Sample records for dns1-c-d-a2bu2 trp4 d-leu5enkephalin

  1. Delta opioid peptide (D-Ala 2, D-Leu 5) enkephalin: linking hibernation and neuroprotection.

    Borlongan, Cesario V; Wang, Yun; Su, Tsung-Ping


    Hibernation is a potential protective strategy for the peripheral, as well as for the central nervous system. A protein factor termed hibernation induction trigger (HIT) was found to induce hibernation in summer-active ground squirrels. Purification of HIT yielded an 88-kD peptide that is enriched in winter hibernators. Partial sequence of the 88-kD protein indicates that it may be related to the inhibitor of metalloproteinase. Using opioid receptor antagonists to elucidate the mechanisms of HIT, it was found that HIT targeted the delta opioid receptors. Indeed, delta opioid (D-Ala 2, D-Leu 5) enkephalin (DADLE) was shown to induce hibernation. Specifically, HIT and DADLE were found to prolong survival of peripheral organs, such as the lung, the heart, liver, and kidney preserved en bloc or as a single preparation. In addition, DADLE has been recently demonstrated to promote survival of neurons in the central nervous system. Exposure to DADLE dose-dependently enhanced cell viability of cultured primary rat fetal dopaminergic cells. Subsequent transplantation of these DADLE-treated dopaminergic cells into the Parkinsonian rat brain resulted in a two-fold increase in surviving grafted cells. Interestingly, delivery of DADLE alone protected against dopaminergic depletion in a rodent model of Parkinson s disease. Similarly, DADLE blocked and reversed the dopaminergic terminal damage induced by methamphetamine (METH). Such neuroprotective effects of DADLE against METH neurotoxicity was accompanied by attenuation of mRNA expressions of a tumor necrosis factor p53 and an immediate early gene c-fos. In parallel to these beneficial effects of DADLE on the dopaminergic system, DADLE also ameliorated the neuronal damage induced by ischemia-reperfusion following a transient middle cerebral artery occlusion. In vitro replication of this ischemia cell death by serum-deprivation of PC12 cells revealed that DADLE exerted neuroprotection in a naltrexone-sensitive manner. These

  2. The transient receptor potential, TRP4, cation channel is a novel member of the family of calmodulin binding proteins.

    Trost, C; Bergs, C; Himmerkus, N; Flockerzi, V


    The mammalian gene products, transient receptor potential (trp)1 to trp7, are related to the Drosophila TRP and TRP-like ion channels, and are candidate proteins underlying agonist-activated Ca(2+)-permeable ion channels. Recently, the TRP4 protein has been shown to be part of native store-operated Ca(2+)-permeable channels. These channels, most likely, are composed of other proteins in addition to TRP4. In the present paper we report the direct interaction of TRP4 and calmodulin (CaM) by: (1...

  3. Visualization of μ1 opiate receptors in rat brain by using a computerized autoradiographic subtraction technique

    Goodman, R.R.; Pasternak, G.W.


    The authors have developed a quantitative computerized subtraction technique to demonstrate in rat brain the regional distribution of μ 1 sites, a common very-high-affinity binding site for both morphine and the enkephalins. Low concentrations of [D-Ala 2 , D-Leu 5 ]enkephalin selectively inhibit the μ 1 binding of [ 3 H]dihydromorphine, leaving μ 2 -sites, while low morphine concentrations eliminate the μ 1 binding of [ 3 H][D-Ala 2 , D-Leu 5 ]enkephalin, leaving sigma sites. Thus, quantitative differences between images of sections incubated in the presence and absence of these low concentrations of unlabeled opioid represent μ 1 binding sites. The regional distributions of μ 1 sites labeled with [ 3 H]dihydromorphine were quite similar to those determined by using [ 3 H][D-Ala 2 , D-Leu 5 ]enkephalin. High levels of μ 1 binding were observed in the periaqueductal gray, medial thalamus, and median raphe, consistent with the previously described role of μ 1 sites in analgesia. Other regions with high levels of μ 1 binding include the nucleus accumbens, the clusters and subcallosal streak of the striatum, hypothalamus, medial habenula, and the medial septum/diagonal band region. The proportion of total specific binding corresponding to μ 1 sites varied among the regions, ranging from 14% to 75% for [ 3 H][D-Ala 2 , D-Leu 5 ]enkephalin and 20% to 52% for [ 3 H]dihydromorphine

  4. The distribution of multiple opiate receptors in bovine brain

    Ninkovic, M.; Hunt, S.P.; Emson, P.C.; Iversen, L.L.


    The distribution of μ and delta opiate receptors in bovine brain has been investigated using the selective radioligands [ 3 H]morphine and D-[ 3 H]Ala 2 , D-Leu 5 -enkephalin. Their distributions were found to vary independently through different brain areas with up to a 10-fold difference between the ratio of μ to delta binding sites for the substantia nigra and the dentate gyrus of the hippocampus. (Auth.)

  5. Selective localization of different types of opiate receptors in hippocampus as revealed by in vitro autoradiography

    Duka, T.; Wuester, M.; Schubert, P.; Stoiber, R.; Herz, A.


    The visualization of opiate binding sites within the hippocampus of the rat has been achieved by means of an in vitro autoradiography. In line with the concept of multiple opiate receptors, different opioid agonists revealed a particular distribution pattern. Whereas the selective delta-receptor agonist [ 3 H]D-Ala 2 , D-Leu 5 -enkephalin specifically labelled binding sites in the CA 2 area, [ 3 H]etorphine grains displayed a uniform dense distribution throughout the pyramidal cell layers from CA 1 to CA 4 . (Auth.)

  6. Site-directed alkylation of multiple opioid receptors. I. Binding selectivity

    James, I.F.; Goldstein, A.


    A method for measuring and expressing the binding selectivity of ligands for mu, delta, and kappa opioid binding sites is reported. Radioligands are used that are partially selective for these sites in combination with membrane preparations enriched in each site. Enrichment was obtained by treatment of membranes with the alkylating agent beta-chlornaltrexamine in the presence of appropriate protecting ligands. After enrichment for mu receptors, [ 3 H] dihydromorphine bound to a single type of site as judged by the slope of competition binding curves. After enrichment for delta or kappa receptors, binding sites for [ 3 H] [D-Ala2, D-Leu5]enkephalin and [3H]ethylketocyclazocine, respectively, were still not homogeneous. There were residual mu sites in delta-enriched membranes but no evidence for residual mu or delta sites in kappa-enriched membranes were found. This method was used to identify ligands that are highly selective for each of the three types of sites

  7. Suppression of guinea pig ileum induced contractility by plasma albumin of hibernators

    Bruce, David S.; Ambler, Douglas L.; Henschel, Timothy M.; Oeltgen, Peter R.; Nilekani, Sita P.; Amstrup, Steven C.


    Previous studies suggest that hibernation may be regulated by internal opioids and that the putative “hibernation induction trigger” (HIT) may itself be an opioid. This study examined the effect of plasma albumin (known to bind HIT) on induced contractility of the guinea pig ileum muscle strip. Morphine (400 nM) depressed contractility and 100 nM naloxone restored it. Ten milligrams of lyophilized plasma albumin fractions from hibernating ground squirrels, woodchucks, black bears, and polar bears produced similar inhibition, with partial reversal by naloxone. Five hundredths mg of d-Ala2-d-Leu5-enkephalin (DADLE) also inhibited contractility and naloxone reversed it. Conclusions are that hibernating individuals of these species contain an HIT substance that is opioid in nature and summer animals do not; an endogenous opioid similar to leu-enkephalin may be the HIT compound or give rise to it.

  8. Fluoxetine, a selective inhibitor of serotonin uptake, potentiates morphine analgesia without altering its discriminative stimulus properties or affinity for opioid receptors

    Hynes, M.D.; Lochner, M.A.; Bemis, K.G.; Hymson, D.L.


    The analgesic effect of morphine in the rat tail jerk assay was enhanced by the serotonin uptake inhibitor, fluoxetine. Tail jerk latency was not affected by fluoxetine alone. Morphine's affinity for opioid receptors labeled in vitro with 3 H-naloxone or 3 H-D-Ala 2 -D-Leu 5 -enkephalin was not altered by fluoxetine, which has no affinity for these sites at concentrations as high as 1000 nM. In rats trained to discriminate morphine from saline, fluoxetine at doses of 5 or 10 mg/kg were recognized as saline. Increasing the fluoxetine dose to 20 mg/kg did not result in generalization to either saline or morphine. The dose response curve for morphine generalization was not significantly altered by fluoxetine doses of 5 or 10 mg/kg. Those rats treated with the combination of morphine and 20 mg/kg of fluoxetine did not exhibit saline or morphine appropriate responding. Fluoxetine potentiates the analgesic properties of morphine without enhancing its affinity for opioid receptors or its discriminative stimulus properties. 30 references, 2 figures, 2 tables

  9. The effect of various opiate receptor agonists on the seizure threshold in the rat. Is dynorphin an endogenous anticonvulsant?

    Przewłocka, B; Stala, L; Lasoń, W; Przewłocki, R


    The effects of various opiate receptor agonists on the seizure threshold after an intravenous infusion of pentylenetetrazol were investigated in rats. The mu- and epsilon-receptor agonists, morphine (20-40 micrograms) and beta-endorphin (5-10 micrograms) show proconvulsant properties towards clonic and tonic seizures. The delta-receptor agonist (D-Ala2,D-Leu5-enkephalin, DADL 5-40 micrograms) and alpha-neoendorphin (20-40 micrograms) show pro- and anticonvulsant properties towards clonic and tonic seizures, respectively. Anticonvulsant properties of DADL are possibly due to its action on the spinal cord, since after the intrathecal injection this effect is still observed. Similarities between DADL and alpha-neoendorphin suggest that they may act through the same receptor. The kappa-receptor agonist dynorphin A (5-20 micrograms) and its degradation-resistant analogue D-Arg-dynorphin1-13 (10 micrograms) show significant anticonvulsant properties. Our present results suggest that the kappa-receptor agonist dynorphin may act physiologically as an endogenous anticonvulsant, in contrast to other opioid peptides.

  10. Opioid modulation of GABA release in the rat inferior colliculus

    Forge Andrew


    Full Text Available Abstract Background The inferior colliculus, which receives almost all ascending and descending auditory signals, plays a crucial role in the processing of auditory information. While the majority of the recorded activities in the inferior colliculus are attributed to GABAergic and glutamatergic signalling, other neurotransmitter systems are expressed in this brain area including opiate peptides and their receptors which may play a modulatory role in neuronal communication. Results Using a perfusion protocol we demonstrate that morphine can inhibit KCl-induced release of [3H]GABA from rat inferior colliculus slices. DAMGO ([D-Ala(2, N-Me-Phe(4, Gly(5-ol]-enkephalin but not DADLE ([D-Ala2, D-Leu5]-enkephalin or U69593 has the same effect as morphine indicating that μ rather than δ or κ opioid receptors mediate this action. [3H]GABA release was diminished by 16%, and this was not altered by the protein kinase C inhibitor bisindolylmaleimide I. Immunostaining of inferior colliculus cryosections shows extensive staining for glutamic acid decarboxylase, more limited staining for μ opiate receptors and relatively few neurons co-stained for both proteins. Conclusion The results suggest that μ-opioid receptor ligands can modify neurotransmitter release in a sub population of GABAergic neurons of the inferior colliculus. This could have important physiological implications in the processing of hearing information and/or other functions attributed to the inferior colliculus such as audiogenic seizures and aversive behaviour.

  11. Opioid modulation of GABA release in the rat inferior colliculus

    Tongjaroenbungam, Walaiporn; Jongkamonwiwat, Nopporn; Cunningham, Joanna; Phansuwan-Pujito, Pansiri; Dodson, Hilary C; Forge, Andrew; Govitrapong, Piyarat; Casalotti, Stefano O


    Background The inferior colliculus, which receives almost all ascending and descending auditory signals, plays a crucial role in the processing of auditory information. While the majority of the recorded activities in the inferior colliculus are attributed to GABAergic and glutamatergic signalling, other neurotransmitter systems are expressed in this brain area including opiate peptides and their receptors which may play a modulatory role in neuronal communication. Results Using a perfusion protocol we demonstrate that morphine can inhibit KCl-induced release of [3H]GABA from rat inferior colliculus slices. DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin) but not DADLE ([D-Ala2, D-Leu5]-enkephalin or U69593 has the same effect as morphine indicating that μ rather than δ or κ opioid receptors mediate this action. [3H]GABA release was diminished by 16%, and this was not altered by the protein kinase C inhibitor bisindolylmaleimide I. Immunostaining of inferior colliculus cryosections shows extensive staining for glutamic acid decarboxylase, more limited staining for μ opiate receptors and relatively few neurons co-stained for both proteins. Conclusion The results suggest that μ-opioid receptor ligands can modify neurotransmitter release in a sub population of GABAergic neurons of the inferior colliculus. This could have important physiological implications in the processing of hearing information and/or other functions attributed to the inferior colliculus such as audiogenic seizures and aversive behaviour. PMID:15353008

  12. Opiate-induced seizures: a study of mu and delta specific mechanisms.

    Snead, O C


    Two groups of experiments were conducted to determine if morphine- and enkephalin-induced seizures are specifically mediated by the mu and delta receptor, respectively. In the first experiments, designed to assess the ontogeny of mu- or delta-seizures, rats from 6 h to 85 days of age received implanted cortical and depth electrodes as well as an indwelling cannula in the lateral ventricle. Various amounts of the mu-receptor agonists, morphine and morphiceptin, and the delta agonists, D-Ala2-D-Leu5-enkephalin (DADL) and Tyr-D-Ser-Gly-Phe-Leu-Thr (DSLET), were then administered intracerebroventricularly (icv) with continuous EEG monitoring. The second experiments entailed use of the nonspecific opiate antagonist, naloxone, as well as the specific delta antagonist, ICI 154,129, against seizures induced by icv-administered morphine, morphiceptin, DADL, or DSLET. Both morphine and morphiceptin produced electrical seizure activity in rats as young as 5 days after birth. The drugs produced similar seizure activity in terms of electrical morphology, observed behavior, ontogeny, threshold dose, and reversibility with small doses of naloxone. In the pharmacologic experiments, icv naloxone blocked all opiate-induced seizures. ICI 154,129 blocked DSLET seizure, had little effect on enkephalin or DADL seizures, and no effect on morphine or morphiceptin seizures. These data indicate that DSLET seizures are delta-specific but that all other opiate-induced seizures studied may involve multiple opiate receptor-mediated mechanisms.

  13. Autoradiographic analysis of mu1, mu2, and delta opioid binding in the central nervous system of C57BL/6BY and CXBK (opioid receptor-deficient) mice

    Moskowitz, A.S.


    In this study the authors used semi-quantitative in vitro autoradiography to compare the levels of binding to central mu 1 , mu 2 , and delta opioid sites in two strains of mice, C57BL/6BY and CXBK. The CXBK strain is known to be deficient in whole brain opioid binding sites and to be less sensitive than the C57 strain to the analgesic and locomotor stimulatory effects of opiates and opioids. Delta sites were visualized using [ 3 H][D-Ala 2 -D-Leu 5 ]-enkephalin (DADL) plus a low concentration of morphine, total mu sites (mu 1 and mu 2 ) were visualized using [ 3 H]dihydromorphine (DHM), and mu 2 sites were visualized using [ 3 H]DHM plus a low concentration of DADL. Binding to mu 1 sites was determined by subtracting mu 2 binding from total mu binding. The authors found that the two strains did not consistently differ in the levels of delta sites. The CXBK strain, however, either had less or the same amount of mu binding as the C57 strain in all areas studied. The CXBK strain was especially deficient in mu 1 binding, particularly in areas involved in pain processing. (Auth.)

  14. Sigma and opioid receptors in human brain tumors

    Thomas, G.E.; Szuecs, M.; Mamone, J.Y.; Bem, W.T.; Rush, M.D.; Johnson, F.E.; Coscia, C.J.


    Human brain tumors and nude mouse-borne human neuroblastomas and gliomas were analyzed for sigma and opioid receptor content. Sigma binding was assessed using [ 3 H] 1, 3-di-o-tolylguanidine (DTG), whereas opioid receptor subtypes were measured with tritiated forms of the following: μ, [D-ala 2 , mePhe 4 , gly-ol 5 ] enkephalin (DAMGE); κ, ethylketocyclazocine (EKC) or U69,593; δ, [D-pen 2 , D-pen 5 ] enkephalin (DPDPE) or [D-ala 2 , D-leu 5 ] enkephalin (DADLE) with μ suppressor present. Binding parameters were estimated by homologous displacement assays followed by analysis using the LIGAND program. Sigma binding was detected in 15 of 16 tumors examined with very high levels found in a brain metastasis from an adenocarcinoma of lung and a human neuroblastoma (SK-N-MC) passaged in nude mice. κ opioid receptor binding was detected in 4 of 4 glioblastoma multiforme specimens and 2 of 2 human astrocytoma cell lines tested but not in the other brain tumors analyzed

  15. Combined autoradiographic-immunocytochemical analysis of opioid receptors and opioid peptide neuronal systems in brain

    Lewis, M.E.; Khachaturian, H.; Watson, S.J.


    Using adjacent section autoradiography-immunocytochemistry, the distribution of (TH)naloxone binding sites was studied in relation to neuronal systems containing (Leu)enkephalin, dynorphin A, or beta-endorphin immunoreactivity in rat brain. Brain sections from formaldehyde-perfused rats show robust specific binding of (TH)naloxone, the pharmacological (mu-like) properties of which appear unaltered. In contrast, specific binding of the delta ligand (TH)D-Ala2,D-Leu5-enkephalin was virtually totally eliminated as a result of formaldehyde perfusion. Using adjacent section analysis, the authors have noted associations between (TH)naloxone binding sites and one, two, or all three opioid systems in different brain regions; however, in some areas, no apparent relationship could be observed. Within regions, the relationship was complex. The complexity of the association between (TH)naloxone binding sites and the multiple opioid systems, and previous reports of co-localization of mu and kappa receptors in rat brain, are inconsistent with a simple-one-to-one relationship between a given opioid precursor and opioid receptor subtype. Instead, since differential processing of the three precursors gives rise to peptides of varying receptor subtype potencies and selectivities, the multiple peptide-receptor relationships may point to a key role of post-translational processing in determining the physiological consequences of opioid neurotransmission.

  16. Complete Analysis of a Biologically Active Tetrapeptide: A Project Utilizing Thin-Layer Chromatography and Tandem Quadrupole Mass Spectrometry

    Lefevre, Joseph W.; Dodsworth, David W.


    The biologically active tetrapeptide d-Ala-Gly-l-Phe-d-Leu ([des-Tyr1-d-Ala2-d-Leu5]enkephalin) was analyzed for its amino acid content and stereochemistry by normal and reversed-phase thin-layer chromatography (TLC), and its sequence was determined by tandem quadrupole mass spectrometry. The project involved sequential N-dansylation of a portion of the tetrapeptide, hydrolysis, isolation, and identification of the N-terminal amino acid as dansyl-alanine by comparison with standards using normal-phase TLC. A second portion of the tetrapeptide was hydrolyzed and the resulting four free amino acids were converted to their corresponding dansyl derivatives and purified by preparative normal-phase TLC. The three dansyl amino acids not identified previously were identified by TLC. The stereochemistry of each was determined by comparison with dansyl-dl-amino acid standards using reversed-phase TLC in the presence of ß-cyclodextrin, a chiral mobile phase additive. Finally, the correct amino acid sequence was determined by tandem quadrupole mass spectrometry. This project gives students valuable experience in microscale synthesis, both normal and reversed-phase TLC, stereochemical analysis, and mass spectrometry.

  17. Change in the properties of the opiate receptors of the brain under conditions of habituation of rats to morphine

    Zaitsev, S.V.; Sergeeva, M.G.; Chichenkov, O.N.; Petrov, V.E.; Varfolomeev, S.D.


    The influence of prolonged administration of morphine on the properties of the opiate receptors of the rat brain was investigated. For this purpose they conducted an analysis of the isotherms of binding of labeled μ-, σ-, and chi-ligands: morphine, D-Ala 2 , D-Leu 5 -enkephalin, and ethylketocyclazocin, with membrane preparations of the brains of rats tolerant to morphine, as well as the control animals. For a quantitative determination of the dissociation constants of the ligand-receptor complexes (K) and the concentration of the reagents ([Q]), they used differential method and the method of simulation modeling. It was shown that the values of K and [Q] for individual animals are subjected to substantial dispersion, whereas the ratios [Q]/K undergo minor individual fluctuations, both in the control group and in the group of rats tolerant to morphine. This permits the ratio [Q]/K to be singled out as one of the main parameters for comparing the properties of opiate receptors of various groups of animals. Using this criterion, as well as the method of simulated modeling, it was shown that the development of tolerance is accompanied by a change in the properties of the δ-receptors (the ratio [Q]/K decreases by a factor of more than two). In contrast to the δ-receptors, no significant influence of the tolerance on the properties of the μ- and chi-receptors, as well as the ultrahigh-affinity ligand binding sites, was detected

  18. Light microscopic autoradiographic localization of mu and delta opioid binding sites in the mouse central nervous system

    Moskowitz, A.S.; Goodman, R.R.


    Much work has been done on opioid systems in the rat CNS. Although the mouse is widely used in pharmacological studies of opioid action, little has been done to characterize opioid systems in this species. In the present study the distribution of mu and delta opioid binding sites in the mouse CNS was examined using a quantitative in vitro autoradiography procedure. Tritiated dihydromorphine was used to visualize mu sites and [3H-d-Ala2-d-Leu5]enkephalin with a low concentration of morphine was used to visualize delta sites. Mu and delta site localizations in the mouse are very similar to those previously described in the rat (Goodman, R.R., S.H. Snyder, M.J. Kuhar, and W.S. Young, 3d (1980) Proc. Natl. Acad. Sci. U.S.A. 77:6239-6243), with certain exceptions and additions. Mu and delta sites were observed in sensory processing areas, limbic system, extrapyramidal motor system, and cranial parasympathetic system. Differential distributions of mu and delta sites were noted in many areas. Mu sites were prominent in laminae I, IV, and VI of the neocortex, in patches in the striatum, and in the ventral pallidum, nucleus accumbens, medial and midline thalamic nuclei, medial habenular nucleus, interpeduncular nucleus, and laminae I and II of the spinal cord. In contrast, delta sites were prominent in all laminae of the neocortex, olfactory tubercle, diffusely throughout the striatum, and in the basal, lateral, and cortical nuclei of the amygdala. The determination of the differential distributions of opioid binding sites should prove useful in suggesting anatomical substrates for the actions of opiates and opioids

  19. Correlation between the distribution of 3H-labelled enkephalin in rat brain and the anatomical regions involved in enkephalin-induced seizures.

    Haffmans, J; Blankwater, Y J; Ukponmwan, O E; Zijlstra, F J; Vincent, J E; Hespe, W; Dzoljic, M R


    The correlation between the distribution of the intraventricularly (i.v.t.) administered delta agonist [3H](D-ala2,D-leu5)-enkephalin ([3H]DADL) and the anatomical regions involved in enkephalin-induced seizures has been studied in rat by using an autoradiographic method and recording of the electromyogram (EMG) and the electroencephalogram (EEG). The results indicate that within 10 min, the radioactivity of the intraventricularly administered drug reached all parts of the ventricular system, including the central canal of the spinal cord. However, within 2.5 min after the intraventricular administration of [3H]DADL, which corresponds to the onset of DADL-induced seizures, the substance appeared mainly in the left lateral ventricle and occasionally in the third ventricle. During the first 2.5 min the substance penetrated regularly into the surrounding periventricular tissue of the striatum, septum and hippocampus to a depth of about 100 microns. The most intensive and long-lasting epileptic discharges, exceeding 30 min were observed in the hippocampus, in contrast to the mild and short-lasting electrophysiological responses of the septum and corpus striatum. The experiments suggest that the short onset of enkephalin-induced excitatory phenomena is due to the rapid distribution and penetration of the substance in the surrounding periventricular tissue. According to these data, it is proposed that activation of delta opiate receptors, localized within the first 100 microns of the periventricular tissue, mainly in the hippocampus, is essential for the triggering of endorphin-induced seizure activity.

  20. Photoaffinity labeling of opiate receptors using intrinsically photoactive 3H-opiates

    Kooper, G.N.; Levinson, N.R.; Copeland, C.F.; Bowen, W.D.


    Opiate receptors in rat and cow brain membranes have been labeled irreversibly using the intrinsic photolability of 3H-opiates. Membranes were incubated with 3H-ligand and then irradiated with UV light of 254 nm. Nonspecific binding was determined in the presence of 10 microM unlabeled levallorphan. Irreversible binding was defined as binding which survived heat or acid denaturation of membranes. Specific incorporation of label into denatured samples was observed only when unbound or loosely bound 3H-ligand was washed free from the membranes prior to irradiation. There was a general correlation between photosensitivity of the 3H-ligand and its ability to photolabel receptors. Hence, photolabeling presumably results by covalent attachment of highly reactive species generated during photochemical decomposition of ligand. With 3H-etorphine, optimal irradiation time was 5 min. In addition to 3H-etorphine, receptors could be labeled irreversibly with 3H-oxymorphone, 3H-dihydromorphine, and 3H-ethylketocyclazocine. Of the specific binding present in irradiated, nondenatured samples, 45-60% remained attached to receptors upon denaturation. 3H-Ethylketocyclazocine exhibited an 86% yield of incorporation. Signal-to-noise levels of 50-80% could be achieved in denatured samples. Therefore, this method provides a means of covalently labeling opiate receptors in high yield and with high signal-to-noise ratios. The opioid peptides, 3H-D-Ala2,D-Leu5-enkephalin, 3H-D-Ser2,Leu5,Thr6-enkephalin, 3H-D-Ala2,Met5-enkephalin amide, and 3H-D-Ala2,N-MePhe4,Gly-ol5-enkephalin, as well as the benzomorphan, 3H-bremazocine, apparently lack the structural characteristics which allow photolabeling

  1. Peptidergic modulation of efferent sympathetic neurons in intrathoracic ganglia regulating the canine heart.

    Armour, J A


    When either substance P or vasoactive intestinal peptide was injected into an acutely decentralized intrathoracic sympathetic ganglion, short-lasting augmentation of cardiac chronotropism and inotropism was induced. These augmentations were induced before the fall in systemic arterial pressure occurred which was a consequence of these peptides leaking into the systemic circulation in enough quantity to alter peripheral vascular resistance directly. When similar volumes of normal saline were injected into an intrathoracic ganglion, no significant cardiac changes were induced. When substance P or vasoactive intestinal peptide was administered into an intrathoracic ganglion, similar cardiac augmentations were induced either before or after the intravenous administration of hexamethonium. In contrast, when these peptides were injected into an intrathoracic ganglion in which the beta-adrenergic blocking agent timolol (0.1 mg/0.1 ml of normal saline) had been administered no cardiac augmentation occurred. These data imply that in the presence of beta-adrenergic blockade intraganglionic administration of substance P or vasoactive intestinal peptide does not modify enough intrathoracic neurons to alter cardiac chronotropism and inotropism detectably. When neuropeptide Y was injected into an intrathoracic ganglion, no cardiac changes occurred. However, when cardiac augmentations were induced by sympathetic preganglionic axon stimulation these were enhanced following the intraganglionic administration of neuropeptide Y. As this effect occurred after timolol was administered into the ipsilateral ganglia, but not after intravenous administration of hexamethonium, it is proposed that the effects of neuropeptide Y are dependent upon functioning intrathoracic ganglionic nicotinic cholinergic synaptic mechanisms. Intravenous administration of either morphine or [D-ala2,D-leu5]enkephalin acetate did not alter the capacity of the preganglionic sympathetic axons to augment the heart

  2. Co-localization of hypocretin-1 and leucine-enkephalin in hypothalamic neurons projecting to the nucleus of the solitary tract and their effect on arterial pressure.

    Ciriello, J; Caverson, M M; McMurray, J C; Bruckschwaiger, E B


    Experiments were done to investigate whether hypothalamic hypocretin-1 (hcrt-1; orexin-A) neurons that sent axonal projections to cardiovascular responsive sites in the nucleus of the solitary tract (NTS) co-expressed leucine-enkephalin (L-Enk), and to determine the effects of co-administration of hcrt-1 and D-Ala2,D-Leu5-Enkephalin (DADL) into NTS on mean arterial pressure (MAP) and heart rate. In the first series, in the Wistar rat the retrograde tract-tracer fluorogold (FG) was microinjected (50nl) into caudal NTS sites at which L-glutamate (0.25 M; 10 nl) elicited decreases in MAP and where fibers hcrt-1 immunoreactive fibers were observed that also contained L-Enk immunoreactivity. Of the number of hypothalamic hcrt-1 immunoreactive neurons identified ipsilateral to the NTS injection site (1207 ± 78), 32.3 ± 2.3% co-expressed L-Enk immunoreactivity and of these, 2.6 ± 1.1% were retrogradely labeled with FG. Hcrt-1/L-Enk neurons projecting to NTS were found mainly within the perifornical region. In the second series, the region of caudal NTS found to contain axons that co-expressed hcrt-1 and L-Enk immunoreactivity was microinjected with a combination of hcrt-1 and DADL in α-chloralose anesthetized Wistar rats. Microinjection of DADL into NTS elicited depressor and bradycardia responses similar to those elicited by microinjection of hcrt-1. An hcrt-1 injection immediately after the DADL injection elicited an almost twofold increase in the magnitude of the depressor and bradycardia responses compared to those elicited by hcrt-1 alone. Prior injections of the non-specific opioid receptor antagonist naloxone or the specific opioid δ-receptor antagonist ICI 154,129 significantly attenuated the cardiovascular responses to the combined hcrt-1-DADL injections. Taken together, these data suggest that activation of hypothalamic-opioidergic neuronal systems contribute to the NTS hcrt-1 induced cardiovascular responses, and that this descending hypothalamo

  3. Opioid-induced preconditioning: recent advances and future perspectives.

    Peart, Jason N; Gross, Eric R; Gross, Garrett J


    . Physiol. 285, H1435-H1443], the CNS [Borlongan, C.V., Wang, Y., Su, T.P., 2005. Delta opioid peptide (d-ala 2, d-leu 5) enkephalin: linking hiberation and neuroprotection. Front Biosci. 9, 3392-3398] and the myocardium [Romano, M.A., Seymour, E.M., Berry, J.A., McNish, R.A., Bolling, S.F., 2004. Relative contribution of endogenous opioids to myocardial ischemic tolerance. J Surg Res. 118, 32-37; Peart, J.N., Gross, G.J., 2004a. Exogenous activation of delta- and kappa-opioid receptors affords cardioprotection in isolated murine heart. Basic Res Cardiol. 99(1), 29-37]. For the purpose of this review, we will focus primarily on the protective effects of opioids against post-reperfusion myocardial stunning and infarction.

  4. Effects of casoxin 4 on morphine inhibition of small animal intestinal contractility and gut transit in the mouse

    Glen S Patten


    Full Text Available Glen S Patten1,2, Richard J Head1, Mahinda Y Abeywardena1,21CSIRO Preventative Health National Research Flagship, Adelaide, Australia; 2CSIRO Food and Nutritional Sciences, Adelaide, AustraliaBackground and aims: Chronic opioid analgesia has the debilitating side-effect of constipation in human patients. The major aims of this study were to: 1 characterize the opioid-specific antagonism of morphine-induced inhibition of electrically driven contraction of the small intestine of mice, rats, and guinea pigs; and 2 test if the oral delivery of small milk-derived opioid antagonist peptides could block morphine-induced inhibition of intestinal transit in mice.Methods: Mouse, rat, and guinea pig intact ileal sections were electrically stimulated to contract and inhibited with morphine in vitro. Morphine inhibition was then blocked by opioid subtype antagonists in the mouse and guinea pig. Using a polymeric dye, Poly R-478, the opioid antagonists casoxin 4 and lactoferroxin A were tested orally for blocking activity of morphine inhibition of gut transit in vivo by single or double gavage techniques.Results: The guinea pig tissue was more sensitive to morphine inhibition compared with the mouse or the rat (IC50 [half maximal inhibitory concentration] values as nmol/L ± SEM were 34 ± 3, 230 ± 13, and 310 ± 14 respectively (P < 0.01. The inhibitory influence of opioid agonists (IC50 in electrically driven ileal mouse preparations were DADLE ([D-Ala2, D-Leu5]-enkephalin ≥ met-enkephalin ≥ dynorphin A ≥ DAMGO ([D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin > morphine > morphiceptin as nmol/L 13.9, 17.3, 19.5, 23.3, 230, and 403 respectively. The mouse demonstrated predominantly Κ- and δ-opioid receptor activity with a smaller µ-opioid receptor component. Both mouse and guinea pig tissue were sensitive to casoxin 4 antagonism of morphine inhibition of contraction. In contrast to naloxone, relatively high oral doses of the µ-opioid receptor antagonists

  5. Cation-pi interactions stabilize the structure of the antimicrobial peptide indolicidin near membranes: molecular dynamics simulations

    Khandelia, Himanshu; Kaznessis, Yiannis N


    We implemented molecular dynamics simulations of the 13-residue antimicrobial peptide indolicidin (ILPWKWPWWPWRR-NH2) in dodecylphosphocholine (DPC) and sodium dodecyl sulfate (SDS) micelles. In DPC, a persistent cation-pi interaction between TRP11 and ARG13 defined the structure of the peptide...... near the interface. A transient cation-pi interaction was also observed between TRP4 and the choline group on DPC lipids. We also implemented simulation of a mutant of indolicidin in the DPC micelle where TRP11 was replaced by ALA11. As a result of the mutation, the boat-shaped conformation is lost...... and the structure becomes significantly less defined. On the basis of this evidence, we argue that cation-pi interactions determine the experimentally measured, well-defined boat-shaped structure of indolicidin. In SDS, the lack of such interactions and the electrostatic binding of the terminal arginine residues...

  6. Identification and characterization of two dermorphins from skin extracts of the Amazonian frog Phyllomedusa bicolor.

    Mignogna, G; Severini, C; Simmaco, M; Negri, L; Erspamer, G F; Kreil, G; Barra, D


    Skin extracts of South American hylid frogs of the subfamily Phyllomedusinae contain dermorphins and deltorphins, opioid heptapeptides highly selective for either mu or delta receptors. In all these peptides, a D-amino acid is present in the second position. The structure of the precursors for Ala-deltorphins was recently deduced from cloned cDNAs derived from skin of Phyllomedusa bicolor (Richter et al. (1990) Proc. Natl. Acad. Sci. USA 87, 4836-4839). From the amino acid sequence of these precursors, the existence of three peptides related to dermorphin could be predicted. From methanol extracts of skin of Ph. bicolor we have isolated two of these peptides, [Lys7]dermorphin-OH and [Trp4,Asn7]dermorphin-OH. The biological activity of these new dermorphins and their amidated counterparts is presented.

  7. Dermorphin-related peptides from the skin of Phyllomedusa bicolor and their amidated analogs activate two mu opioid receptor subtypes that modulate antinociception and catalepsy in the rat.

    Negri, L; Erspamer, G F; Severini, C; Potenza, R L; Melchiorri, P; Erspamer, V


    Three naturally occurring dermorphin-like peptides from the skin of the frog Phyllomedusa bicolor, the related carboxyl-terminal amides, and some substituted analogs were synthesized, their binding profiles to opioid receptors were determined, and their biological activities were studied in isolated organ preparations and intact animals. The opioid binding profile revealed a very high selectivity of these peptides for mu sites and suggested the existence of two receptor subtypes, of high and low affinity. The peptides tested acted as potent mu opioid agonists on isolated organ preparations. They were several times more active in inhibiting electrically evoked contractions in guinea pig ileum than in mouse vas deferens. When injected into the lateral brain ventricle or peritoneum of rats, the high-affinity-site-preferring ligand, [Lys7-NH2]dermorphin, behaved as a potent analgesic agent. By contrast, the low-affinity-site-preferring ligand, [Trp4,Asn7-NH2]dermorphin, produced a weak antinociception but an intense catalepsy.

  8. cDNAs encoding [D-Ala2]deltorphin precursors from skin of Phyllomedusa bicolor also contain genetic information for three dermorphin-related opioid peptides.

    Richter, K; Egger, R; Negri, L; Corsi, R; Severini, C; Kreil, G


    We present the structure of four precursors for [D-Ala2]deltorphins I and II as deduced from cDNAs cloned from skin of the frog Phyllomedusa bicolor. These contain the genetic information for one copy of [D-Ala2]deltorphin II and zero, one, or three copies of [D-Ala2]deltorphin I. In each case, the D-alanine of the end product is encoded by a normal GCG codon for L-alanine. In addition, the existence of three peptides related to dermorphin was predicted from the amino acid sequence of the precursors. These peptides were synthesized with a D-alanine in position 2 and their pharmacological properties were tested. Two of them, [Lys7]dermorphin-OH and [Trp4,Asn7]dermorphin-OH, were found to have roughly the same affinity and selectivity for mu-type opioid receptors as dermorphin.

  9. Discovery of melanocortin ligands via a double simultaneous substitution strategy based on the Ac-His-DPhe-Arg-Trp-NH2 template.

    Todorovic, Aleksandar; Lensing, Cody J; Holder, Jerry Ryan; Scott, Joseph W; Sorensen, Nicholas B; Haskell-Luevano, Carrie


    The melanocortin system regulates an array of diverse physiological functions including pigmentation, feeding behavior, energy homeostasis, cardiovascular regulation, sexual function, and steroidogenesis. Endogenous melanocortin agonist ligands all possess the minimal messaging tetrapeptide sequence His-Phe-Arg-Trp. Based on this endogenous sequence, the Ac-His1-DPhe2-Arg3-Trp4-NH 2 tetrapeptide has previously been shown to be a useful scaffold when utilizing traditional positional scanning approaches to modify activity at the various melanocortin receptors (MC1-5R). The study reported herein was undertaken to evaluate a double simultaneous substitution strategy as an approach to further diversify the Ac-His1-DPhe2-Arg3-Trp4-NH 2 tetrapeptide with concurrent introduction of natural and unnatural amino acids at positions 1, 2, or 4 as well as an octanoyl residue at the N-terminus. The designed library includes the following combinations: (A) double simultaneous substitution at capping group position (Ac) together with position 1, 2, or 4, (B) double simultaneous substitution at position 1 and 2, (C) double simultaneous substitution at position 1 and 4, and (D) double simultaneous substitution at position 2 and 4. Several lead ligands with unique pharmacologies were discovered in the current study including antagonists targeting the neuronal mMC3R with minimal agonist activity and ligands with selective profiles for the various melanocortin subtypes. The results suggest that the double simultaneous substitution strategy is a suitable approach in altering melanocortin receptor potency, selectivity, or converting agonists into antagonists and vice versa.

  10. Eco-Friendly Insecticide Discovery via Peptidomimetics: Design, Synthesis, and Aphicidal Activity of Novel Insect Kinin Analogues.

    Zhang, Chuanliang; Qu, Yanyan; Wu, Xiaoqing; Song, Dunlun; Ling, Yun; Yang, Xinling


    Insect kinin neuropeptides are pleiotropic peptides that are involved in the regulation of hindgut contraction, diuresis, and digestive enzyme release. They share a common C-terminal pentapeptide sequence of Phe(1)-Xaa(2)-Yaa(3)-Trp(4)-Gly(5)-NH2 (where Xaa(2) = His, Asn, Phe, Ser, or Tyr; Yaa(3) = Pro, Ser, or Ala). Recently, the aphicidal activity of insect kinin analogues has attracted the attention of researchers. Our previous work demonstrated that the sequence-simplified insect kinin pentapeptide analogue Phe-Phe-[Aib]-Trp-Gly-NH2 could retain good aphicidal activity and be the lead compound for the further discovery of eco-friendly insecticides which encompassed a broad array of biochemicals derived from micro-organisms and other natural sources. Using the peptidomimetics strategy, we chose Phe-Phe-[Aib]-Trp-Gly-NH2 as the lead compound, and we designed and synthesized three series, including 31 novel insect kinin analogues. The aphicidal activity of the new analogues against soybean aphid was determined. The results showed that all of the analogues exhibited aphicidal activity. Of particular interest was the analogue II-1, which exhibited improved aphicidal activity with an LC50 of 0.019 mmol/L compared with the lead compound (LC50 = 0.045 mmol/L) or the commercial insecticide pymetrozine (LC50 = 0.034 mmol/L). This suggests that the analogue II-1 could be used as a new lead for the discovery of potential eco-friendly insecticides.

  11. Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non-homologous end joining-mediated integrative transformation with genes from Saccharomyces cerevisiae.

    Yarimizu, Tohru; Nonklang, Sanom; Nakamura, Junpei; Tokuda, Shuya; Nakagawa, Takaaki; Lorreungsil, Sasithorn; Sutthikhumpha, Surasit; Pukahuta, Charida; Kitagawa, Takao; Nakamura, Mikiko; Cha-Aim, Kamonchai; Limtong, Savitree; Hoshida, Hisashi; Akada, Rinji


    The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus. Copyright © 2013 John Wiley & Sons, Ltd.

  12. Chymotrypsinogen C Genetic Variants, Including c.180TT, Are Strongly Associated With Chronic Pancreatitis in Pediatric Patients.

    Grabarczyk, Alicja Monika; Oracz, Grzegorz; Wertheim-Tysarowska, Katarzyna; Kujko, Aleksandra Anna; Wejnarska, Karolina; Kolodziejczyk, Elwira; Bal, Jerzy; Koziel, Dorota; Kowalik, Artur; Gluszek, Stanislaw; Rygiel, Agnieszka Magdalena


    Genetic studies in adults/adolescent patients with chronic pancreatitis (CP) identified chymotrypsinogen C (CTRC) genetic variants but their association with CP risk has been difficult to replicate. To evaluate the risk of CP associated with CTRC variants in CP pediatric patients-control study. The distribution of CTRC variants in CP pediatric cohort (n = 136, median age at CP onset 8 years) with no history of alcohol/smoking abuse was compared with controls (n = 401, median age 45). We showed that p.Arg254Trp (4.6%) and p.Lys247_Arg254del (5.3%) heterozygous mutations are frequent and significantly associated with CP risk in pediatric patients (odds ratio [OR] = 19.1; 95% CI 2.8-160; P = 0.001 and OR = 5.5; 95% CI 1.6-19.4; P = 0.001, respectively). For the first time, we demonstrated that the c.180TT genotype of common p.Gly60Gly variant is strong, an independent CP risk factor (OR = 23; 95% CI 7.7-70; P A variant, both CA and AA genotype, is significantly underrepresented in CP compared with controls (15% vs 35%; OR = 0.33; 95% CI 0.19-0.59; P risk factors. The c.493+51C>A variant may play a protective role against CP development.

  13. Mutations in POGLUT1, Encoding Protein O-Glucosyltransferase 1, Cause Autosomal-Dominant Dowling-Degos Disease

    Basmanav, F. Buket; Oprisoreanu, Ana-Maria; Pasternack, Sandra M.; Thiele, Holger; Fritz, Günter; Wenzel, Jörg; Größer, Leopold; Wehner, Maria; Wolf, Sabrina; Fagerberg, Christina; Bygum, Anette; Altmüller, Janine; Rütten, Arno; Parmentier, Laurent; El Shabrawi-Caelen, Laila; Hafner, Christian; Nürnberg, Peter; Kruse, Roland; Schoch, Susanne; Hanneken, Sandra; Betz, Regina C.


    Dowling-Degos disease (DDD) is an autosomal-dominant genodermatosis characterized by progressive and disfiguring reticulate hyperpigmentation. We previously identified loss-of-function mutations in KRT5 but were only able to detect pathogenic mutations in fewer than half of our subjects. To identify additional causes of DDD, we performed exome sequencing in five unrelated affected individuals without mutations in KRT5. Data analysis identified three heterozygous mutations from these individuals, all within the same gene. These mutations, namely c.11G>A (p.Trp4∗), c.652C>T (p.Arg218∗), and c.798-2A>C, are within POGLUT1, which encodes protein O-glucosyltransferase 1. Further screening of unexplained cases for POGLUT1 identified six additional mutations, as well as two of the above described mutations. Immunohistochemistry of skin biopsies of affected individuals with POGLUT1 mutations showed significantly weaker POGLUT1 staining in comparison to healthy controls with strong localization of POGLUT1 in the upper parts of the epidermis. Immunoblot analysis revealed that translation of either wild-type (WT) POGLUT1 or of the protein carrying the p.Arg279Trp substitution led to the expected size of about 50 kDa, whereas the c.652C>T (p.Arg218∗) mutation led to translation of a truncated protein of about 30 kDa. Immunofluorescence analysis identified a colocalization of the WT protein with the endoplasmic reticulum and a notable aggregating pattern for the truncated protein. Recently, mutations in POFUT1, which encodes protein O-fucosyltransferase 1, were also reported to be responsible for DDD. Interestingly, both POGLUT1 and POFUT1 are essential regulators of Notch activity. Our results furthermore emphasize the important role of the Notch pathway in pigmentation and keratinocyte morphology. PMID:24387993

  14. Mobilisation and remobilisation of a large archetypal pathogenicity island of uropathogenic Escherichia coli in vitro support the role of conjugation for horizontal transfer of genomic islands

    Hochhut Bianca


    Full Text Available Abstract Background A substantial amount of data has been accumulated supporting the important role of genomic islands (GEIs - including pathogenicity islands (PAIs - in bacterial genome plasticity and the evolution of bacterial pathogens. Their instability and the high level sequence similarity of different (partial islands suggest an exchange of PAIs between strains of the same or even different bacterial species by horizontal gene transfer (HGT. Transfer events of archetypal large genomic islands of enterobacteria which often lack genes required for mobilisation or transfer have been rarely investigated so far. Results To study mobilisation of such large genomic regions in prototypic uropathogenic E. coli (UPEC strain 536, PAI II536 was supplemented with the mobRP4 region, an origin of replication (oriVR6K, an origin of transfer (oriTRP4 and a chloramphenicol resistance selection marker. In the presence of helper plasmid RP4, conjugative transfer of the 107-kb PAI II536 construct occured from strain 536 into an E. coli K-12 recipient. In transconjugants, PAI II536 existed either as a cytoplasmic circular intermediate (CI or integrated site-specifically into the recipient's chromosome at the leuX tRNA gene. This locus is the chromosomal integration site of PAI II536 in UPEC strain 536. From the E. coli K-12 recipient, the chromosomal PAI II536 construct as well as the CIs could be successfully remobilised and inserted into leuX in a PAI II536 deletion mutant of E. coli 536. Conclusions Our results corroborate that mobilisation and conjugal transfer may contribute to evolution of bacterial pathogens through horizontal transfer of large chromosomal regions such as PAIs. Stabilisation of these mobile genetic elements in the bacterial chromosome result from selective loss of mobilisation and transfer functions of genomic islands.

  15. Nitrite reduction on a multimetallic porphyrin/polyoxotungstate layer-by-layer modified electrodes

    García, Macarena; Honores, Jessica; Quezada, Diego; Díaz, Carlos; Dreyse, Paulina; Celis, Freddy; Kubiak, Clifford P.; Canzi, Gabriele; Guzmán, Fernando


    Electro and photoelectrochemical reduction of nitrite in aqueous solution was studied using a multielectrocatalysts modified ITO electrode. ITO modification was carried out using the layer-by-layer (LBL) method, where sequential electrostatic assemblies were formed using a μ-(meso-5,10,15,20-tetra(pirydil)porphyrin)tetrakis{bis(bipyridine)chloride ruthenium (II)} [MTRP] n+ , coordinated in its central cavity with Mn(III), Zn(II) or Ni(II) as a cationic layer, and polyoxotungstate [SiW 12 O 40 ] 4− as the anionic layer. Electrochemical measurements and UV–vis spectroscopy were used to monitor the modification process. Optimal results were obtained when three layers were deposited onto the ITO surface and were stable in aqueous solution. The order of the multilayer formation was explored by comparing a modified electrode where [Zn(II)TRP] 4+ was the outermost layer with an electrode where [SiW 12 O 40 ] 4− was the outer layer. Results show that the best performing electrode is one with [SiW 12 O 40 ] 4− as the outer layer. Nitrite reduction on these electrode surfaces was studied in dark conditions and under light irradiation. Potential controlled electrolysis experiments were also performed, finding hydroxylamine, hydrazine and ammonia as the reduction products in dark conditions. Under light irradiation, only hydrazine and ammonia were found and, we observed an increase in the amount of obtained product. In this case, the electrolysis was carried out 150 mV less and half of time than in dark conditions. These results show that the combination of light and potential give rise to an improvement in the electrocatalytic properties of the modified electrodes. Continuous photolysis and IR spectroelectrochemical experiments were carried out to determinate the nature of this phenomena, evidencing the formation of an intermediary species between nitrite and [Mn(III)TRP] 5+.