Release of proenkephalin-derived opioid peptides from rat striatum in vitro and their rapid degradation |
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Authors: | G Patey A Cupo H Mazarguil J L Morgat J Rossier |
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Institution: | 1. Laboratoire de Physiologie Nerveuse, Centre National de la Recherche Scientifique, 91190 Gif-sur-Yvette, France;2. Centre d''Immunologie CNRS-INSERM, Marseille Luminy, France;3. Pharmacologie et Toxicologie Fondamentales, Centre National de la Recherche Scientifique, Toulouse, France;4. Service de Biochimie, Departement de Biologie, CEN de Saclay, Gif-sur-Yvette, France |
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Abstract: | In a previous paper we demonstrated that the heptapeptide Met]enkephalyl-Arg6-Phe7 was released from rat striatal slices by high K+ concentration and rapidly degraded by peptidases, even in the presence of the neutral endopeptidase 24.11 ("enkephalinase")-inhibitor, thiorphan (0.1 microM), the angiotensin-converting enzyme inhibitor, captopril (1 microM), and the aminopeptidase inhibitor, bestatin (20 microM). In this study the pattern of degradation of exogenous 3H]heptapeptide by rat striatal slices has been studied. The angiotensin-converting enzyme and aminopeptidase(s) were partly responsible for this degradation. In addition an enzymatic activity that cleaved the Phe4-Met5 bond was involved in the degradation of the heptapeptide by striatal slices. This activity was inhibited by the dipeptide Leu-Arg (1 mM) and the tripeptide Leu-Arg-Leu (1 mM). The simultaneous presence of thiorphan (0.1 microM), captopril (1 microM), bestatin (20 microM) and Leu-Arg (1 mM) almost completely inhibited the degradation of 3H]heptapeptide by striatal slices. In the presence of these peptidase inhibitors a concomitant release of Met]enkephalin, the heptapeptide Met]enkephalyl-Arg6-Phe7 and the octapeptide Met]enkephalyl-Arg6-Gly7-Leu8 was evoked by KCl or veratridine. The K+-evoked release was by a Ca2+-dependent mechanism and the release evoked by veratridine was blocked by tetrodotoxin. In both cases the ratio of Met]enkephalin to heptapeptide amounts released was close to that found in their common precursor, proenkephalin. Thus the enkephalinergic neuron appears to be capable of synthesizing, from a unique precursor, four different putative opioid neurotransmitters, namely Met]enkephalin, Leu]enkephalin, the heptapeptide Met]enkephalyl-Arg6-Phe7 and the octapeptide Met]enkephalyl-Arg6-Gly7-Leu8, to store these peptides and to release them upon depolarization. |
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Keywords: | BSA bovine serum albumin HPLC high performance liquid chromatography PBS phosphate-buffered saline RIA radioimmunoassay TEAF triethylamine-formate buffer |
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