Résumé :
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Communication n° 306 Growth hormone secretagogues (GHS) stimulate GH release through the activation of a pituitary receptor. GHS binding sites were also found in various tissues including skeletal muscle. We recently showed that in vitro application of L-163,255, a nonpeptidic GHS, increased the cytosolic calcium concentration ([Ca2+]i) in skeletal muscle fibers, but the underlying mechanisms are still poorly understood (Pierno et al., Br J Pharmacol 139: 575-84, 2003). The ability of L-163,255 and ghrelin, the peptidic endogenous ligand for GHS receptor, to trigger variation in [Ca2+]i was studied in Fura-2-loaded skeletal muscle fibers mechanically isolated from the rat extensor digitorum longus (EDL) muscle. The two compounds induce changes in resting calcium level following biphasic dose-response relationships. Resting [Ca2+]i decreased when the GHS was applied at the lowest concentrations tested (<0.5µM for ghrelin and <5 µM for L-163,255), and slowly recovered to control value in ~30 min after drug removal. In contrast, above 0.5 µM ghrelin or 5 µM L-163,255, the GHS induced a significant [Ca2+]i increase characterized by a slow, 5-min long, rising phase reaching a plateau that lasted at least 30 min under continuous drug application. [Ca2+]i rapidly recovered to control value in ~1 min after drug removal. Although maximal calcium increase had similar amplitude value for both GHS tested, addition of 500 µM were necessary to obtain such a response for L-163,255 whereas 10 µM were sufficient for the endogenous ligand. Accordingly the whole L-163,255 dose-response curve was shifted rightward as compared to the ghrelin one. Calcium influx, as assessed by the manganese quenching technique, was not modified by L-163,255 or ghrelin application. Moreover, the GHS-induced [Ca2+]i increase was not blocked by nifedipine, by external calcium withdrawal nor by ruthenium red, suggesting that GHS mobilize intracellular calcium stores independently of ryanodine receptors. Surprisingly, the GHS-receptor antagonist [DLys-3]-GHRP-6 did not block the GHS calcium response but induced a [Ca2+]i increase similar to that observed for GHS. This finding suggests that ghrelin exerts important biological activities in skeletal muscle through the activation of one or more GHS-receptor subtypes different from the pituitary one. (Supported by Italian MIUR-FIRB RBNE01XMP4).
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