The peak H+-evoked current was potentiated by membrane stretch (which occurs in ischaemia when [K+]o rises) and by arachidonic acid (which is released when [Ca2+]i rises in ischaemia). Arachidonic acid increased to 77% the fraction of cells showing a sustained current evoked by acid pH. The ASIC currents were also potentiated by lactate (which is released when metabolism becomes anaerobic in ischaemia) and by FMRFamide (which may mimic the action of related mammalian RFamide transmitters). These data reinforce suggestions of a mechanosensory aspect to ASIC channel function, and show that the activation of ASICs reflects the integration of multiple signals which are present during ischaemia. PMID:12205186

  19. Neomycin damage and regeneration of hair cells in both mechanoreceptor and electroreceptor lateral line organs of the larval Siberian sturgeon (Acipenser baerii).

    Science.gov (United States)

    Fan, Chunxin; Zou, Sha; Wang, Jian; Zhang, Bo; Song, Jiakun

    2016-05-01

    The lateral line found in some amphibians and fishes has two distinctive classes of sensory organs: mechanoreceptors (neuromasts) and electroreceptors (ampullary organs). Hair cells in neuromasts can be damaged by aminoglycoside antibiotics and they will regenerate rapidly afterward. Aminoglycoside sensitivity and the capacity for regeneration have not been investigated in ampullary organs. We treated Siberian sturgeon (Acipenser baerii) larvae with neomycin and observed loss and regeneration of sensory hair cells in both organs by labeling with DASPEI and scanning electron microscopy (SEM). The numbers of sensory hair cells in both organs were reduced to the lowest levels at 6 hours posttreatment (hpt). New sensory hair cells began to appear at 12 hpt and were regenerated completely in 7 days. To reveal the possible mechanism for ampullary hair cell regeneration, we analyzed cell proliferation and the expression of neural placodal gene eya1 during regeneration. Both cell proliferation and eya1 expression were concentrated in peripheral mantle cells and both increased to the highest level at 12 hpt, which is consistent with the time course for regeneration of the ampullary hair cells. Furthermore, we used Texas Red-conjugated gentamicin in an uptake assay following pretreatment with a cation channel blocker (amiloride) and found that entry of the antibiotic was suppressed in both organs. Together, our results indicate that ampullary hair cells in Siberian sturgeon larvae can be damaged by neomycin exposure and they can regenerate rapidly. We suggest that the mechanisms for aminoglycoside uptake and hair cell regeneration are conserved for mechanoreceptors and electroreceptors. J. Comp. Neurol. 524:1443-1456, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  20. Successful protocol of anaesthesia for measuring transepithelial nasal potential difference in spontaneously breathing mice.

    Science.gov (United States)

    Leal, T; Lebacq, J; Vanbinst, R; Lederman, Ch; De Kock, M; Wallemacq, P

    2006-01-01

    Numerous difficulties arise during in vivo measurements of transepithelial nasal potential difference (PD) in mice, such as inadequate duration and depth of anaesthesia, bronchoaspiration of solutions perfused in the nose, and respiratory and/or cardiovascular depression. Anaesthesia was induced in adult C57 mice with intraperitoneal injection of a combination of fentanyl, droperidol and medetomidine, each of these at either a small dose (0.20, 10 and 0.33 mg/kg, respectively) or at a large dose (0.40, 20 and 0.40 mg/kg, respectively), combined with a fixed dose of 0.375 microg clonidine. In order to establish a pharmacokinetic-pharmacodynamic relationship, blood concentrations of the first three drugs were measured in 24 animals by liquid-chromatography tandem mass spectrometry. At the end of the experiment, naloxone, a competitive morphinic antagonist, and atipamezole, an alpha-2 adrenergic antagonist, were administered. Bronchoaspiration was prevented by tilting the animal head downwards and by absorbing the excess fluid from the opposite nostril and from the oral cavity. Optimal assessment of anaesthesia associated with regular respiration, loss of blink, pupillary and pedal withdrawal reflexes was obtained with doses of fentanyl, droperidol and medetomidine corresponding to 0.20, 20 and 0.40 mg/kg, respectively. Blood concentrations of fentanyl around 17 ng/mL induced loss of respiratory efforts and were followed by death during the experiment. Integrity of ion transport was demonstrated under continuous perfusion by successive depolarization after amiloride and repolarization after chloride-free solution. The combination investigated in this study lead to adequate surgical anaesthesia (stage III, plane 2) for prolonged nasal PD measurements in spontaneously breathing mice.

  1. Increase vs. decrease of calcium uptake by isolated heart cells induced by H2O2 vs. HOCl

    International Nuclear Information System (INIS)

    Kaminishi, T.; Matsuoka, T.; Yanagishita, T.; Kako, K.J.

    1989-01-01

    Adult rat heart myocytes were labeled rapidly with exogenous [45Ca2+]. Addition of 2.5 mM H2O2 to the heart cell suspension raised the content of rapidly exchangeable intracellular Ca2+ twofold, whereas addition of 1-30 mM HOCl decreased the Ca2+ content. The H2O2-induced increase in Ca2+ content was dependent on the medium Na+, pH, and temperature but was not significantly affected by addition of verapamil, diltiazem, amiloride, or 3-aminobenzamide. The [3H]ouabain binding to myocytes was suppressed by H2O2, whereas the Ca2+ efflux from myocytes was not influenced. An uncoupler, carbonyl cyanide m-chlorophenylhydrazone, reduced Ca2+ content, implying that the H2O2-induced change in Ca2+ content was not directly related to ATP depletion. On the other hand, the H2O2-induced Ca2+ accumulation in myocytes was prevented by deferoxamine or o-phenanthroline. These results suggest that H2O2 inhibited Na+-K+-ATPase, resulting in an increase in intracellular Na+ concentration and stimulation of sarcolemmal Na+-Ca2+ exchange activity, which caused a transient net Ca2+ influx into myocytes. By contrast, HOCl decreased the Ca2+ content of the rapidly exchangeable pool below control levels and this action of HOCl was antagonized by 1,4-dithiothreitol. HOCl accelerated Ca2+ efflux from myocytes. Ca2+ uptake and Ca2+-ATPase of the isolated sarcoplasmic reticular (SR) fraction were highly sensitive to the action of HOCl. Ca2+ uptake by intracellular sites, studied with myocytes permeabilized with digitonin, was inhibited by both H2O2 and HOCl. Thus these results suggest that HOCl inhibits the SR Ca2+ pump, resulting in the observed acceleration of Ca2+ efflux from and decline in Ca2+ content of myocytes

  2. Elevated levels of plasminogen activators in the pathogenesis of delayed radiation damage in rat cervical spinal cord in vivo

    International Nuclear Information System (INIS)

    Sawaya, R.; Rayford, A.; Kono, S.; Rao, J.S.; Ang, K.K.; Feng, Y.; Stephens, L.C.

    1994-01-01

    The pathophysiology of the cellular basis of radiation-induced demyelination and white-matter necrosis of the central nervous system (CNS) is poorly understood. Preliminary data suggest that tissue damage is partly mediated through changes in the proteolytic enzymes. In this study, we irradiated rat cervical spinal cords with single doses of 24 Gy of 18 MV photons or 20 MeV electrons and measured the levels of plasminogen activators at days 2, 7, 30, 60, 90, 120, 130 and 145 after irradiation, using appropriate controls at each time. Fibrin zymography revealed fibrinolytic bands representing molecular weights of 68,000 and 48,000 in controls and irradiated samples; these bands increased significantly at days 120, 130 and 145 after irradiation. Inhibition of these enzymatic bands with specific antibodies against tissue-type plasminogen activator (tPA) and amiloride, an inhibitor for urokinase plasminogen activator (uPA), confirmed that these bands were tPA and uPA. Enzymatic levels quantified by densitometry showed a twofold elevation in the levels of tPA and more than a tenfold increase in uPA after 120 days' irradiation. Activity of uPA was increased threefold by day 2 and increased steadily with time compared to nonirradiated control samples. Enzyme-linked immunosorbent assay (ELISA) also showed a threefold increase in the tPA content in the extracts of irradiated rat cervical spinal cords at days 120, 130 and 145. This study adds additional information to the proposed role of plasminogen activators in the pathogenic pathways of radiation damage in the CNS. 38 refs., 6 figs

  3. Elevated levels of plasminogen activators in the pathogenesis of delayed radiation damage in rat cervical spinal cord in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Sawaya, R.; Rayford, A.; Kono, S.; Rao, J.S.; Ang, K.K.; Feng, Y.; Stephens, L.C. [Univ. of Texas, Houston, TX (United States)

    1994-06-01

    The pathophysiology of the cellular basis of radiation-induced demyelination and white-matter necrosis of the central nervous system (CNS) is poorly understood. Preliminary data suggest that tissue damage is partly mediated through changes in the proteolytic enzymes. In this study, we irradiated rat cervical spinal cords with single doses of 24 Gy of 18 MV photons or 20 MeV electrons and measured the levels of plasminogen activators at days 2, 7, 30, 60, 90, 120, 130 and 145 after irradiation, using appropriate controls at each time. Fibrin zymography revealed fibrinolytic bands representing molecular weights of 68,000 and 48,000 in controls and irradiated samples; these bands increased significantly at days 120, 130 and 145 after irradiation. Inhibition of these enzymatic bands with specific antibodies against tissue-type plasminogen activator (tPA) and amiloride, an inhibitor for urokinase plasminogen activator (uPA), confirmed that these bands were tPA and uPA. Enzymatic levels quantified by densitometry showed a twofold elevation in the levels of tPA and more than a tenfold increase in uPA after 120 days` irradiation. Activity of uPA was increased threefold by day 2 and increased steadily with time compared to nonirradiated control samples. Enzyme-linked immunosorbent assay (ELISA) also showed a threefold increase in the tPA content in the extracts of irradiated rat cervical spinal cords at days 120, 130 and 145. This study adds additional information to the proposed role of plasminogen activators in the pathogenic pathways of radiation damage in the CNS. 38 refs., 6 figs.

  4. Nebulizer performance: AFLM study. Association Française de Lutte contre la Mucoviscidose.

    Science.gov (United States)

    Faurisson, F; Dessanges, J F; Grimfeld, A; Beaulieu, R; Kitzis, M D; Peytavin, G; Lefebvre, J P; Farinotti, R; Sautegeau, A

    1995-01-01

    This study was conducted by the AFLM order to determine the performance characteristics of 12 commercially available nebulizers (6 ultrasonic and 6 jet) used in the treatment of cystic fibrosis (CF). The nebulizers were connected to a circuit which simulated the ventilation of a CF child and CF adult, and were tested using three drug solutions: tobramycin (T), colistin (C), and amiloride (A). Nebulizer performance was evaluated according to the volume of drug solution delivered in 10 min during the simulated inspiratory phase (VI), drug granulometry (G%), drug concentration modification in the nebulizer reservoir (delta C), and percentage of efficiently aerosolized drug EA%). The ultrasonic devices delivered a significantly higher VI than the jet nebulizers (p < 0.0001) for all three study drug. Ventilation rate did not influence VI. Regarding granulometry, higher percentages of T and A were found to be contained in droplets ranging from 0.5 to 5.0 micron following ultrasonic nebulization. Drug concentration modifications were independent of the nebulizer used but were influenced by drug type; overconcentrations of T and A were observed (delta C = +10.5 +/- 18.6 and +13.4 +/- 8.9%, respectively). On average, the ultrasonic devices achieved a higher EA% than the jet nebulizers (17.3 +/- 6.7 and 9.7 +/- 9.6%, respectively). This study highlights the significant variability in performance of different nebulizer types and empahsizes the importance of accurately testing nebulizers prior to clinical use so that the most efficacious nebulizer/drug combinations may be prescribed.

  5. Role of intracellular calcium in cellular volume regulation

    International Nuclear Information System (INIS)

    Wong, S.M.; Chase, H.S. Jr.

    1986-01-01

    We investigated the role of intracellular calcium in epithelial cell volume regulation using cells isolated from the toad urinary bladder. A suspension of cells was prepared by treatment of the bladder with collagenase followed by ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid. The cells retained their ion-transporting capabilities: ouabain (1 mM) and amiloride (10 microM) inhibited cellular uptake of 86 Rb and 22 Na, respectively. Using a Coulter counter to measure cellular volume, we found that we could swell cells either by reducing the extracellular osmolality or by adding the permeant solute urea (45 mM) isosmotically. Under both conditions, cells first swelled and then returned to their base-line volume, in spite of the continued presence of the stimulus to swell. Volume regulation was inhibited when cells were swelled at low extracellular [Ca] (100 nM) and was retarded in cells preloaded with the calcium buffer quin 2. Swelling increased the intracellular free calcium concentration ([Ca]i), as measured by quin 2 fluorescence: [Ca]i increased 35 +/- 9 nM (n = 6) after hypotonic swelling and 42 +/- 3 nM (n = 3) after urea swelling. Reducing extracellular [Ca] to less than 100 nM prevented the swelling-induced increase in [Ca]i, suggesting that the source of the increase in [Ca]i was extracellular. This result was confirmed in measurements of cellular uptake of 45Ca: the rate of uptake was significantly higher in swollen cells compared with control (1.1 +/- 0.2 vs. 0.4 +/- 0.1 fmol . cell-1 X 5 min-1). Our experiments provide the first demonstration that cellular swelling increases [Ca]i. This increase is likely to play a critical role in cellular volume regulation

  6. EBIO, an agent causing maintained epithelial chloride secretion by co-ordinate actions at both apical and basolateral membranes.

    Science.gov (United States)

    MacVinish, L J; Keogh, J; Cuthbert, A W

    2001-01-01

    The effect of 1-ethyl-2-benzimidazolone (EBIO) on electrogenic chloride secretion in murine colonic and nasal epithelium was investigated by the short-circuit technique. In the colon, EBIO produces a sustained current increase in the presence of amiloride, which is sensitive to furosemide. In nasal epithelium EBIO causes only a small, transient current increase. Sustained increases in current were obtained in response to forskolin in both epithelia. To examine the mechanisms by which EBIO increases chloride secretion, the effects on intracellular mediators were measured in colonic crypts. There was no effect on [Ca(2+)]i but cAMP content was increased, more so in the presence of IBMX, indicating a direct effect on adenylate cyclase. In colonic epithelia in which the apical surface was permeabilized by nystatin, and the tissue subjected to an apical to basolateral K(+) gradient, EBIO caused a current increase that was entirely sensitive to charybdotoxin (ChTX). In similarly permeabilized colons Br-cAMP caused a current increase that was entirely sensitive to 293B. Thus EBIO increases chloride secretion in the colon by coordinated actions at both the apical and basolateral faces of the cells. These include direct and indirect actions on Ca(2+)-sensitive and cAMP-sensitive K(+) channels respectively, and indirect actions on the basolateral cotransporter and apical CFTR chloride channels via cAMP. In CF colonic epithelia EBIO did not evoke chloride secretion. It is not clear why the nasal epithelium responds poorly to EBIO whereas it gives a sustained response to the related compound chlorzoxazone.

  7. Subcellular localization and displacement by diuretics of the peripheral benzodiazepine binding site (PBS) from rat kidney

    Energy Technology Data Exchange (ETDEWEB)

    Lukeman, S.; Fanestil, D.

    1986-03-05

    Although the PBS has been identified in many organs, its function and cellular location are speculative. Using rapid filtration, binding of (/sup 3/H)RO 5-4864 (*RO) (.75 nM) was assessed in four subcellular fractions (.3 mg/ml) derived from depapillated rat kidney by differential centrifugation: N (450g x 2 min), O (13,000 x 10), P (105,000 x 30), and S. The binding distribution was: N-18%, O-74%, P-6%, and S-2%. Marker enzyme analysis revealed that O was enriched in mitochondria (M), lysosomes (L), peroxisomes (P), and endoplasmic reticulum (ER), but not plasma membrane, and that N contained small amounts (10-15%) of markers for the above. Repeated washing of O removed ER enzymes but preserved *RO binding. O was further fractionated with centrifugation (57,000g x 4 hr) on a linear sucrose gradient (18-65%); *RO binding then comigrated with M but not P and L markers. Centrifugation of isolated M (5500 x 10 min) on another linear sucrose gradient (37-65%) gave low and high density bands, which contained 65% and 35% of *RO binding activity, resp. *RO binding in O was specific, saturable, reversible, and inhibited by diuretics. Inhibitors with the highest potency were indacrinone (K/sub d/ = 35 ..mu..M), hydrochlorothiazide (100 ..mu..M), and ethacrynic acid (325 ..mu..M). Low potency inhibitors (K/sub d/ greater than or equal to 1 mM) included amiloride, triamterene, furosemide, bumetanide, and ozolinone.

  8. Effects of thyroid hormone on Na+-K+ transport in resting and stimulated rat skeletal muscle

    International Nuclear Information System (INIS)

    Everts, M.E.; Clausen, T.

    1988-01-01

    The effects of hypothyroidism and 3,5,3'-triiodothyronine (T 3 ) treatment on passive Na + -K + fluxes and Na + -K + pump concentration were investigated in isolated rat muscle. Within 12 h after a single dose of T 3 (20 μg/100 g body wt), K + efflux had increased by 21% in soleus and by 20% in extensor digitorum longus muscle. In the presence of ouabain, even larger effects were observed. These changes were associated with a 12% rise in amiloride-suppressible Na + influx but no significant increase in [ 3 H]ouabain binding site concentration. After 3 days of T 3 treatment, the stimulating effect on K + efflux and Na + influx in soleus reached a plateau ∼80 and 40% above control levels, respectively, whereas the maximum increase in [ 3 H]ouabain binding site concentration (103%) was only fully developed after 8 days. Hypothyroidism decreased 86 Rb efflux by 30%. The efflux of K + and the influx of Na + per contraction (both ∼7 nmol/g wet wt) as well as the net loss of K + induced by electrical stimulation were unaffected by T 3 treatment. The rise in resting K + efflux after 12-24 h of T 3 treatment could be partly blocked by dantrolene or trifluoroperazine, indicating that an increase in the cytoplasmic Ca 2+ concentration may contribute to the early rise in K + efflux. It is concluded that the early rise in the resting passive leaks of Na + and K + induced by T 3 is a major driving force for Na + -K + pump synthesis

  9. Lipopolysaccharide hyperpolarizes guinea pig airway epithelium by increasing the activities of the epithelial Na(+) channel and the Na(+)-K(+) pump.

    Science.gov (United States)

    Dodrill, Michael W; Fedan, Jeffrey S

    2010-10-01

    Earlier, we found that systemic administration of lipopolysaccharide (LPS; 4 mg/kg) hyperpolarized the transepithelial potential difference (V(t)) of tracheal epithelium in the isolated, perfused trachea (IPT) of the guinea pig 18 h after injection. As well, LPS increased the hyperpolarization component of the response to basolateral methacholine, and potentiated the epithelium-derived relaxing factor-mediated relaxation responses to hyperosmolar solutions applied to the apical membrane. We hypothesized that LPS stimulates the transepithelial movement of Na(+) via the epithelial sodium channel (ENaC)/Na(+)-K(+) pump axis, leading to hyperpolarization of V(t). LPS increased the V(t)-depolarizing response to amiloride (10 μM), i.e., offset the effect of LPS, indicating that Na(+) transport activity was increased. The functional activity of ENaC was measured in the IPT after short-circuiting the Na(+)-K(+) pump with basolateral amphotericin B (7.5 μM). LPS had no effect on the hyperpolarization response to apical trypsin (100 U/ml) in the Ussing chamber, indicating that channel-activating proteases are not involved in the LPS-induced activation of ENaC. To assess Na(+)-K(+) pump activity in the IPT, ENaC was short-circuited with apical amphotericin B. The greater V(t) in the presence of amphotericin B in tracheas from LPS-treated animals compared with controls revealed that LPS increased Na(+)-K(+) pump activity. This finding was confirmed in the Ussing chamber by inhibiting the Na(+)-K(+) pump via extracellular K(+) removal, loading the epithelium with Na(+), and observing a greater hyperpolarization response to K(+) restoration. Together, the findings of this study reveal that LPS hyperpolarizes the airway epithelium by increasing the activities of ENaC and the Na(+)-K(+) pump.

  10. Characterization of thyroid hormone effects on Na-K pump and membrane potential of cultured rat skeletal myotubes

    International Nuclear Information System (INIS)

    Brodie, C.; Sampson, S.R.

    1988-01-01

    The purpose of this study was to characterize the effects of thyroid hormone on the Na-K pump and resting membrane potential (EM) of rat skeletal myotubes in culture. Myotubes were obtained from fetal (19-21 day) or neonatal rats (1-2 day) by serial trypsinization and maintained in culture for up to 10 days. Cells were treated with T4 or T3 on day 6 or 7, and measurements were made of EM, [ 3 H]ouabain binding, and ouabain-sensitive 86 Rb uptake at various times thereafter. Hormone treatment increased the values of all three variables within 24 h, plateau levels being attained by 48-72 h. Cycloheximide and actinomycin D totally blocked the effects of thyroid hormone when added together to the cells, thus suggesting that protein synthesis is necessary for the effects of these hormones. Scatchard analysis showed that the new receptors have lower ouabain affinity than those in control. Blockade of spontaneously occurring action potentials with tetrodotoxin, which blocks voltage-dependent Na channels, or Na/H antiporter with amiloride, abolished the hormone effects seen after 24 h and significantly reduced those obtained after 48 h of hormone treatment. The results demonstrate that thyroid hormone-induced increased amount and activity of the electrogenic Na-K pump in cultured myotubes occurs, at least in part, in response to an initial effect to increase Na influx. Moreover, the findings are consistent with the concept that the Na-K pump plays an important role in regulation of EM in this preparation

  11. Bumetanide increases Cl--dependent short-circuit current in late distal colon: Evidence for the presence of active electrogenic Cl- absorption.

    Science.gov (United States)

    Tang, Lieqi; Fang, Xiefan; Winesett, Steven P; Cheng, Catherine Y; Binder, Henry J; Rivkees, Scott A; Cheng, Sam X

    2017-01-01

    Mammalian colonic epithelia consist of cells that are capable of both absorbing and secreting Cl-. The present studies employing Ussing chamber technique identified two opposing short-circuit current (Isc) responses to basolateral bumetanide in rat distal colon. Apart from the transepithelial Cl--secretory Isc in early distal colon that was inhibited by bumetanide, bumetanide also stimulated Isc in late distal colon that had not previously been identified. Since bumetanide inhibits basolateral Na+-K+-2Cl- cotransporter (NKCC) in crypt cells and basolateral K+-Cl- cotransporter (KCC) in surface epithelium, we proposed this stimulatory Isc could represent a KCC-mediated Cl- absorptive current. In support of this hypothesis, ion substitution experiments established Cl- dependency of this absorptive Isc and transport inhibitor studies demonstrated the involvement of an apical Cl- conductance. Current distribution and RNA sequencing analyses revealed that this Cl- absorptive Isc is closely associated with epithelial Na+ channel (ENaC) but is not dependent on ENaC activity. Thus, inhibition of ENaC by 10 μM amiloride or benzamil neither altered the direction nor its activity. Physiological studies suggested that this Cl- absorptive Isc senses dietary Cl- content; thus when dietary Cl- was low, Cl- absorptive Isc was up-regulated. In contrast, when dietary Cl- was increased, Cl- absorptive Isc was down-regulated. We conclude that an active Cl- extrusion mechanism exists in ENaC-expressing late distal colon and likely operates in parallel with ENaC to facilitate NaCl absorption.

  12. Sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured rat caput epididymal epithelium.

    Science.gov (United States)

    Zuo, Wu-Lin; Li, Sheng; Huang, Jie-Hong; Yang, Deng-Liang; Zhang, Geng; Chen, Si-Liang; Ruan, Ye-Chun; Ye, Ke-Nan; Cheng, Christopher H K; Zhou, Wen-Liang

    2011-01-01

    The epithelium lining the epididymis provides an optimal acidic fluid microenvironment in the epididymal tract that enable spermatozoa to complete the maturation process. The present study aims to investigate the functional role of Na(+)/HCO(3)(-) cotransporter in the pH regulation in rat epididymis. Immunofluorescence staining of pan cytokeratin in the primary culture of rat caput epididymal epithelium showed that the system was a suitable model for investigating the function of epididymal epithelium. Intracellular and apical pH were measured using the fluorescent pH sensitive probe carboxy-seminaphthorhodafluor-4F acetoxymethyl ester (SNARF-4F) and sparklet pH electrode respectively to explore the functional role of rat epididymal epithelium. In the HEPES buffered Krebs-Henseleit (KH) solution, the intracellular pH (pHi) recovery from NH(4)Cl induced acidification in the cultured caput epididymal epithelium was completely inhibited by amiloride, the inhibitor of Na(+)/H(+) exchanger (NHE). Immediately changing of the KH solution from HEPES buffered to HCO(3)(-) buffered would cause another pHi recovery. The pHi recovery in HCO(3)(-) buffered KH solution was inhibited by 4, 4diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), the inhibitor of HCO(3)(-) transporter or by removal of extracellular Na(+). The extracellular pH measurement showed that the apical pH would increase when adding DIDS to the apical side of epididymal epithelial monolayer, however adding DIDS to the basolateral side had no effect on apical pH. The present study shows that sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured caput epididymal epithelium.

  13. High salt loading induces urinary storage dysfunction via upregulation of epithelial sodium channel alpha in the bladder epithelium in Dahl salt-sensitive rats.

    Science.gov (United States)

    Yamamoto, Seiji; Hotta, Yuji; Maeda, Kotomi; Kataoka, Tomoya; Maeda, Yasuhiro; Hamakawa, Takashi; Shibata, Yasuhiro; Sasaki, Shoichi; Ugawa, Shinya; Yasui, Takahiro; Kimura, Kazunori

    2017-11-01

    We aimed to investigate whether high salt intake affects bladder function via epithelial sodium channel (ENaC) by using Dahl salt-resistant (DR) and salt-sensitive (DS) rats. Bladder weight of DR + high-salt diet (HS, 8% NaCl) and DS + HS groups were significantly higher than those of DR + normal-salt diet (NS, 0.3% NaCl) and DS + NS groups after one week treatment. We thereafter used only DR + HS and DS + HS group. Systolic and diastolic blood pressures were significantly higher in DS + HS group than in DR + HS group after the treatment period. Cystometrogram showed the intercontraction intervals (ICI) were significantly shorter in DS + HS group than in DR + HS group during infusion of saline. Subsequent infusion of amiloride significantly prolonged ICI in DS + HS group, while no intra-group difference in ICI was observed in DR + HS group. No intra- or inter-group differences in maximum intravesical pressure were observed. Protein expression levels of ENaCα in the bladder were significantly higher in DS + HS group than in DR + HS group. ENaCα protein was localized at bladder epithelium in both groups. In conclusion, high salt intake is considered to cause urinary storage dysfunction via upregulation of ENaC in the bladder epithelium with salt-sensitive hypertension, suggesting that ENaC might be a candidate for therapeutic target for urinary storage dysfunction. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  14. Endocytic mechanism of internalization of dietary peptide lunasin into macrophages in inflammatory condition associated with cardiovascular disease.

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    Anthony Cam

    Full Text Available Cardiovascular disease (CVD is the leading cause of death in the United States. Diet influences risk factors associated with CVD and atherosclerosis, a major vascular disease that arises from inflammation. Lunasin, a peptide derived from plant foods such as soybeans, contains a unique Arg-Gly-Asp cell-adhesion motif and inhibits the pathways involved in the inflammatory cascade. The objective was to determine the mechanism by which lunasin is internalized into human THP-1 macrophages, investigate the expression of endocytic membrane proteins in inflammatory conditions and to identify the pathways involved. While lipopolysaccharide (10 nM, vitronectin (130 nM and a combination of these two molecules enhanced lunasin uptake and increased basal αVβ3 integrin expression, lunasin reduced αVβ3 expression by 25.5, 26.8 and 49.2%, respectively. The pretreatment of cells with brefeldin A (71 µM, an inhibitor of protein trafficking, inhibited lunasin internalization by up to 99.8%. Lunasin increased caveolin-1 expression by up to 204.8%, but did not modulate clathrin. The pretreatment of macrophages with nystatin (54 µM, an inhibitor of caveolae-dependent endocytosis, reduced lunasin internalization. The presence of amantadine (1 mM and amiloride (1 mM, inhibitors of clathrin-mediated endocytosis and macropinocytosis, abolished lunasin cell entry. Lunasin elicited a transient reduction in intracellular levels of Ca²⁺ in LPS-induced macrophages. The results suggest that internalization of lunasin into macrophages is amplified in inflammatory conditions and is primarily mediated by endocytic mechanisms that involve integrin signaling, clathrin-coated structures and macropinosomes. Lunasin may be responsible for attenuation of CVD risk factors by interacting with pathways involved in endocytosis and inflammation.

  15. Endocytic mechanism of internalization of dietary peptide lunasin into macrophages in inflammatory condition associated with cardiovascular disease.

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    Cam, Anthony; Sivaguru, Mayandi; Gonzalez de Mejia, Elvira

    2013-01-01

    Cardiovascular disease (CVD) is the leading cause of death in the United States. Diet influences risk factors associated with CVD and atherosclerosis, a major vascular disease that arises from inflammation. Lunasin, a peptide derived from plant foods such as soybeans, contains a unique Arg-Gly-Asp cell-adhesion motif and inhibits the pathways involved in the inflammatory cascade. The objective was to determine the mechanism by which lunasin is internalized into human THP-1 macrophages, investigate the expression of endocytic membrane proteins in inflammatory conditions and to identify the pathways involved. While lipopolysaccharide (10 nM), vitronectin (130 nM) and a combination of these two molecules enhanced lunasin uptake and increased basal αVβ3 integrin expression, lunasin reduced αVβ3 expression by 25.5, 26.8 and 49.2%, respectively. The pretreatment of cells with brefeldin A (71 µM), an inhibitor of protein trafficking, inhibited lunasin internalization by up to 99.8%. Lunasin increased caveolin-1 expression by up to 204.8%, but did not modulate clathrin. The pretreatment of macrophages with nystatin (54 µM), an inhibitor of caveolae-dependent endocytosis, reduced lunasin internalization. The presence of amantadine (1 mM) and amiloride (1 mM), inhibitors of clathrin-mediated endocytosis and macropinocytosis, abolished lunasin cell entry. Lunasin elicited a transient reduction in intracellular levels of Ca²⁺ in LPS-induced macrophages. The results suggest that internalization of lunasin into macrophages is amplified in inflammatory conditions and is primarily mediated by endocytic mechanisms that involve integrin signaling, clathrin-coated structures and macropinosomes. Lunasin may be responsible for attenuation of CVD risk factors by interacting with pathways involved in endocytosis and inflammation.

  16. Dissecting Bacterial Cell Wall Entry and Signaling in Eukaryotic Cells: an Actin-Dependent Pathway Parallels Platelet-Activating Factor Receptor-Mediated Endocytosis.

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    Loh, Lip Nam; Gao, Geli; Tuomanen, Elaine I

    2017-01-03

    The Gram-positive bacterial cell wall (CW) peptidoglycan-teichoic acid complex is released into the host environment during bacterial metabolism or death. It is a highly inflammatory Toll-like receptor 2 (TLR2) ligand, and previous in vivo studies have demonstrated its ability to recapitulate pathological features of pneumonia and meningitis. We report that an actin-dependent pathway is involved in the internalization of the CW by epithelial and endothelial cells, in addition to the previously described platelet-activating factor receptor (PAFr)-dependent uptake pathway. Unlike the PAFr-dependent pathway, which is mediated by clathrin and dynamin and does not lead to signaling, the alternative pathway is sensitive to 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and engenders Rac1, Cdc42, and phosphatidylinositol 3-kinase (PI3K) signaling. Upon internalization by this macropinocytosis-like pathway, CW is trafficked to lysosomes. Intracellular CW trafficking is more complex than previously recognized and suggests multiple points of interaction with and without innate immune signaling. Streptococcus pneumoniae is a major human pathogen infecting the respiratory tract and brain. It is an established model organism for understanding how infection injures the host. During infection or bacterial growth, bacteria shed their cell wall (CW) into the host environment and trigger inflammation. A previous study has shown that CW enters and crosses cell barriers by interacting with a receptor on the surfaces of host cells, termed platelet-activating factor receptor (PAFr). In the present study, by using cells that are depleted of PAFr, we identified a second pathway with features of macropinocytosis, which is a receptor-independent fluid uptake mechanism by cells. Each pathway contributes approximately the same amount of cell wall trafficking, but the PAFr pathway is silent, while the new pathway appears to contribute to the host inflammatory response to CW insult. Copyright © 2017

  17. Acid extrusion via blood-brain barrier causes brain alkalosis and seizures after neonatal asphyxia.

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    Helmy, Mohamed M; Ruusuvuori, Eva; Watkins, Paul V; Voipio, Juha; Kanold, Patrick O; Kaila, Kai

    2012-11-01

    Birth asphyxia is often associated with a high seizure burden that is predictive of poor neurodevelopmental outcome. The mechanisms underlying birth asphyxia seizures are unknown. Using an animal model of birth asphyxia based on 6-day-old rat pups, we have recently shown that the seizure burden is linked to an increase in brain extracellular pH that consists of the recovery from the asphyxia-induced acidosis, and of a subsequent plateau level well above normal extracellular pH. In the present study, two-photon imaging of intracellular pH in neocortical neurons in vivo showed that pH changes also underwent a biphasic acid-alkaline response, resulting in an alkaline plateau level. The mean alkaline overshoot was strongly suppressed by a graded restoration of normocapnia after asphyxia. The parallel post-asphyxia increase in extra- and intracellular pH levels indicated a net loss of acid equivalents from brain tissue that was not attributable to a disruption of the blood-brain barrier, as demonstrated by a lack of increased sodium fluorescein extravasation into the brain, and by the electrophysiological characteristics of the blood-brain barrier. Indeed, electrode recordings of pH in the brain and trunk demonstrated a net efflux of acid equivalents from the brain across the blood-brain barrier, which was abolished by the Na/H exchange inhibitor, N-methyl-isobutyl amiloride. Pharmacological inhibition of Na/H exchange also suppressed the seizure activity associated with the brain-specific alkalosis. Our findings show that the post-asphyxia seizures are attributable to an enhanced Na/H exchange-dependent net extrusion of acid equivalents across the blood-brain barrier and to consequent brain alkalosis. These results suggest targeting of blood-brain barrier-mediated pH regulation as a novel approach in the prevention and therapy of neonatal seizures.

  18. Acid sensing ion channel 1 in lateral hypothalamus contributes to breathing control.

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    Nana Song

    Full Text Available Acid-sensing ion channels (ASICs are present in neurons and may contribute to chemoreception. Among six subunits of ASICs, ASIC1 is mainly expressed in the central nervous system. Recently, multiple sites in the brain including the lateral hypothalamus (LH have been found to be sensitive to extracellular acidification. Since LH contains orexin neurons and innervates the medulla respiratory center, we hypothesize that ASIC1 is expressed on the orexin neuron and contributes to acid-induced increase in respiratory drive. To test this hypothesis, we used double immunofluorescence to determine whether ASIC1 is expressed on orexin neurons in the LH, and assessed integrated phrenic nerve discharge (iPND in intact rats in response to acidification of the LH. We found that ASIC1 was co-localized with orexinA in the LH. Microinjection of acidified artificial cerebrospinal fluid increased the amplitude of iPND by 70% (pH 7.4 v.s. pH 6.5:1.05±0.12 v.s. 1.70±0.10, n = 6, P<0.001 and increased the respiratory drive (peak amplitude of iPND/inspiratory time, PA/Ti by 40% (1.10±0.23 v.s. 1.50±0.38, P<0.05. This stimulatory effect was abolished by blocking ASIC1 with a nonselective inhibitor (amiloride 10 mM, a selective inhibitor (PcTX1, 10 nM or by damaging orexin neurons in the LH. Current results support our hypothesis that the orexin neuron in the LH can exert an excitation on respiration via ASIC1 during local acidosis. Since central acidification is involved in breathing dysfunction in a variety of pulmonary diseases, understanding its underlying mechanism may improve patient management.

  19. A comparison between diuretics and angiotensin-receptor blocker agents in patients with stage I hypertension (PREVER-treatment trial: study protocol for a randomized double-blind controlled trial

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    Figueiredo Neto José A

    2011-02-01

    Full Text Available Abstract Background Cardiovascular disease is the leading cause of death in Brazil, and hypertension is its major risk factor. The benefit of its drug treatment to prevent major cardiovascular events was consistently demonstrated. Angiotensin-receptor blockers (ARB have been the preferential drugs in the management of hypertension worldwide, despite the absence of any consistent evidence of advantage over older agents, and the concern that they may be associated with lower renal protection and risk for cancer. Diuretics are as efficacious as other agents, are well tolerated, have longer duration of action and low cost, but have been scarcely compared with ARBs. A study comparing diuretic and ARB is therefore warranted. Methods/design This is a randomized, double-blind, clinical trial, comparing the association of chlorthalidone and amiloride with losartan as first drug option in patients aged 30 to 70 years, with stage I hypertension. The primary outcomes will be variation of blood pressure by time, adverse events and development or worsening of microalbuminuria and of left ventricular hypertrophy in the EKG. The secondary outcomes will be fatal or non-fatal cardiovascular events: myocardial infarction, stroke, heart failure, evidence of new subclinical atherosclerosis and sudden death. The study will last 18 months. The sample size will be of 1200 participants for group in order to confer enough power to test for all primary outcomes. The project was approved by the Ethics committee of each participating institution. Discussion The putative pleiotropic effects of ARB agents, particularly renal protection, have been disputed, and they have been scarcely compared with diuretics in large clinical trials, despite that they have been at least as efficacious as newer agents in managing hypertension. Even if the null hypothesis is not rejected, the information will be useful for health care policy to treat hypertension in Brazil. Clinical trials

  20. Metabolic evidence that serosal sodium does not recycle through the active transepithelial transport pathway of toad bladder.

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    Canessa, M; Labarca, P; Leaf, A

    1976-12-25

    The possibility that sodium from the serosal bathing medium "back diffuses" into the active sodium transport pool within the mucosal epithelial cell of the isolated toad bladder was examined by determining the effect on the metabolism of the tissue of removing sodium from the serosal medium. It was expected that if recycling of serosal sodium did occur through the active transepithelial transport pathway of the isolated toad bladder, removal of sodium from the serosal medium would reduce the rate of CO2 production by the tissue and enhance of stoichiometric ratio of sodium ions transported across the bladder per molecula of sodium transport dependent CO2 produced simultaneously by the bladder (JNa/JCO2). The data revealed no significant change in this ratio (17.19 with serosal sodium and 16.13 after replacing serosal sodium with choline). Further, when transepithelial sodium transport was inhibited (a) by adding amiloride to the mucosal medium, or (b) by removing sodium from the mucosal medium, subsequent removal of sodium from the serosal medium, or (c) addition of ouabain failed to depress the basal rate of CO2 production by the bladder [(a)rate of basal, nontransport related, CO2 production (JbCO2) equals 1.54 +/- 0.52 with serosal sodium and 1.54 +/- 0.37 without serosal sodium; (b) Jb CO2 equals 2.18 +/- 0.21 with serosal sodium and 2.09 +/- 0.21 without serosal sodium; (c) 1.14 +/- 0.26 without ouabain and 1.13 +/- 0.25 with ouabain; unite of JbCO2 are nmoles mg d.w.-1 min-1]. The results support the hypothesis that little, if any, recycling of serosal sodium occurs in the total bladder.

  1. First Line Treatment of Meniere’s Disease

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    Anup Acharya

    2016-12-01

    Full Text Available Introduction: There is no consensus on the first line medical treatment of Meniere disease to produce symptomatic improvement and slow the disease progress. Dietary salt restriction, diuretics, and vasodilators like betahistine are among the first line drugs that have been used for long. There is lack of evidence due to paucity of quality studies to support their effectiveness and advocate their use. This study is done to evaluate the effectiveness of three first line treatment of Meniere disease i.e. salt restriction, oral diuretics, and betahistine. Methods: Double blind randomized controlled trial was carried out in out-patient clinic of Ear Nose and Throat department of Lumbini Medical College Teaching Hospital. Cases were randomized into three groups; dietary salt restriction, diuretics as amiloride and furosemide, and vasodilator as betahistine. Pre and post treatment evaluation was done in terms of number and severity of vertigo, tinnitus, and hearing outcome. Results: There were a total of 97 cases with F:M ratio of 1.1:1. Mean age of patients was 47.86 yr (SD=12.7. Twenty-nine (30% were treated with dietary sodium restriction alone (Group A, 35 (36% were treated with diuretics (Group B and the rest 33 (34% were treated with vasodilator (betahistine, Group C. There was no significant difference in hearing outcome in any group. Tinnitus was significantly improved in Group B. Number of vertigo attack was significantly decreased in Group B and Group C. Severity of vertigo was significantly decreased in Group B. Conclusion: Dietary salt restriction alone was not effective in controlling any aspect of the disease whereas diuretics were effective in reducing tinnitus and number and severity of vertigo. Betahistine was effective in reducing the number of vertigo attacks but not effective on other aspects of the disease.

  2. Angiotensin 2 directly increases rabbit renal brush-border membrane sodium transport: Presence of local signal transduction system

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    Morduchowicz, G.A.; Sheikh-Hamad, D.; Dwyer, B.E.; Stern, N.; Jo, O.D.; Yanagawa, N. (Sepulveda Veterans Administration, CA (USA))

    1991-05-01

    In the present study, the authors have examined the direct actions of angiotensin II (AII) in rabbit renal brush border membrane (BBM) where binding sites for AII exist. Addition of AII (10(-11)-10(-7) M) was found to stimulate 22Na+ uptake by the isolated BBM vesicles directly. All did not affect the Na(+)-dependent BBM glucose uptake, and the effect of AII on BBM 22Na+ uptake was inhibited by amiloride, suggesting the involvement of Na+/H+ exchange mechanism. BBM proton permeability as assessed by acridine orange quenching was not affected by AII, indicating the direct effect of AII on Na+/H+ antiport system. In search of the signal transduction mechanism, it was found that AII activated BBM phospholipase A2 (PLA) and that BBM contains a 42-kDa guanine nucleotide-binding regulatory protein (G-protein) that underwent pertussis toxin (PTX)-catalyzed ADP-ribosylation. Addition of GTP potentiated, while GDP-beta S or PTX abolished, the effects of AII on BBM PLA and 22Na+ uptake, suggesting the involvement of G-protein in AII's actions. On the other hand, inhibition of PLA by mepacrine prevented AII's effect on BBM 22Na+ uptake, and activation of PLA by mellitin or addition of arachidonic acid similarly enhanced BBM 22Na+ uptake, suggesting the role of PLA activation in mediating AII's effect on BBM 22Na+ uptake. In summary, results of the present study show a direct stimulatory effect of AII on BBM Na+/H+ antiport system, and suggest the presence of a local signal transduction system involving G-protein mediated PLA activation.

  3. The plant-derived glucocorticoid receptor agonist Endiandrin A acts as co-stimulator of colonic epithelial sodium channels (ENaC via SGK-1 and MAPKs.

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    Dana Kuntzsch

    Full Text Available In a search for secondary plant compounds that bind to the glucocorticoid receptor (GR, the cyclobutane lignan endiandrin A was discovered from the rainforest tree Endiandra anthropophagorum Domin. Our present study aims to characterize the effect of endiandrin A on GR-dependent induction of colonic sodium transport. The effect of endiandrin A was analyzed in GR-expressing colonic HT-29/B6 cells (HT-29/B6-GR. GR transactivation and subcellular localization were investigated by reporter gene assay and immunofluorescence. Epithelial sodium channel (ENaC was analyzed by qRT-PCR and by measuring amiloride-sensitive short-circuit current (I(sc in Ussing chambers. Endiandrin A (End A has been identified as GR receptor binder. However, it did not cause significant GR transactivation as pGRE-luciferase activity was only 7% of that of the maximum effect of dexamethasone. Interestingly, endiandrin A had a significant impact on dexamethasone-dependent sodium absorption in cells co-exposed to tumor necrosis factor (TNF-α. This was in part due to up-regulation of β- and γ-ENaC subunit expression. Endiandrin A potentiated GR-mediated transcription by increasing GR protein expression and phosphorylation. It inhibited c-Jun N-terminal kinase (JNK activation induced by dexamethasone and/or TNF-α and increased levels of GR localized to the nucleus. Additionally, endiandrin A increased the serum- and glucocorticoid-induced kinase (sgk-1 via activation of p38. Finally, the regulation of ENaC function by endiandrin A was confirmed in rat native colon. In conclusion, endiandrin A potentiates glucocorticoid-driven activation of colonic epithelial sodium channels via JNK inhibition and p38 activation due to transcriptional up-regulation of β- and γ-ENaC-subunits along with induction of sgk-1.

  4. Cytotoxic mechanisms of Zn2+ and Cd2+ involve Na+/H+ exchanger (NHE) activation by ROS

    International Nuclear Information System (INIS)

    Koutsogiannaki, Sophia; Evangelinos, Nikolaos; Koliakos, George; Kaloyianni, Martha

    2006-01-01

    The signaling mechanism induced by cadmium (Cd) and zinc (Zn) in gill cells of Mytilus galloprovincialis was investigated. Both metals cause an increase in ·O 2 - production, with Cd to be more potent (216 ± 15%) than Zn (150 ± 9.5%), in relation to control value (100%). The metals effect was reversed after incubation with the amiloride analogue, EIPA, a selective Na + /H + exchanger (NHE) inhibitor as well as in the presence of calphostin C, a protein kinase C (PKC) inhibitor. The heavy metals effect on ·O 2 - production was mediated via the interaction of metal ions with α 1 - and β-adrenergic receptors, as shown after incubation with their respective agonists and antagonists. In addition, both metals caused an increase in intracellular pH (pHi) of gill cells. EIPA together with either metal significantly reduced the effect of each metal treatment on pHi. Incubation of gill cells with the oxidants rotenone, antimycin A and pyruvate caused a significant increase in pHi (ΔpHi 0.830, 0.272 and 0.610, respectively), while in the presence of the anti-oxidant N-acetyl cysteine (NAC) a decrease in pHi (ΔpHi -0.090) was measured, indicating that change in reactive oxygen species (ROS) production by heavy metals affects NHE activity. When rosiglitazone was incubated together with either heavy metal a decrease in O 2 - production was observed. Our results show a key role of NHE in the signal transduction pathway induced by Zn and Cd in gill cells, with the involvement of ROS, PKC, adrenergic and PPAR-γ receptors. In addition, differences between the two metals concerning NHE activation, O 2 - production and interaction with adrenergic receptors were observed

  5. Macropinocytosis is responsible for the uptake of pathogenic and non-pathogenic mycobacteria by B lymphocytes (Raji cells

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    García-Pérez Blanca Estela

    2012-10-01

    Full Text Available Abstract Background The classical roles of B cells include the production of antibodies and cytokines and the generation of immunological memory, these being key factors in the adaptive immune response. However, their role in innate immunity is currently being recognised. Traditionally, B cells have been considered non-phagocytic cells; therefore, the uptake of bacteria by B cells is not extensively documented. In this study, we analysed some of the features of non-specific bacterial uptake by B lymphocytes from the Raji cell line. In our model, B cells were infected with Mycobacterium tuberculosis (MTB, Mycobacterium smegmatis (MSM, and Salmonella typhimurium (ST. Results Our observations revealed that the Raji B cells were readily infected by the three bacteria that were studied. All of the infections induced changes in the cellular membrane during bacterial internalisation. M. smegmatis and S. typhimurium were able to induce important membrane changes that were characterised by abundant filopodia and lamellipodia formation. These membrane changes were driven by actin cytoskeletal rearrangements. The intracellular growth of these bacteria was also controlled by B cells. M. tuberculosis infection also induced actin rearrangement-driven membrane changes; however, the B cells were not able to control this infection. The phorbol 12-myristate 13-acetate (PMA treatment of B cells induced filopodia and lamellipodia formation, the production of spacious vacuoles (macropinosomes, and the fluid-phase uptake that is characteristic of macropinocytosis. S. typhimurium infection induced the highest fluid-phase uptake, although both mycobacteria also induced fluid uptake. A macropinocytosis inhibitor such as amiloride was used and abolished the bacterial uptake and the fluid-phase uptake that is triggered during the bacterial infection. Conclusions Raji B cells can internalise S. typhimurium and mycobacteria through an active process, such as

  6. Neurotransmitter modulation of extracellular H+ fluxes from isolated retinal horizontal cells of the skate

    Science.gov (United States)

    Molina, Anthony J A; Verzi, Michael P; Birnbaum, Andrea D; Yamoah, Ebenezer N; Hammar, Katherine; Smith, Peter J S; Malchow, Robert Paul

    2004-01-01

    Self-referencing H+-selective microelectrodes were used to measure extracellular H+ fluxes from horizontal cells isolated from the skate retina. A standing H+ flux was detected from quiescent cells, indicating a higher concentration of free hydrogen ions near the extracellular surface of the cell as compared to the surrounding solution. The standing H+ flux was reduced by removal of extracellular sodium or application of 5-(N-ethyl-N-isopropyl) amiloride (EIPA), suggesting activity of a Na+–H+ exchanger. Glutamate decreased H+ flux, lowering the concentration of free hydrogen ions around the cell. AMPA/kainate receptor agonists mimicked the response, and the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) eliminated the effects of glutamate and kainate. Metabotropic glutamate agonists were without effect. Glutamate-induced alterations in H+ flux required extracellular calcium, and were abolished when cells were bathed in an alkaline Ringer solution. Increasing intracellular calcium by photolysis of the caged calcium compound NP-EGTA also altered extracellular H+ flux. Immunocytochemical localization of the plasmalemma Ca2+–H+-ATPase (PMCA pump) revealed intense labelling within the outer plexiform layer and on isolated horizontal cells. Our results suggest that glutamate modulation of H+ flux arises from calcium entry into cells with subsequent activation of the plasmalemma Ca2+–H+-ATPase. These neurotransmitter-induced changes in extracellular pH have the potential to play a modulatory role in synaptic processing in the outer retina. However, our findings argue against the hypothesis that hydrogen ions released by horizontal cells normally act as the inhibitory feedback neurotransmitter onto photoreceptor synaptic terminals to create the surround portion of the centre-surround receptive fields of retinal neurones. PMID:15272044

  7. Experimental Study of the Effects of EIPA, Losartan, and BQ-123 on Electrophysiological Changes Induced by Myocardial Stretch.

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    Chorro, Francisco J; Canto, Irene Del; Brines, Laia; Such-Miquel, Luis; Calvo, Conrado; Soler, Carlos; Zarzoso, Manuel; Trapero, Isabel; Tormos, Álvaro; Such, Luis

    2015-12-01

    Mechanical response to myocardial stretch has been explained by various mechanisms, which include Na(+)/H(+) exchanger activation by autocrine-paracrine system activity. Drug-induced changes were analyzed to investigate the role of these mechanisms in the electrophysiological responses to acute myocardial stretch. Multiple epicardial electrodes and mapping techniques were used to analyze changes in ventricular fibrillation induced by acute myocardial stretch in isolated perfused rabbit hearts. Four series were studied: control (n = 9); during perfusion with the angiotensin receptor blocker losartan (1 μM, n = 8); during perfusion with the endothelin A receptor blocker BQ-123 (0.1 μM, n = 9), and during perfusion with the Na(+)/H(+) exchanger inhibitor EIPA (5-[N-ethyl-N-isopropyl]-amiloride) (1 μM, n = 9). EIPA attenuated the increase in the dominant frequency of stretch-induced fibrillation (control=40.4%; losartan=36% [not significant]; BQ-123=46% [not significant]; and EIPA=22% [P<.001]). During stretch, the activation maps were less complex (P<.0001) and the spectral concentration of the arrhythmia was greater (greater regularity) in the EIPA series: control=18 (3%); EIPA = 26 (9%) (P < .02); losartan=18 (5%) (not significant); and BQ-123=18 (4%) (not significant). The Na(+)/H(+) exchanger inhibitor EIPA attenuated the electrophysiological effects responsible for the acceleration and increased complexity of ventricular fibrillation induced by acute myocardial stretch. The angiotensin II receptor antagonist losartan and the endothelin A receptor blocker BQ-123 did not modify these effects. Copyright © 2014 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.

  8. fMLP-Induced IL-8 Release Is Dependent on NADPH Oxidase in Human Neutrophils

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    María A. Hidalgo

    2015-01-01

    Full Text Available N-Formyl-methionyl-leucyl-phenylalanine (fMLP and platelet-activating factor (PAF induce similar intracellular signalling profiles; but only fMLP induces interleukin-8 (IL-8 release and nicotinamide adenine dinucleotide phosphate reduced (NADPH oxidase activity in neutrophils. Because the role of ROS on IL-8 release in neutrophils is until now controversial, we assessed if NADPH oxidase is involved in the IL-8 secretions and PI3K/Akt, MAPK, and NF-κB pathways activity induced by fMLP. Neutrophils were obtained from healthy volunteers. IL-8 was measured by ELISA, IL-8 mRNA by qPCR, and ROS production by luminol-amplified chemiluminescence, reduction of ferricytochrome c, and FACS. Intracellular pH changes were detected by spectrofluorescence. ERK1/2, p38 MAPK, and Akt phosphorylation were analysed by immunoblotting and NF-κB was analysed by immunocytochemistry. Hydroxy-3-methoxyaceto-phenone (HMAP, diphenyleneiodonium (DPI, and siRNA Nox2 reduced the ROS and IL-8 release in neutrophils treated with fMLP. HMAP, DPI, and amiloride (a Na+/H+ exchanger inhibitor inhibited the Akt phosphorylation and did not affect the p38 MAPK and ERK1/2 activity. DPI and HMAP reduced NF-κB translocation induced by fMLP. We showed that IL-8 release induced by fMLP is dependent on NADPH oxidase, and ROS could play a redundant role in cell signalling, ultimately activating the PI3K/Akt and NF-κB pathways in neutrophils.

  9. An Examination of the Role of L-Glutamate and Inosine 5'-Monophosphate in Hedonic Taste-Guided Behavior by Mice Lacking the T1R1 + T1R3 Receptor.

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    Blonde, Ginger D; Spector, Alan C

    2017-06-01

    The heterodimeric T1R1 + T1R3 receptor is considered critical for normal signaling of L-glutamate and 5'-ribonucleotides in the oral cavity. However, some taste-guided responsiveness remains in mice lacking one subunit of the receptor, suggesting that other receptors are sufficient to support some behaviors. Here, mice lacking both receptor subunits (KO) and wild-type (WT, both n = 13) mice were tested in a battery of behavioral tests. Mice were trained and tested in gustometers with a concentration series of Maltrin-580, a maltodextrin, in a brief-access test (10-s trials) as a positive control. Similar tests followed with monosodium glutamate (MSG) with and without the ribonucleotide inosine 5'-monophosphate (IMP), but always in the presence of the epithelial sodium channel blocker amiloride (A). Brief-access tests were repeated following short-term (30-min) and long-term (48-h) exposures to MSG + A + IMP and were also conducted with sodium gluconate replacing MSG. Finally, progressive ratio tests were conducted with Maltrin-580 or MSG + A + IMP, to assess appetitive behavior while minimizing satiation. Overall, MSG generated little concentration-dependent responding in either food-restricted WT or KO mice, even in combination with IMP. However, KO mice licked less to the amino acid stimuli, a measure of consummatory behavior in the brief-access tests. In contrast, both groups initiated a similar number of trials and had a similar breakpoint in the progressive ratio task, both measures of appetitive (approach) behavior. Collectively, these results suggest that while the T1R1 + T1R3 receptor is necessary for consummatory responding to MSG (+IMP), other receptors are sufficient to maintain appetitive responding to this "umami" stimulus complex in food-restricted mice. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  10. Herpes simplex virus internalization into epithelial cells requires Na+/H+ exchangers and p21-activated kinases but neither clathrin- nor caveolin-mediated endocytosis.

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    Devadas, Deepika; Koithan, Thalea; Diestel, Randi; Prank, Ute; Sodeik, Beate; Döhner, Katinka

    2014-11-01

    Herpes simplex virus 1 (HSV-1) is an alphaherpesvirus that has been reported to infect some epithelial cell types by fusion at the plasma membrane but others by endocytosis. To determine the molecular mechanisms of productive HSV-1 cell entry, we perturbed key endocytosis host factors using specific inhibitors, RNA interference (RNAi), or overexpression of dominant negative proteins and investigated their effects on HSV-1 infection in the permissive epithelial cell lines Vero, HeLa, HEp-2, and PtK2. HSV-1 internalization required neither endosomal acidification nor clathrin- or caveolin-mediated endocytosis. In contrast, HSV-1 gene expression and internalization were significantly reduced after treatment with 5-(N-ethyl-N-isopropyl)amiloride (EIPA). EIPA blocks the activity of Na(+)/H(+) exchangers, which are plasma membrane proteins implicated in all forms of macropinocytosis. HSV-1 internalization furthermore required the function of p21-activated kinases that contribute to macropinosome formation. However, in contrast to some forms of macropinocytosis, HSV-1 did not enlist the activities of protein kinase C (PKC), tyrosine kinases, C-terminal binding protein 1, or dynamin to activate its internalization. These data suggest that HSV-1 depends on Na(+)/H(+) exchangers and p21-activated kinases either for macropinocytosis or for local actin rearrangements required for fusion at the plasma membrane or subsequent passage through the actin cortex underneath the plasma membrane. After initial replication in epithelial cells, herpes simplex viruses (HSVs) establish latent infections in neurons innervating these regions. Upon primary infection and reactivation from latency, HSVs cause many human skin and neurological diseases, particularly in immunocompromised hosts, despite the availability of effective antiviral drugs. Many viruses use macropinocytosis for virus internalization, and many host factors mediating this entry route have been identified, although the

  11. Modulation of the cost of pHi regulation during metabolic depression: a (31)P-NMR study in invertebrate (Sipunculus nudus) isolated muscle.

    Science.gov (United States)

    Pörtner, H O; Bock, C; Reipschläger, A

    2000-08-01

    Extracellular acidosis has been demonstrated to play a key role in the process of metabolic depression under long-term environmental stress, exemplified in the marine invertebrate Sipunculus nudus. These findings led to the hypothesis that acid-base regulation is associated with a visible cost depending on the rate and mode of H(+)-equivalent ion exchange. To test this hypothesis, the effects of different ion-transport inhibitors on the rate of pH recovery during hypercapnia, on energy turnover and on steady-state acid-base variables were studied in isolated body wall musculature of the marine worm Sipunculus nudus under control conditions (pHe 7.90) and during steady-state extracellular acidosis (pHe 7.50 or 7.20) by in vivo (31)P-NMR and oxygen consumption analyses. During acute hypercapnia (2 % CO(2)), recovery of pHi was delayed at pHe 7.5 compared with pHe 7.9. Inhibition of the Na(+)/H(+)-exchanger by 5-(N,N-dimethyl)-amiloride (DMA) at pHe 7.5 delayed recovery even further. This effect was much smaller at pHe 7.9. Inhibition of anion exchange by the addition of the transport inhibitor 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) prevented pH recovery at pHe 7.5 and delayed recovery at pHe 7.9, in accordance with an effect on Na(+)-dependent Cl(-)/HCO(3)(-) exchange. The effects of ouabain, DIDS and DMA on metabolic rate were reduced at low pHe, thereby supporting the conclusion that acidosis caused the ATP demand of Na(+)/K(+)-ATPase to fall. This reduction occurred via an inhibiting effect on both Na(+)/H(+)- and Na(+)-dependent Cl(-)/HCO(3)(-) (i.e. Na(+)/H(+)/Cl(-)/HCO(3)(-)) exchange in accordance with a reduction in the ATP demand for acid-base regulation during metabolic depression. Considering the ATP stoichiometries of the two exchangers, metabolic depression may be supported by the predominant use of Na(+)/H(+)/Cl(-)/HCO(3)(-) exchange under conditions of extracellular acidosis.

  12. Potential role of sodium-proton exchangers in the low concentration arsenic trioxide-increased intracellular pH and cell proliferation.

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    Carmen Aravena

    Full Text Available Arsenic main inorganic compound is arsenic trioxide (ATO presented in solution mainly as arsenite. ATO increases intracellular pH (pHi, cell proliferation and tumor growth. Sodium-proton exchangers (NHEs modulate the pHi, with NHE1 playing significant roles. Whether ATO-increased cell proliferation results from altered NHEs expression and activity is unknown. We hypothesize that ATO increases cell proliferation by altering pHi due to increased NHEs-like transport activity. Madin-Darby canine kidney (MDCK cells grown in 5 mmol/L D-glucose-containing DMEM were exposed to ATO (0.05, 0.5 or 5 µmol/L, 0-48 hours in the absence or presence of 5-N,N-hexamethylene amiloride (HMA, 5-100 µmol/L, NHEs inhibitor, PD-98059 (30 µmol/L, MAPK1/2 inhibitor, Gö6976 (10 µmol/L, PKCα, βI and μ inhibitor, or Schering 28080 (10 µmol/L, H(+/K(+ATPase inhibitor plus concanamycin (0.1 µmol/L, V type ATPases inhibitor. Incorporation of [(3H]thymidine was used to estimate cell proliferation, and counting cells with a hemocytometer to determine the cell number. The pHi was measured by fluorometry in 2,7-bicarboxyethyl-5,6-carboxyfluorescein loaded cells. The Na(+-dependent HMA-sensitive NHEs-like mediated proton transport kinetics, NHE1 protein abundance in the total, cytoplasm and plasma membrane protein fractions, and phosphorylated and total p42/44 mitogen-activated protein kinases (p42/44(mapk were also determined. Lowest ATO (0.05 µmol/L, ~0.01 ppm used in this study increased cell proliferation, pHi, NHEs-like transport and plasma membrane NHE1 protein abundance, effects blocked by HMA, PD-98059 or Gö6976. Cell-buffering capacity did not change by ATO. The results show that a low ATO concentration increases MDCK cells proliferation by NHEs (probably NHE1-like transport dependent-increased pHi requiring p42/44(mapk and PKCα, βI and/or μ activity. This finding could be crucial in diseases where uncontrolled cell growth occurs, such as tumor growth, and

  13. Plasminogen-dependent and -independent proteolytic activity of murine endothelioma cells with targeted inactivation of fibrinolytic genes.

    Science.gov (United States)

    Lijnen, H R; Wagner, E F; Collen, D

    1997-02-01

    Plasminogen-dependent and -independent proteolytic activity of marine endothelioma (End) cells that were derived from mice with targeted inactivation of the tissue-type plasminogen activator (t-PA-/-), urokinase-type plasminogen activator (u-PA-/-) or plasminogen activator inhibitor-1 (PAI-1-/-) genes was studied with the use of fibrin and extracellular matrix degradation assays. In a buffer milieu, the activation rate of plasminogen (final concentration 0.25 microM) with wild-type and t-PA-/- End cells (3 x 10(4) to 4 x 10(6) cells/ml) was comparable, but it was about 4-fold reduced with u-PA-/- End cells and 3-fold enhanced with PAI-1-/- End cells. Plasminogen activation was markedly reduced by addition of amiloride or of anti-murine u-PA antibodies but not by addition of anti-murine t-PA antibodies, and it was not stimulated by addition of fibrin. Lysis of 125I-fibrin labeled matrix in the presence of plasminogen was comparable with wild-type, t-PA-/- and PAI-1-/- End cells (50% lysis in 3 h with 0.7 to 1.5 x 10(6) cells/ml), but was significantly reduced with u-PA-/- End cells (50% lysis in 20 h with 0.87 x 10(6) cells/ml). Lysis of 3H-proline labeled extracellular matrix in the presence of plasminogen with wild-type, t-PA-/- and PAI-1-/- End cells (20% lysis in 48 h with 3 to 5 x 10(6) cells/ml) was comparable, but it was virtually abolished with u-PA-/- End cells. In the absence of plasminogen, lysis of both the fibrin and the extracellular matrix by all four cell types was drastically reduced and was virtually abolished by addition of phenylmethylsulfonylfluoride or 1,10 phenanthroline. These data indicate that the proteolytic activity of the transformed murine endothelioma cells, measured in plasminogen activation or matrix degradation assays, is essentially u-PA-related and largely plasminogen-dependent.

  14. Characterization of copper transport in gill cells of a mangrove crab Ucides cordatus

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    Sá, M.G. [Biosciences Institute, Department of Physiology, University of São Paulo, Rua do Matão, Travessa 14, 101, São Paulo 05508-900, SP (Brazil); Zanotto, F.P., E-mail: fzanotto@usp.br [Biosciences Institute, Department of Physiology, University of São Paulo, Rua do Matão, Travessa 14, 101, São Paulo 05508-900, SP (Brazil); Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Rua Três de Maio 100, Sao Paulo 04044-020 (Brazil)

    2013-11-15

    Highlights: •Copper transport in gill cells of a mangrove crab Ucides cordatus is dependent of calcium. •Copper transport mechanism is ATP-dependent. •Transport was monitored second by second during 300 s. -- Abstract: The branchial epithelium of crustaceans is exposed to the environment and is the first site affected by metal pollution. The aim of this work was to characterize copper (Cu) transport using a fluorescent dye, Phen Green, in gill cells of a hypo-hyper-regulator mangrove crab Ucides cordatus. The results showed that added extracellular CuCl{sub 2} (0, 0.025, 0.150, 0.275, 0.550 and 1.110 μM) showed typical Michaelis–Menten transport for Cu in anterior and posterior gill cells (V{sub max} for anterior and posterior gills: 0.41 ± 0.12 and 1.76 ± 0.27 intracellular Cu in μM × 22.10{sup 4} cells{sup −1} × 300 s{sup −1} respectively and K{sub m} values: 0.44 ± 0.04 and 0.32 ± 0.13 μM, respectively). Intracellular Cu was significantly higher for posterior gill cells compared to anterior gill cells, suggesting differential accumulation for each gill type. Extracellular Ca at 20 mM decreased cellular Cu transport for both anterior and posterior gill cells. Nifedipine and verapamil, calcium channel inhibitors from plasma membrane, decreased Cu transport and affected K{sub m} for both gills. These results could be due to a competition between Cu and Ca. Amiloride, a Na/Ca exchanger inhibitor, as well as bafilomycin, a proton pump inhibitor, caused a decrease of intracellular Cu compared to control. Ouabain and KB-R 7943, acting on Na homeostasis, similarly decreased intracellular Cu in both gill cells. Besides that, gill cells exposed to ATP and Cu simultaneously, showed an increase in intracellular copper, which was inhibited by vanadate, an inhibitor of P-type ATPase. These results suggest either the presence of a Cu-ATPase in crab gill cells, responsible for Cu influx, or the effect of a change in electrochemical membrane potential that

  15. Colonic epithelial ion transport is not affected in patients with diverticulosis

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    Tilotta Maria C

    2007-09-01

    Full Text Available Abstract Background Colonic diverticular disease is a bothersome condition with an unresolved pathogenesis. It is unknown whether a neuroepithelial dysfunction is present. The aim of the study was two-fold; (1 to investigate colonic epithelial ion transport in patients with diverticulosis and (2 to adapt a miniaturized Modified Ussing Air-Suction (MUAS chamber for colonic endoscopic biopsies. Methods Biopsies were obtained from the sigmoid part of the colon. 86 patients were included. All patients were referred for colonoscopy on suspicion of neoplasia and they were without pathological findings at colonoscopy (controls except for diverticulosis in 22 (D-patients. Biopsies were mounted in MUAS chambers with an exposed area of 5 mm2. Electrical responses to various stimulators and inhibitors of ion transport were investigated together with histological examination. The MUAS chamber was easy to use and reproducible data were obtained. Results Median basal short circuit current (SCC was 43.8 μA·cm-2 (0.8 – 199 for controls and 59.3 μA·cm-2 (3.0 – 177.2 for D-patients. Slope conductance was 77.0 mS·cm-2 (18.6 – 204.0 equal to 13 Ω·cm2 for controls and 96.6 mS·cm-2 (8.4 – 191.4 equal to 10.3 Ω·cm2 for D-patients. Stimulation with serotonin, theophylline, forskolin and carbachol induced increases in SCC in a range of 4.9 – 18.6 μA·cm-2, while inhibition with indomethacin, bumetanide, ouabain and amiloride decreased SCC in a range of 6.5 – 27.4 μA·cm-2, and all with no significant differences between controls and D-patients. Histological examinations showed intact epithelium and lamina propria before and after mounting for both types of patients. Conclusion We conclude that epithelial ion transport is not significantly altered in patients with diverticulosis and that the MUAS chamber can be adapted for studies of human colonic endoscopic biopsies.

  16. Bioelectric patterning during oogenesis: stage-specific distribution of membrane potentials, intracellular pH and ion-transport mechanisms in Drosophila ovarian follicles.

    Science.gov (United States)

    Krüger, Julia; Bohrmann, Johannes

    2015-01-16

    Bioelectric phenomena have been found to exert influence on various developmental and regenerative processes. Little is known about their possible functions and the cellular mechanisms by which they might act during Drosophila oogenesis. In developing follicles, characteristic extracellular current patterns and membrane-potential changes in oocyte and nurse cells have been observed that partly depend on the exchange of protons, potassium ions and sodium ions. These bioelectric properties have been supposed to be related to various processes during oogenesis, e. g. pH-regulation, osmoregulation, cell communication, cell migration, cell proliferation, cell death, vitellogenesis and follicle growth. Analysing in detail the spatial distribution and activity of the relevant ion-transport mechanisms is expected to elucidate the roles that bioelectric phenomena play during oogenesis. To obtain an overview of bioelectric patterning along the longitudinal and transversal axes of the developing follicle, the spatial distributions of membrane potentials (Vmem), intracellular pH (pHi) and various membrane-channel proteins were studied systematically using fluorescent indicators, fluorescent inhibitors and antisera. During mid-vitellogenic stages 9 to 10B, characteristic, stage-specific Vmem-patterns in the follicle-cell epithelium as well as anteroposterior pHi-gradients in follicle cells and nurse cells were observed. Corresponding distribution patterns of proton pumps (V-ATPases), voltage-dependent L-type Ca(2+)-channels, amiloride-sensitive Na(+)-channels and Na(+),H(+)-exchangers (NHE) and gap-junction proteins (innexin 3) were detected. In particular, six morphologically distinguishable follicle-cell types are characterized on the bioelectric level by differences concerning Vmem and pHi as well as specific compositions of ion channels and carriers. Striking similarities between Vmem-patterns and activity patterns of voltage-dependent Ca(2+)-channels were found, suggesting

  17. Cyclic-AMP regulates postnatal development of neural and behavioral responses to NaCl in rats.

    Science.gov (United States)

    Qian, Jie; Mummalaneni, Shobha; Phan, Tam-Hao T; Heck, Gerard L; DeSimone, John A; West, David; Mahavadi, Sunila; Hojati, Deanna; Murthy, Karnam S; Rhyu, Mee-Ra; Spielman, Andrew I; Özdener, Mehmet Hakan; Lyall, Vijay

    2017-01-01

    During postnatal development rats demonstrate an age-dependent increase in NaCl chorda tympani (CT) responses and the number of functional apical amiloride-sensitive epithelial Na+ channels (ENaCs) in salt sensing fungiform (FF) taste receptor cells (TRCs). Currently, the intracellular signals that regulate the postnatal development of salt taste have not been identified. We investigated the effect of cAMP, a downstream signal for arginine vasopressin (AVP) action, on the postnatal development of NaCl responses in 19-23 day old rats. ENaC-dependent NaCl CT responses were monitored after lingual application of 8-chlorophenylthio-cAMP (8-CPT-cAMP) under open-circuit conditions and under ±60 mV lingual voltage clamp. Behavioral responses were tested using 2 bottle/24h NaCl preference tests. The effect of [deamino-Cys1, D-Arg8]-vasopressin (dDAVP, a specific V2R agonist) was investigated on ENaC subunit trafficking in rat FF TRCs and on cAMP generation in cultured adult human FF taste cells (HBO cells). Our results show that in 19-23 day old rats, the ENaC-dependent maximum NaCl CT response was a saturating sigmoidal function of 8-CPT-cAMP concentration. 8-CPT-cAMP increased the voltage-sensitivity of the NaCl CT response and the apical Na+ response conductance. Intravenous injections of dDAVP increased ENaC expression and γ-ENaC trafficking from cytosolic compartment to the apical compartment in rat FF TRCs. In HBO cells dDAVP increased intracellular cAMP and cAMP increased trafficking of γ- and δ-ENaC from cytosolic compartment to the apical compartment 10 min post-cAMP treatment. Control 19-23 day old rats were indifferent to NaCl, but showed clear preference for appetitive NaCl concentrations after 8-CPT-cAMP treatment. Relative to adult rats, 14 day old rats demonstrated significantly less V2R antibody binding in circumvallate TRCs. We conclude that an age-dependent increase in V2R expression produces an AVP-induced incremental increase in cAMP that modulates

  18. Cyclic-AMP regulates postnatal development of neural and behavioral responses to NaCl in rats.

    Directory of Open Access Journals (Sweden)

    Jie Qian

    Full Text Available During postnatal development rats demonstrate an age-dependent increase in NaCl chorda tympani (CT responses and the number of functional apical amiloride-sensitive epithelial Na+ channels (ENaCs in salt sensing fungiform (FF taste receptor cells (TRCs. Currently, the intracellular signals that regulate the postnatal development of salt taste have not been identified. We investigated the effect of cAMP, a downstream signal for arginine vasopressin (AVP action, on the postnatal development of NaCl responses in 19-23 day old rats. ENaC-dependent NaCl CT responses were monitored after lingual application of 8-chlorophenylthio-cAMP (8-CPT-cAMP under open-circuit conditions and under ±60 mV lingual voltage clamp. Behavioral responses were tested using 2 bottle/24h NaCl preference tests. The effect of [deamino-Cys1, D-Arg8]-vasopressin (dDAVP, a specific V2R agonist was investigated on ENaC subunit trafficking in rat FF TRCs and on cAMP generation in cultured adult human FF taste cells (HBO cells. Our results show that in 19-23 day old rats, the ENaC-dependent maximum NaCl CT response was a saturating sigmoidal function of 8-CPT-cAMP concentration. 8-CPT-cAMP increased the voltage-sensitivity of the NaCl CT response and the apical Na+ response conductance. Intravenous injections of dDAVP increased ENaC expression and γ-ENaC trafficking from cytosolic compartment to the apical compartment in rat FF TRCs. In HBO cells dDAVP increased intracellular cAMP and cAMP increased trafficking of γ- and δ-ENaC from cytosolic compartment to the apical compartment 10 min post-cAMP treatment. Control 19-23 day old rats were indifferent to NaCl, but showed clear preference for appetitive NaCl concentrations after 8-CPT-cAMP treatment. Relative to adult rats, 14 day old rats demonstrated significantly less V2R antibody binding in circumvallate TRCs. We conclude that an age-dependent increase in V2R expression produces an AVP-induced incremental increase in c

  19. The Epithelial Sodium Channel α subunit (α ENaC alternatively spliced form "b" in Dahl rats: What's next?

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    Shehata Marlene F

    2010-07-01

    Full Text Available Abstract Background The amiloride-sensitive Epithelial Sodium Channel (ENaC is critical in maintaining Na+ balance, extracellular fluid volume and long term blood pressure control. ENaC is composed of three main subunits α, β, & γ. While α ENaC is critical for channel functionality, β & γ ENaC maximize channel function. To date, there are four alternatively spliced forms of the α subunit of ENaC (α ENaC-a, -b, -c, & -d that have been published in rats, in addition to the major α ENaC transcript. While α ENaC-a, -c & -d transcripts are low abundance transcripts compared to full-length α ENaC, α ENaC-b is a higher abundance and salt-sensitive transcript compared to full-length α ENaC. Presentation of the hypothesis α ENaC-b protein, which is preferentially produced in Dahl R rats, to a greater extent on high salt diet, exerts a dominant negative effect on full-length α ENaC subunit by physically binding to and trapping full-length α ENaC subunit in the endoplasmic reticulum, and finally accelerating full-length α ENaC proteolytic degradation in a dose-dependent manner. Testing the hypothesis 1 To examine the mRNA and protein abundance of α ENaC-b relative to α ENaC full-length in kidney, lung, and taste tissues of Dahl rats. 2 To compare the expression (mRNA and protein of α ENaC-b in kidneys of Dahl S and R rats on regular and high salt diet. 3 To examine the putative binding of α ENaC-b proteins to full-length α ENaC in vitro and to determine the impact of such binding on full-length α ENaC expression in vitro. Implications of the hypothesis Our studies will be the first to demonstrate the over-expression of salt-sensitive α ENaC-b spliced form in kidney tissues of Dahl R rats at the expense of full-length α ENaC. The current proposal will provide highly novel insights into the putative mechanisms leading to ENaC hypoactivity in high-salt-fed Dahl R rats. Finally, findings from the present proposal will uncover a new

  20. Effects of SM-20550, a selective Na+-H+ exchange inhibitor, on the ion transport of myocardial mitochondria.

    Science.gov (United States)

    Hotta, Y; Ishikawa, N; Ohashi, N; Matsui, K

    2001-03-01

    The effect of a novel Na+-H+ exchange inhibitor, SM-20550 [N-(aminoiminomethyl)-1,4-dimethyl-1H-indole-2-carboxamide methanesulfonate] (SM) on the ion transport of myocardial mitochondria was studied using ion fluorometry and superfusion techniques. Isolated mitochondria from the guinea-pig heart were pre-loaded with fluoroprobes of either BCECF AM for H+, SBFI AM for Na+ or fura-2 AM for Ca2+. Initially, the treated mitochondria were superfused with a normal medium (MOPS-buffer, pH 7.4, 24 degrees C), subsequently fluorometric experiments on the Na+, H+, Ca2+ mobilization across the mitochondrial membrane were performed. The intramitochondrial pH (pHm) was increased by the superfusion of Na+ at physiological cytosolic concentrations of 10 mM, indicating the existence of a Na+-H+ exchange in mitochondrial membranes. The Na+ induced elevation of pH was dose-dependently inhibited by SM 1 microM (delta pHm; 45% as drug-free 100%), and 10 microM (delta pHm; 70%), as observed in our experiments with the myocardial sarcolemmal membrane. The selective Na+-H+ exchange inhibitor SM reduced such pHm elevations more markedly than that of EIPA [5-(N-ethyl-N-isopropyl) amiloride]. The Na+-H+ exchange inhibitors, SM and EIPA suppressed the intramitochondrial Ca2+ elevation ([Ca2+]m) brought on by external Ca2+ concentration changes: The pretreatment with SM 1 microM, 10 microM and EIPA 10 microM reduced the [Ca2+]m influx by 28.3, 56.5 and 63%, respectively. Additionally, the [Ca2+]m elevation induced by acidification of the perfusate was reduced by the prior infusion of SM and EIPA. Pretreatment of mitochondria with SM or EIPA which had beneficial effects on the left ventricular developed pressure (LVDP) in the ischemia-reperfusion injury of Langendorff hearts, reduced the intramitochondrial Na+ and pHm levels, indicating interplay of the inhibitory mechanism of Ca2+-uptake into mitochondria coupled with Na+-H+ exchange. These findings suggested that protective effects of Na

  1. Hyperosmotic perfusion of the beating rat heart and the role of the Na+/K+/2Cl- co-transporter and the Na+/H+ exchanger.

    Science.gov (United States)

    Falck, G; Schjøtt, J; Bruvold, M; Krane, J; Skarra, S; Jynge, P

    2000-02-01

    The aim of the present study was to investigate the role of the Na+/K+/2Cl- co-transporter and the Na+/H+ exchanger on contractile function and electrolyte regulation during hyperosmotic perfusion of the heart. Langendorff perfused rat hearts were subjected to hyperosmolal perfusion in 10-min intervals. Perfusates were made hyperosmotic by adding mannitol to the buffer (370, 450 and 600 mOsmol/kg H2O). Cardiac contractile function was monitored with a balloon in the left ventricle (LV) coupled to a pressure transducer. Cardiac effluent was sampled repeatedly throughout and after hyperosmotic perfusion and analysed for content of Na+, K+, and Cl-. All three hyperosmotic perfusates initially reduced LV developed pressure (LVDP), but for 370 and 450 mOsmol/kg H2O, LVDP recovered to baseline within 4 min of perfusion. With 600 mOsmol/kg H2O, LVDP recovered slowly and was 50% below baseline after 10 min of hyperosmotic perfusion. Inhibition of the Na+/H+ exchanger with 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and 3-methylsulfonyl-4-piperidinobenzoyl-guanidine methanesulfonate (HOE 694) abolished the recovery of LVDP to the 600 mOsmol/kg H2O perfusate, whereas inhibition of the Na+/K+/2Cl- co-transporter had no impact on LVDP. Potassium was taken up by the heart during hyperosmotic perfusion and this uptake was significantly reduced with inhibition of the Na+/H+ exchanger. Intracellular pH was assessed with 31p magnetic resonance spectroscopy and hyperosmolality induced a significant alkalosis that was dependent upon the Na+/H+ exchanger. The rat heart responds to moderate elevations in osmolality with a transient reduction in contractile function, whereas an elevation of 300 mOsmol/kg H2O persistently reduces contractile function. The Na+/H+ exchanger, but not the Na+/K+/2Cl- co-transporter, is of importance in contractile recovery and electrolyte regulation during hyperosmotic perfusion in the rat heart.

  2. El litio y su relación con la acuaporina-2 y el canal de sodio ENaC

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    Luciano Galizia

    2012-04-01

    Full Text Available Desde hace más de cuarenta años que el litio es usado para el tratamiento de la enfermedad bipolar; recientes estudios sugieren también su utilidad en el trastorno cognitivo mínimo tipo amnésico. El litio es filtrado en el glomérulo y un 65-75% del mismo es reabsorbido en el túbulo contorneado proximal y en el asa ascendente de Henle por el transportador Na+, K+, 2Cl- y vía paracelular. Una pequeña fracción del litio entra en las células principales del túbulo colector por medio del canal epitelial de sodio sensible al amiloride (ENaC localizado en la membrana apical de la célula. Luego de 10- 20 años de tratamiento con litio los enfermos pueden desarrollar poliuria, acidosis tubular e insuficiencia renal crónica que puede terminar en una forma de diabetes que no responde a la arginina vasopresina llamada diabetes insípida nefrogénica. Se cree que estas fallas renales son consecuencias de una reducción en el número de moléculas de acuaporina 2 en la membrana apical. Las causas para esto son complejas. El litio es un poderoso inhibidor de la isoforma beta de la enzima glicógeno sintetasa quinasa y esto está asociado a una menor actividad de la adenilato ciclasa que lleva a una disminución en la concentración intracelular de cAMP. Esto finalmente interferiría con la síntesis de nuevas moléculas de acuaporina 2 y con el tráfico de ellas desde la zona subapical de la célula hacia la membrana celular, causando la disminución en la reabsorción de agua en la parte distal del nefrón.

  3. Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production.

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    Ann L Wozniak

    2010-09-01

    Full Text Available The hepatitis C virus (HCV p7 protein is critical for virus production and an attractive antiviral target. p7 is an ion channel when reconstituted in artificial lipid bilayers, but channel function has not been demonstrated in vivo and it is unknown whether p7 channel activity plays a critical role in virus production. To evaluate the contribution of p7 to organelle pH regulation and virus production, we incorporated a fluorescent pH sensor within native, intracellular vesicles in the presence or absence of p7 expression. p7 increased proton (H(+ conductance in vesicles and was able to rapidly equilibrate H(+ gradients. This conductance was blocked by the viroporin inhibitors amantadine, rimantadine and hexamethylene amiloride. Fluorescence microscopy using pH indicators in live cells showed that both HCV infection and expression of p7 from replicon RNAs reduced the number of highly acidic (pH<5 vesicles and increased lysosomal pH from 4.5 to 6.0. These effects were not present in uninfected cells, sub-genomic replicon cells not expressing p7, or cells electroporated with viral RNA containing a channel-inactive p7 point mutation. The acidification inhibitor, bafilomycin A1, partially restored virus production to cells electroporated with viral RNA containing the channel inactive mutation, yet did not in cells containing p7-deleted RNA. Expression of influenza M2 protein also complemented the p7 mutant, confirming a requirement for H(+ channel activity in virus production. Accordingly, exposure to acid pH rendered intracellular HCV particles non-infectious, whereas the infectivity of extracellular virions was acid stable and unaffected by incubation at low pH, further demonstrating a key requirement for p7-induced loss of acidification. We conclude that p7 functions as a H(+ permeation pathway, acting to prevent acidification in otherwise acidic intracellular compartments. This loss of acidification is required for productive HCV infection

  4. Comparison of single and combination diuretics on glucose tolerance (PATHWAY-3): protocol for a randomised double-blind trial in patients with essential hypertension.

    Science.gov (United States)

    Brown, Morris J; Williams, Bryan; MacDonald, Thomas M; Caulfield, Mark; Cruickshank, J Kennedy; McInnes, Gordon; Sever, Peter; Webb, David J; Salsbury, Jackie; Morant, Steve; Ford, Ian

    2015-08-07

    Thiazide diuretics are associated with increased risk of diabetes mellitus. This risk may arise from K(+)-depletion. We hypothesised that a K(+)-sparing diuretic will improve glucose tolerance, and that combination of low-dose thiazide with K(+)-sparing diuretic will improve both blood pressure reduction and glucose tolerance, compared to a high-dose thiazide. This is a parallel-group, randomised, double-blind, multicentre trial, comparing hydrochlorothiazide 25-50 mg, amiloride 10-20 mg and combination of both diuretics at half these doses. A single-blind placebo run-in of 1 month is followed by 24 weeks of blinded active treatment. There is forced dose-doubling after 3 months. The Primary end point is the blood glucose 2 h after oral ingestion of a 75 g glucose drink (OGTT), following overnight fasting. The primary outcome is the difference between 2 h glucose at weeks 0, 12 and 24. Secondary outcomes include the changes in home systolic blood pressure (BP) and glycated haemoglobin and prediction of response by baseline plasma renin. Eligibility criteria are: age 18-79, systolic BP on permitted background treatment ≥ 140 mm Hg and home BP ≥ 130 mm Hg and one component of the metabolic syndrome additional to hypertension. Principal exclusions are diabetes, estimated-glomerular filtration rate 200 mm Hg or DBP >120 mm Hg (box 2). The sample size calculation indicates that 486 patients will give 80% power at α=0.01 to detect a difference in means of 1 mmol/L (SD=2.2) between 2 h glucose on hydrochlorothiazide and comparators. PATHWAY-3 was approved by Cambridge South Ethics Committee, number 09/H035/19. The trial results will be published in a peer-reviewed scientific journal. Eudract number 2009-010068-41 and clinical trials registration number: NCT02351973. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  5. Action of cholera toxin in the intestinal epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Hyun, C.S.

    1982-01-01

    The primary event in the action of cholera toxin on the isolated chick intestinal epithelial cell is its interaction with a large number of high affinity binding sites in the cell membrane. Binding of /sup 125/I-labeled toxin is rapid, temperature-dependent, reversible, and saturable over a wide range of concentrations and includes only a small contribution from nonspecific sites. A characteristic lag phase of 10 min occurs following the complete binding of toxin before any increase in cellular cAMP levels can be detected. The response (elevation of cellular cAMP) is linear with time for 40 to 50 min and causes a six- to eight-fold increase over control levels (10 to 15 picomole cAMP/mg cellular protein) at steady state. cAMP and agents that increase cAMP production inhibit Cl/sup -/-independent Na/sup +/ influx into the isolated enterocytes whereas chlorpromazine (CPZ) which completely abolishes toxin-induced elevation of cAMP both reverses and prevents the cAMP-mediated inhibition of Na/sup +/ entry. Correlation between cellular cAMP levels and the magnitude of Na/sup +/ influx provides evidence for a cAMP-mediated control of intestinal Na/sup +/ uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT on Na/sup +/ during induction of intestinal secretion. The effect of cAMP on Na/sup +/ but not Cl/sup -/ influx preparations can be partially explained in terms of a cAMP-regulated Na/sup +//H/sup +/ neutral exchange system. Data on the coupling relationship between Na/sup +/ transport and the intra- and extracellular pH in the enterocytes show that an amiloride-sensitive electroneutral Na/sup +//H/sup +/ exchange process occurs. This coupling between Na/sup +/ and H/sup +/ is partially inhibited by CT and dbcAMP, suggesting that the Na/sup +//H/sup +/ exchange may be a cAMP-regulated process. 31 references, 32 figures, 5 tables.

  6. Cell Surface THY-1 Contributes to Human Cytomegalovirus Entry via a Macropinocytosis-Like Process.

    Science.gov (United States)

    Li, Qingxue; Fischer, Elizabeth; Cohen, Jeffrey I

    2016-11-01

    Previously we showed that THY-1 has a critical role in the initial stage of infection of certain cell types with human cytomegalovirus (HCMV) and that THY-1 is important for HCMV-mediated activation of phosphatidylinositol 3-kinase (PI3K)/Akt during virus entry. THY-1 is known to interact with integrins and is a major cargo protein of clathrin-independent endocytic vesicles. Since macropinocytosis involves integrin signaling, is PI3K/Akt dependent, and is a clathrin-independent endocytic process, we determined whether THY-1 has a role in HCMV entry by macropinocytosis. Using electron microscopy in two cell lines that support HCMV infection in a THY-1-dependent manner, we found that HCMV enters these cells by a macropinocytosis-like process. THY-1 associated with HCMV virions on the cell surface and colocalized with virus inside macropinosomes. 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) and soluble THY-1 blocked HCMV infection in the cell lines by ≥80% and 60%, respectively. HCMV entry into the cells triggered increased influx of extracellular fluid, a marker of macropinocytosis, and this increased fluid uptake was inhibited by EIPA and by soluble THY-1. Blocking actin depolymerization, Na + /H + exchange, PI3K, and Pak1 kinase, which are critical for macropinocytosis, impaired HCMV infection. Neither internalized HCMV virions nor THY-1 in virus-infected cells colocalized with transferrin as determined by confocal microscopy, indicating that clathrin-mediated endocytosis was not involved in THY-1-associated virus entry. These results suggest that HCMV has adapted to utilize THY-1, a cargo protein of clathrin-independent endocytotic vesicles, to facilitate efficient entry into certain cell types by a macropinocytosis-like process. Human cytomegalovirus (HCMV) infects over half of the population and is the most common infectious cause of birth defects. The virus is the most important infection occurring in transplant recipients. The mechanism of how HCMV enters cells

  7. Macropinocytosis is the entry mechanism of amphotropic murine leukemia virus.

    Science.gov (United States)

    Rasmussen, Izabela; Vilhardt, Frederik

    2015-02-01

    The entry mechanism of murine amphotropic retrovirus (A-MLV) has not been unambiguously determined. We show here that A-MLV is internalized not by caveolae or other pinocytic mechanisms but by macropinocytosis. Thus, A-MLV infection of mouse embryonic fibroblasts deficient for caveolin or dynamin, and NIH 3T3 cells knocked down for caveolin expression, was unaffected. Conversely, A-MLV infection of NIH 3T3 and HeLa cells was sensitive to amiloride analogues and actin-depolymerizing drugs that interfere with macropinocytosis. Further manipulation of the actin cytoskeleton through conditional expression of dominant positive or negative mutants of Rac1, PAK1, and RhoG, to increase or decrease macropinocytosis, similarly correlated with an augmented or inhibited infection with A-MLV, respectively. The same experimental perturbations affected the infection of viruses that use clathrin-coated-pit endocytosis or other pathways for entry only mildly or not at all. These data agree with immunofluorescence studies and cryo-immunogold labeling for electron microscopy, which demonstrate the presence of A-MLV in protrusion-rich areas of the cell surface and in cortical fluid phase (dextran)-filled macropinosomes, which also account for up to a half of the cellular uptake of the cell surface-binding lectin concanavalin A. We conclude that A-MLV use macropinocytosis as the predominant entry portal into cells. Binding and entry of virus particles into mammalian cells are the first steps of infection. Understanding how pathogens and toxins exploit or divert endocytosis pathways has advanced our understanding of membrane trafficking pathways, which benefits development of new therapeutic schemes and methods of drug delivery. We show here that amphotropic murine leukemia virus (A-MLV) pseudotyped with the amphotropic envelope protein (which expands the host range to many mammalian cells) gains entry into host cells by macropinocytosis. Macropinosomes form as large, fluid

  8. Inhibition of Megakaryocyte Differentiation by Antibody-Drug Conjugates (ADCs) is Mediated by Macropinocytosis: Implications for ADC-induced Thrombocytopenia.

    Science.gov (United States)

    Zhao, Hui; Gulesserian, Sara; Ganesan, Sathish Kumar; Ou, Jimmy; Morrison, Karen; Zeng, Zhilan; Robles, Veronica; Snyder, Josh; Do, Lisa; Aviña, Hector; Karki, Sher; Stover, David R; Doñate, Fernando

    2017-09-01

    Thrombocytopenia is a common adverse event in cancer patients treated with antibody-drug conjugates (ADC), including AGS-16C3F, an ADC targeting ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase-3) and trastuzumab emtansine (T-DM1). This study aims to elucidate the mechanism of action of ADC-induced thrombocytopenia. ENPP3 expression in platelets and megakaryocytes (MK) was investigated and shown to be negative. The direct effect of AGS-16C3F on platelets was evaluated using platelet rich plasma following the expression of platelet activation markers. Effects of AGS-16C3F, T-DM1, and control ADCs on maturing megakaryocytes were evaluated in an in vitro system in which human hematopoietic stem cells (HSC) were differentiated into MKs. AGS-16C3F, like T-DM1, did not affect platelets directly, but inhibited MK differentiation by the activity of Cys-mcMMAF, its active metabolite. FcγRIIA did not appear to play an important role in ADC cytotoxicity to differentiating MKs. AGS-16C3F, cytotoxic to MKs, did not bind to FcγRIIA on MKs. Blocking the interaction of T-DM1 with FcγRIIA did not prevent the inhibition of MK differentiation and IgG1-mcMMAF was not as cytotoxic to MKs despite binding to FcγRIIA. Several lines of evidence suggest that internalization of AGS-16C3F into MKs is mediated by macropinocytosis. Macropinocytosis activity of differentiating HSCs correlated with cell sensitivity to AGS-16C3F. AGS-16C3F was colocalized with a macropinocytosis marker, dextran-Texas Red in differentiating MKs. Ethyl isopropyl amiloride (EIPA), a macropinocytosis inhibitor, blocked internalization of dextran-Texas Red and AGS-16C3F. These data support the notion that inhibition of MK differentiation via macropinocytosis-mediated internalization plays a role in ADC-induced thrombocytopenia. Mol Cancer Ther; 16(9); 1877-86. ©2017 AACR See related article by Zhao et al., p. 1866 . ©2017 American Association for Cancer Research.

  9. Funciones de los canales iónicos CFTR y ENAC en la fibrosis quística

    Directory of Open Access Journals (Sweden)

    Alejandra G. Palma

    2014-04-01

    Full Text Available La fibrosis quística se debe a la ausencia o defecto del canal transmembrana regulador de la fibrosis quística (CFTR, un canal de cloruro codificado en el gen cftr que juega un papel clave en la homeostasis del agua e iones. El CFTR es activado por el AMPc y se localiza en las membranas apicales y basolaterales de las vías aéreas, intestino y glándulas exocrinas. Una de sus funciones primarias en los pulmones es mantener la capa de líquido superficial a través de su función de canal y regular el canal epitelial de sodio sensible al amiloride (ENaC. Se han identificado más de 1900 mutaciones en el gen cftr. La enfermedad se caracteriza por secreciones viscosas en las glándulas exocrinas y por niveles elevados de cloruro de sodio en el sudor. En la fibrosis quística el CFTR no funciona y el ENaC está desregulado; el resultado es un aumento en la reabsorción de sodio y agua con la formación de un líquido viscoso. En las glándulas sudoríparas tanto el Na+ como el Cl- se retienen en el lumen causando una pérdida de electrolitos durante la sudoración y el NaCl se elimina al sudor. Así, los niveles elevados de NaCl son la base del test del sudor inducido por pilocarpina, un método de diagnóstico para la enfermedad. En esta revisión se discuten los movimientos de Cl- y Na+ en las glándulas sudoríparas y pulmón así como el papel del ENaC en la patogénesis de la enfermedad.

  10. Functional TRP and ASIC-like channels in cultured urothelial cells from the rat.

    Science.gov (United States)

    Kullmann, F Aura; Shah, M A; Birder, L A; de Groat, W C

    2009-04-01

    Transient receptor potential (TRP) and acid-sensing ion channels (ASIC) are molecular detectors of chemical, mechanical, thermal, and nociceptive stimuli in sensory neurons. They have been identified in the urothelium, a tissue considered part of bladder sensory pathways, where they might play a role in bladder function. This study investigated functional properties of TRP and ASIC channels in cultured urothelial cells from the rat using patch-clamp and fura 2 Ca(2+) imaging techniques. The TRPV4 agonist 4alpha-phorbol-12,13 didecanoate (4alpha-PDD; 1-5 microM) and the TRPA1/TRPM8 agonist icilin (50-100 microM) elicited transient currents in a high percentage of cells (>70%). 4alpha-PDD responses were suppressed by the TRPV4 antagonist HC-010961 (10 microM). The TRPV1 agonist capsaicin (1-100 microM) and the TRPA1/TRPM8 agonist menthol (5-200 microM) elicited transient currents in a moderate percentage of cells ( approximately 25%). All of these agonists increased intracellular calcium concentration ([Ca(2+)](i)). Most cells responded to more than one TRP agonist (e.g., capsaicin and 4alpha-PDD), indicating coexpression of different TRP channels. In the presence of the TRPV1 antagonist capsazepine (10 microM), changes in pH induced by HCl elicited ionic currents (pH 5.5) and increased [Ca(2+)](i) (pH 6.5) in approximately 50% of cells. Changes in pH using acetic acid (pH 5.5) elicited biphasic-like currents. Responses induced by acid were sensitive to amiloride (10 microM). In summary, urothelial cells express multiple TRP and ASIC channels, whose activation elicits ionic currents and Ca(2+) influx. These "neuron-like" properties might be involved in transmitter release, such as ATP, that can act on afferent nerves or smooth muscle to modulate their responses to different stimuli.

  11. Participation of peripheral TRPV1, TRPV4, TRPA1 and ASIC in a magnesium sulfate-induced local pain model in rat.

    Science.gov (United States)

    Srebro, Dragana; Vučković, Sonja; Prostran, Milica

    2016-12-17

    We previously showed that magnesium sulfate (MS) has systemic antinociceptive and local peripheral pronociceptive effects. The role of transient receptor potential (TRP) channels and acid-sensing ion channels (ASICs) in the mechanism of action of MS has not been investigated in detail. The aim of this study was to explore the participation of TRP channels in the pronociceptive action of MS in rats after its intraplantar injection. The paw withdrawal threshold (PWT) to mechanical stimuli was measured by the electronic von Frey test. Drugs that were tested were either co-administered with an isotonic pH-unadjusted or pH-adjusted solution of MS intraplantarily, or to the contralateral paw to exclude systemic effects. We found that the subcutaneous administration of both pH-adjusted (7.4) and pH-unadjusted (about 6.0) isotonic (6.2% w/v in water) solutions of MS induce the pain at the injection site. The pH-unadjusted MS solution-induced mechanical hyperalgesia decreased in a dose-dependent manner as a consequence of co-injection of capsazepine, a selective TRPV1 antagonist (20, 100 and 500pmol/paw), RN-1734, a selective TRPV4 antagonist (1.55, 3.1 and 6.2μmol/paw), HC-030031, a selective TRPA1 antagonist (5.6, 28.1 and 140nmol/paw), and amiloride hydrochloride, a non-selective ASIC inhibitor (0.83, 2.5 and 7.55μmol/paw). In pH-adjusted MS-induced hyperalgesia, the highest doses of TRPV1, TRPV4 and TRPA1 antagonists displayed effects that were, respectively, either similar, less pronounced or delayed in comparison to the effect induced by administration of the pH-unadjusted MS solution; the ASIC antagonist did not have any effect. These results suggest that the MS-induced local peripheral mechanical hyperalgesia is mediated via modulation of the activity of peripheral TRPV1, TRPV4, TRPA1 and ASICs. Specific local inhibition of TRP channels represents a novel approach to treating local injection-related pain. Copyright © 2016 IBRO. Published by Elsevier Ltd. All

  12. Acid solution is a suitable medium for introducing QX-314 into nociceptors through TRPV1 channels to produce sensory-specific analgesic effects.

    Directory of Open Access Journals (Sweden)

    He Liu

    Full Text Available BACKGROUND: Previous studies have demonstrated that QX-314, an intracellular sodium channel blocker, can enter into nociceptors through capsaicin-activated TRPV1 or permeation of the membrane by chemical enhancers to produce a sensory-selective blockade. However, the obvious side effects of these combinations limit the application of QX-314. A new strategy for targeting delivery of QX-314 into nociceptors needs further investigation. The aim of this study is to test whether acidic QX-314, when dissolves in acidic solution directly, can enter into nociceptors through acid-activated TRPV1 and block sodium channels from the intracellular side to produce a sensory-specific analgesic effect. METHODOLOGY/PRINCIPAL FINDINGS: Acidic solution or noradrenaline was injected intraplantarly to induce acute pain behavior in mice. A chronic constrictive injury model was performed to induce chronic neuropathic pain. A sciatic nerve blockade model was used to evaluate the sensory-specific analgesic effects of acidic QX-314. Thermal and mechanical hyperalgesia were measured by using radiant heat and electronic von Frey filaments test. Spinal Fos protein expression was determined by immunohistochemistry. The expression of p-ERK was detected by western blot assay. Whole cell clamp recording was performed to measure action potentials and total sodium current in rats DRG neurons. We found that pH 5.0 PBS solution induced behavioral hyperalgesia accompanied with the increased expression of spinal Fos protein and p-ERK. Pretreatment with pH 5.0 QX-314, and not pH 7.4 QX-314, alleviated pain behavior, inhibited the increased spinal Fos protein and p-ERK expression induced by pH 5.0 PBS or norepinephrine, blocked sodium currents and abolished the production of action potentials evoked by current injection. The above effects were prevented by TRPV1 channel inhibitor SB366791, but not by ASIC channel inhibitor amiloride. Furthermore, acidic QX-314 employed adjacent to the

  13. Role of peripheral sigma-1 receptors in ischaemic pain: Potential interactions with ASIC and P2X receptors.

    Science.gov (United States)

    Kwon, S G; Roh, D H; Yoon, S Y; Choi, S R; Choi, H S; Moon, J Y; Kang, S Y; Kim, H W; Han, H J; Beitz, A J; Oh, S B; Lee, J H

    2016-04-01

    The role of peripheral sigma-1 receptors (Sig-1Rs) in normal nociception and in pathologically induced pain conditions has not been thoroughly investigated. Since there is mounting evidence that Sig-1Rs modulate ischaemia-induced pathological conditions, we investigated the role of Sig-1Rs in ischaemia-induced mechanical allodynia (MA) and addressed their possible interaction with acid-sensing ion channels (ASICs) and P2X receptors at the ischaemic site. We used a rodent model of hindlimb thrombus-induced ischaemic pain (TIIP) to investigate their role. Western blot was performed to observe changes in Sig-1R expression in peripheral nervous tissues. MA was measured after intraplantar (i.pl.) injections of antagonists for the Sig-1, ASIC and P2X receptors in TIIP rats or agonists of each receptor in naïve rats. Sig-1R expression significantly increased in skin, sciatic nerve and dorsal root ganglia at 3 days post-TIIP surgery. I.pl. injections of the Sig-1R antagonist, BD-1047 on post-operative days 0-3 significantly attenuated the development of MA during the induction phase, but had no effect on MA when given during the maintenance phase (days 3-6 post-surgery). BD-1047 synergistically increased amiloride (an ASICs blocker)- and TNP-ATP (a P2X antagonist)-induced analgesic effects in TIIP rats. In naïve rats, i.pl. injection of Sig-1R agonist PRE-084 alone did not produce MA; but it did induce MA when co-administered with either an acidic pH solution or a sub-effective dose of αβmeATP. Peripheral Sig-1Rs contribute to the induction of ischaemia-induced MA via facilitation of ASICs and P2X receptors. Thus, peripheral Sig-1Rs represent a novel therapeutic target for the treatment of ischaemic pain. © 2015 European Pain Federation - EFIC®

  14. Unbiased View of Synaptic and Neuronal Gene Complement in Ctenophores: Are There Pan-neuronal and Pan-synaptic Genes across Metazoa?

    Science.gov (United States)

    Moroz, Leonid L; Kohn, Andrea B

    2015-12-01

    transcriptomes from 10 different ctenophores did not detect recognized orthologs of synthetic enzymes encoding several classical, low-molecular-weight (neuro)transmitters; glutamate signaling machinery is one of the few exceptions. Novel peptidergic signaling molecules were predicted for ctenophores, together with the diversity of putative receptors including SCNN1/amiloride-sensitive sodium channel-like channels, many of which could be examples of a lineage-specific expansion within this group. In summary, our analysis supports the hypothesis of independent evolution of neurons and, as corollary, a parallel evolution of synapses. We suggest that the formation of synaptic machinery might occur more than once over 600 million years of animal evolution. © The Author 2015. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

  15. Study population and treatment titration in the International Nifedipine GITS Study: Intervention as a Goal in Hypertension Treatment (INSIGHT).

    Science.gov (United States)

    Brown, M J; Castaigne, A; de Leeuw, P W; Mancia, G; Rosenthal, T; Ruilope, L M

    1998-12-01

    To ascertain the baseline characteristics of the high-risk hypertensive patients entering the International Nifedipine GITS Study: Intervention as a Goal in Hypertension Treatment (INSIGHT). To determine the success of single and combination therapy in achieving target blood pressures in such a population. INSIGHT is a double-blind, prospective outcome trial comparing the efficacy of the calcium channel blocker, nifedipine GITS, and the thiazide, co-amilozide, in preventing myocardial infarction and stroke. We recruited 2996 men and 3454 women, aged 55-80 years, with blood pressure during placebo run-in >150/95 mmHg or isolated systolic blood pressure >160 mmHg from nine countries. Treatment allocation to nifedipine GITS 30 mg daily or co-amilozide (hydrochlorothiazide 25 mg/amiloride 5 mg) once daily was performed by minimization rather than randomization to balance additional risk factors. This was followed by four optional increases in treatment: dose-doubling of the primary drug, addition of atenolol 25/50 mg or enalapril 5/10 mg, and then any other hypotensive drug excluding calcium blockers or diuretics. Target blood pressure was 140/90 mmHg or a fall > or = 20/10 mmHg. Blood pressure at randomization was 172+/-15 / 99+/-9 mmHg. Thirteen per cent of the patients were previously untreated. The proportions of each additional risk factors were: smoking > 10/day, 29%; cholesterol > 6.43 mmol/l, 52%; family history of premature myocardial infarction or stroke, 21%; diabetes mellitus 20%; left ventricular hypertrophy, 10%; previous myocardial infarction, other presentations of coronary heart disease, and peripheral vascular disease, each 6%; proteinuria, 3%. Fifty-five per cent of patients had one additional risk factor, whereas 33%, 9% and 3% had two, three or more additional risk factors, respectively. The blood pressure (and falls in blood pressure) at the end of titration and at 1 year after minimization was 139+/-12 / 82+/-7 mmHg (33+/-15 / 17+/-9) in the 5226

  16. Prevention of hypertension in patients with pre-hypertension: protocol for the PREVER-prevention trial

    Directory of Open Access Journals (Sweden)

    Neto José

    2011-03-01

    Full Text Available Abstract Background Blood pressure (BP within pre-hypertensive levels confers higher cardiovascular risk and is an intermediate stage for full hypertension, which develops in an annual rate of 7 out of 100 individuals with 40 to 50 years of age. Non-drug interventions to prevent hypertension have had low effectiveness. In individuals with previous cardiovascular disease or diabetes, the use of BP-lowering agents reduces the incidence of major cardiovascular events. In the absence of higher baseline risk, the use of BP agents reduces the incidence of hypertension. The PREVER-prevention trial aims to investigate the efficacy, safety and feasibility of a population-based intervention to prevent the incidence of hypertension and the development of target-organ damage. Methods This is a randomized, double-blind, placebo-controlled clinical trial, with participants aged 30 to 70 years, with pre-hypertension. The trial arms will be chlorthalidone 12.5 mg plus amiloride 2.5 mg or identical placebo. The primary outcomes will be the incidence of hypertension, adverse events and development or worsening of microalbuminuria and of left ventricular hypertrophy in the EKG. The secondary outcomes will be fatal or non-fatal cardiovascular events: myocardial infarction, stroke, heart failure, evidence of new sub-clinical atherosclerosis, and sudden death. The study will last 18 months. The sample size was calculated on the basis of an incidence of hypertension of 14% in the control group, a size effect of 40%, power of 85% and P alpha of 5%, resulting in 625 participants per group. The project was approved by the Ethics committee of each participating institution. Discussion The early use of blood pressure-lowering drugs, particularly diuretics, which act on the main mechanism of blood pressure rising with age, may prevent cardiovascular events and the incidence of hypertension in individuals with hypertension. If this intervention shows to be effective and safe

  17. Using Drugs to Probe the Variability of Trans-Epithelial Airway Resistance.

    Directory of Open Access Journals (Sweden)

    Kendra Tosoni

    Full Text Available Precision medicine aims to combat the variability of the therapeutic response to a given medicine by delivering the right medicine to the right patient. However, the application of precision medicine is predicated on a prior quantitation of the variance of the reference range of normality. Airway pathophysiology provides a good example due to a very variable first line of defence against airborne assault. Humans differ in their susceptibility to inhaled pollutants and pathogens in part due to the magnitude of trans-epithelial resistance that determines the degree of epithelial penetration to the submucosal space. This initial 'set-point' may drive a sentinel event in airway disease pathogenesis. Epithelia differentiated in vitro from airway biopsies are commonly used to model trans-epithelial resistance but the 'reference range of normality' remains problematic. We investigated the range of electrophysiological characteristics of human airway epithelia grown at air-liquid interface in vitro from healthy volunteers focusing on the inter- and intra-subject variability both at baseline and after sequential exposure to drugs modulating ion transport.Brushed nasal airway epithelial cells were differentiated at air-liquid interface generating 137 pseudostratified ciliated epithelia from 18 donors. A positively-skewed baseline range exists for trans-epithelial resistance (Min/Max: 309/2963 Ω·cm2, trans-epithelial voltage (-62.3/-1.8 mV and calculated equivalent current (-125.0/-3.2 μA/cm2; all non-normal, P<0.001. A minority of healthy humans manifest a dramatic amiloride sensitivity to voltage and trans-epithelial resistance that is further discriminated by prior modulation of cAMP-stimulated chloride transport.Healthy epithelia show log-order differences in their ion transport characteristics, likely reflective of their initial set-points of basal trans-epithelial resistance and sodium transport. Our data may guide the choice of the background set

  18. [Water homeostasis in the living: molecular organization, osmoregulatory reflexes and evolution].

    Science.gov (United States)

    Acher, R

    2002-06-01

    aquaporin 2; 2) the urea transporter UT1; 3) the amiloride-sensitive sodium channel ENaC; 4) the inward-rectifying potassium channel ROM-K1; 5) the chloride channel CFTR. Modulation of each effector is ensured by an A kinase anchoring protein (AKAP). The effectors are transported from cytosol to apical membrane through transport vesicles equipped with membrane fusion proteins (such as VAMP2) that recognize specific apical membrane proteins (such as syntaxins). Water flows into principal cells from collecting duct lumen through AQP2 and flows out to interstitium through AQP3 and AQP4 and finally reachs blood circulation through AQP1 capillaries. Vasopressin orchestrates the live effectors so as to keep water and solutes reabsorptions in balance with volaemia and osmolality set points.

  19. Evidence against the rescue of defective DeltaF508-CFTR cellular processing by curcumin in cell culture and mouse models.

    Science.gov (United States)

    Song, Yuanlin; Sonawane, N D; Salinas, Danieli; Qian, Liman; Pedemonte, Nicoletta; Galietta, Luis J V; Verkman, A S

    2004-09-24

    Curcumin, the yellow colored component of the spice turmeric, has been reported to rescue defective DeltaF508-cystic fibrosis transmembrane conductance regulator (CFTR) cellular processing in homozygous mutant mice, restoring nasal potential differences and improving survival (Egan, M. E., Pearson, M., Weiner, S. A., Rajendran, V., Rubin, D., Glockner-Pagel, J., Canny, S., Du, K., Lukacs, G. L., and Caplan, M. J. (2004) Science 304, 600-602). Because of the implied potential use of curcumin or similar compounds in the therapy of cystic fibrosis caused by the DeltaF508 mutation, we tried to reproduce and extend the pre-clinical data of Egan et al. Fluorometric measurements of iodide influx in Fischer rat thyroid cells expressing DeltaF508-CFTR showed no effect of curcumin (1-40 microm) when added for up to 24 h prior to assay in cells grown at 37 degrees C. Controls, including 27 degrees C rescue and 4 mm phenylbutyrate at 37 degrees C, were strongly positive. Also, curcumin did not increase short circuit current in primary cultures of a human airway epithelium homozygous for DeltaF508-CFTR with a 27 degrees C rescue-positive control. Nasal potential differences in mice were measured in response to topical perfusion with serial solutions containing amiloride, low Cl-, and forskolin. Robust low Cl- and forskolin-induced hyperpolarization of 22 +/- 3 mV was found in wild type mice, with 2.1 +/- 0.4 mV hyperpolarization in DeltaF508 homozygous mutant mice. No significant increase in Cl-/forskolin hyperpolarization was seen in any of the 22 DeltaF508 mice studied using different curcumin preparations and administration regimens, including that used by Egan et al. Assay of serum curcumin by ethyl acetate extraction followed by liquid chromatography/mass spectrometry indicated a maximum serum concentration of 60 nm, well below that of 5-15 microm, where cellular effects by sarcoplasmic/endoplasmic reticulum calcium pump inhibition are proposed to occur. Our results do not

  20. Significance of the neurotensin receptor Na+/H+-exchanger 1 axis in human pancreatic cancer cells

    International Nuclear Information System (INIS)

    Olszewski, U.

    2009-01-01

    bicarbonate buffer. Amiloride-sensitive proton flux effected by NHE1 was stimulated 2 - 2.7-fold by Lys 8 -Ψ-Lys 9 NT(8-13) in BxPC-3 and PANC-1 cells, respectively. NHE1 was phosphorylated in response to the NT analog in BxPC-3, however, not in MIA PaCa-2 cells, and screening of changes in the phosphorylation status of selected proteins in response to Lys 8 -Ψ-Lys 9 NT(8-13) revealed participation of ERK1/2, p38α MAPK and mitogen- and stress-activated kinase 1/2 (MSK1/2) in responses of BxPC-3 and HT-29 cells, whereas Src signaling was stimulated in MIA PaCa-2 cells. Functional involvement of ERK1/2, p38α MAPK and MSK1/2 in stimulation of NHE1 activity by the NT analog was proved by inhibition of these kinases using PD 98059, SC 68376 and dimethyl fumarate (DMF), respectively. Extracellular acidosis stimulates production of interleukin-8 (IL-8), a crucial metastatic factor, in pancreatic cancer cells and, accordingly, Lys 8 -Ψ-Lys 9 NT(8-13) was found to stimulate secretion of IL-8 in BxPC-3 and PANC-1 cells in an amiloride-sensitive manner and to enable PANC-1 cells to migrate through an extracellular matrix gel. Genome-wide microarray analysis revealed distinct alterations in gene expression patterns of BxPC-3 and MIA PaCa-2 cells, with genes upregulated by Lys 8 -Ψ-Lys 9 NT(8-13) in BxPC-3 cells coding for components of the cytoskeleton and cell adhesion, hypoxia-inducible factor-1α and glycolytic enzymes, among others. In conclusion, NT-NTR1 signaling was shown to contribute to the emergence of an increased invasive potential of pancreatic cancer cells by triggering intracellular alkalinization and localized extracellular acidification, activation of stress-associated MSK1/2 signaling and production of IL-8, besides its minor effect on cell proliferation. (author) [de

  1. Caracterización del canal epitelial de sodio en sinciciotrofoblasto de placenta humana preeclamptica Characterization of the epithelial sodium channel in human pre-eclampsia syncytiotrophoblast

    Directory of Open Access Journals (Sweden)

    Silvana del Mónaco

    2006-02-01

    Full Text Available El sinciciotrofoblasto (SCT de placenta humana regula la transferencia de solutos y agua entre la sangre fetal y materna. En el presente trabajo observamos que el canal de sodio ENaC (asociado a cuadros como el síndrome de Liddle y pseudohipoaldosteronismo está presente en la membrana apical del SCT y que la subunidad a del canal tiene una expresión reducida en placentas con hipertensión gestacional (preeclampsia. Realizamos estudios a nivel de expresión de ARN (RT-PCR y a nivel proteico (western blot e inmunohistoquímica. En la línea celular BeWo (modelo de SCT humano el canal se encuentra presente y la expresión del mismo es regulada por las hormonas aldosterona, vasopresina, estradiol y progesterona. Analizamos la actividad del ENaC por electrofisiología y observamos corrientes sensibles a amiloride (10 µM cuando las células BeWo se cultivaron 12 horas con aldosterona (100 nM. Esta corriente presentó una magnitud 20 veces mayor que las corrientes basales, un potencial de reversión cercano a 3 mV y una conductancia de 127 ± 26 pS/pF entre los pulsos de -60 y -140 mV aplicados. Las características de esta corriente son similares a las producidas por ENaC en otros tejidos y evidencian la presencia de un canal funcional. El papel del ENaC en el SCT es poco comprendido, aunque la diferencia de expresión en la preeclampsia podría tener consecuencias para el transporte placentario de agua y iones. Nuestros datos son un aporte para futuros estudios de los mecanismos involucrados en la patofisiología de la preeclampsia.The syncytiotrophoblast (SCT, a multinucleated epithelium forming the outer layer of chorionic villi, acts in human placenta as a transporting barrier regulating the transference of nutrients, solutes and water between maternal and fetal blood. Electrolyte homeostasis and extracellular fluid volume are maintained primarily by regulated Na+ transport. The present study was conducted to analyze the presence of the

  2. Neural influences on human intestinal epithelium in vitro.

    Science.gov (United States)

    Krueger, Dagmar; Michel, Klaus; Zeller, Florian; Demir, Ihsan E; Ceyhan, Güralp O; Slotta-Huspenina, Julia; Schemann, Michael

    2016-01-15

    We present the first systematic and, up to now, most comprehensive evaluation of the basic features of epithelial functions, such as basal and nerve-evoked secretion, as well as tissue resistance, in over 2200 surgical specimens of human small and large intestine. We found no evidence for impaired nerve-evoked epithelial secretion or tissue resistance with age or disease pathologies (stomach, pancreas or colon cancer, polyps, diverticulitis, stoma reversal). This indicates the validity of future studies on epithelial secretion or resistance that are based on data from a variety of surgical specimens. ACh mainly mediated nerve-evoked and basal secretion in the small intestine, whereas vasoactive intestinal peptide and nitric oxide were the primary pro-secretory transmitters in the large intestine. The results of the present study revealed novel insights into regional differences in nerve-mediated secretion in the human intestine and comprise the basis by which to more specifically target impaired epithelial functions in the diseased gut. Knowledge on basic features of epithelial functions in the human intestine is scarce. We used Ussing chamber techniques to record basal tissue resistance (R-basal) and short circuit currents (ISC; secretion) under basal conditions (ISC-basal) and after electrical field stimulation (ISC-EFS) of nerves in 2221 resectates from 435 patients. ISC-EFS was TTX-sensitive and of comparable magnitude in the small and large intestine. ISC-EFS or R-basal were not influenced by the patients' age, sex or disease pathologies (cancer, polyps, diverticulitis). Ion substitution, bumetanide or adenylate cyclase inhibition studies suggested that ISC-EFS depended on epithelial cAMP-driven chloride and bicarbonate secretion but not on amiloride-sensitive sodium absorption. Although atropine-sensitive cholinergic components prevailed for ISC-EFS of the duodenum, jejunum and ileum, PG97-269-sensitive [vasoactive intestinal peptide (VIP) receptor 1

  3. Measles Virus Enters Breast and Colon Cancer Cell Lines through a PVRL4-Mediated Macropinocytosis Pathway.

    Science.gov (United States)

    Delpeut, Sebastien; Sisson, Gary; Black, Karen M; Richardson, Christopher D

    2017-05-15

    Measles virus (MeV) is a member of the family Paramixoviridae that causes a highly contagious respiratory disease but has emerged as a promising oncolytic platform. Previous studies of MeV entry focused on the identification of cellular receptors. However, the endocytic and trafficking pathways utilized during MeV entry remain poorly described. The contribution of each endocytic pathway has been examined in cells that express the MeV receptors SLAM (signaling lymphocyte-activating molecule) and PVRL4 (poliovirus receptor-like 4) (nectin-4). Recombinant MeVs expressing either firefly luciferase or green fluorescent protein together with a variety of inhibitors were used. The results showed that MeV uptake was dynamin independent in the Vero.hPVRL4, Vero.hSLAM, and PVRL4-positive MCF7 breast cancer cell lines. However, MeV infection was blocked by 5-( N -ethyl- N -propyl)amiloride (EIPA), the hallmark inhibitor of macropinocytosis, as well as inhibitors of actin polymerization. By using phalloidin staining, MeV entry was shown to induce actin rearrangements and the formation of membrane ruffles accompanied by transient elevated fluid uptake. Small interfering RNA (siRNA) knockdown of p21-activated kinase 1 (PAK1) demonstrated that MeV enters both Vero.hPVRL4 and Vero.hSLAM cells in a PAK1-independent manner using a macropinocytosis-like pathway. In contrast, MeV entry into MCF7 human breast cancer cells relied upon Rac1 and its effector PAK1 through a PVRL4-mediated macropinocytosis pathway. MeV entry into DLD-1 colon and HTB-20 breast cancer cells also appeared to use the same pathway. Overall, these findings provide new insight into the life cycle of MeV, which could lead to therapies that block virus entry or methods that improve the uptake of MeV by cancer cells during oncolytic therapy. IMPORTANCE In the past decades, measles virus (MeV) has emerged as a promising oncolytic platform. Previous studies concerning MeV entry focused mainly on the identification of

  4. Prevalence and control of hypertension in a Niger Delta semi urban community, Nigeria

    Directory of Open Access Journals (Sweden)

    Suleiman IA

    2013-03-01

    taking their drugs during the survey and only 12.7% (07/55 had regular adherence to medication and adequate BP control was achieved in 7.3% (04/55. Majority of the patients on drugs (21.8% (12/55 were either taking methydopa as monotherapy or in combination with amiloride and hydrochlorothiazide. Other drugs being taken by patients include lisinopril, propranolol, amlodipine, atenolol, nifedipine and low dose aspirin. Conclusion: The prevalence of hypertension in the semi urban community is 15.0% with a pre-hypertension in another 23.5%. There was poor control of blood pressure among previously hypertensive patients.

  5. El litio y su relación con la acuaporina-2 y el canal de sodio ENaC Lithium and its relation with the epithelial sodium channel and aquaporin-2

    Directory of Open Access Journals (Sweden)

    Luciano Galizia

    2012-04-01

    Full Text Available Desde hace más de cuarenta años que el litio es usado para el tratamiento de la enfermedad bipolar; recientes estudios sugieren también su utilidad en el trastorno cognitivo mínimo tipo amnésico. El litio es filtrado en el glomérulo y un 65-75% del mismo es reabsorbido en el túbulo contorneado proximal y en el asa ascendente de Henle por el transportador Na+, K+, 2Cl- y vía paracelular. Una pequeña fracción del litio entra en las células principales del túbulo colector por medio del canal epitelial de sodio sensible al amiloride (ENaC localizado en la membrana apical de la célula. Luego de 10- 20 años de tratamiento con litio los enfermos pueden desarrollar poliuria, acidosis tubular e insuficiencia renal crónica que puede terminar en una forma de diabetes que no responde a la arginina vasopresina llamada diabetes insípida nefrogénica. Se cree que estas fallas renales son consecuencias de una reducción en el número de moléculas de acuaporina 2 en la membrana apical. Las causas para esto son complejas. El litio es un poderoso inhibidor de la isoforma beta de la enzima glicógeno sintetasa quinasa y esto está asociado a una menor actividad de la adenilato ciclasa que lleva a una disminución en la concentración intracelular de cAMP. Esto finalmente interferiría con la síntesis de nuevas moléculas de acuaporina 2 y con el tráfico de ellas desde la zona subapical de la célula hacia la membrana celular, causando la disminución en la reabsorción de agua en la parte distal del nefrón.For more than 40 years lithium has been used to treat bipolar disorder and recent trials suggest a potential efficacy also in the treatment of the amnestic mild cognitive impairment. Lithium is filtered by the glomerulus and 65% - 75% of the filtered amount is reabsorbed along the proximal tubule and in the thick ascending limb of Henle's loop by the Na+, K+, 2Cl- transporter and via paracellular. A small fraction of lithium is reabsorbed in

  6. Targeting Cells With MR Imaging Probes: Cellular Interaction And Intracellular Magnetic Iron Oxide Nanoparticles Uptake In Brain Capillary Endothelial and Choroidal Plexus Epithelial Cells

    Science.gov (United States)

    Cambianica, I.; Bossi, M.; Gasco, P.; Gonzalez, W.; Idee, J. M.; Miserocchi, G.; Rigolio, R.; Chanana, M.; Morjan, I.; Wang, D.; Sancini, G.

    2010-10-01

    microscopy and flow cytometry we studied the cell uptake of magnetic SLNs derivatized with a fluorescent reporter molecule and of L-DOPA-TRITC coated NPs. Inhibition of the caveolae-mediated pathway by preincubation with filipin and nystatin did not modify the cellular uptake of these NPs in both cell lines. Furthermore a mild decrease of the NPs cell uptake was obtained after chlorpromazine and NaN3 pretreatment, which interferes with clathrin and energy-dependent endocytosis, and cytochalasin and amiloride pretreatment which interfere with macropinocytosis. NPs particle size as such can strongly affect the efficiency of cellular uptake and the mode of endocytosis. Considering that our L-DOPA and magnetic SLNs display a medium hydrodynamic size of 120 nm with a polydispersity index of 0.3, we can assume that the cell uptake process of these NPs may develop, depending the particle size, both via clathrin mediated endocytosis and macropinocytosis and only to less extent via the pathway of caveolae-mediated endocytosis. Taken together these results let us to conclude that SLNs iron loaded and iron based L-DOPA coated NPs are internalized into brain endothelial and choroidal plexus epithelial cells and this might provide the first step of an intracellular trafficking to transport these NPs between blood and brain.