The triakontatetraneuropeptide TTN increases [Ca2+](i) in rat astrocytes through activation of peripheral-type benzodiazepine receptors

Astrocytes synthesize a series of regulatory peptides called endozepines, w hich act as endogenous ligands of benzodiazepine receptors. We have recentl y shown that one of these endozepines, the triakontatetraneuropeptide TTN, stimulates DNA synthesis in astroglial cells. The purpose of the present st udy was to determine the mechanism of action of TTN on cultured rat astrocy tes. Binding of the peripheral-type benzodiazepine receptor ligand [H-3]Ro5 -4864 to intact astrocytes was displaced by TTN, whereas its C-terminal fra gment (TTN[17-34], the octadecaneuropeptide ODN) did not compete for [H-3]R o5-4864 binding. Microfluorimetric measurement of cytosolic calcium concent rations ([Ca2+](i)) with the fluorescent probe indo-1 showed that TTN (10(- 10) to 10(-6) M) provokes a concentration-dependent increase in [Ca2+](i) i n cultured astrocytes. Simultaneous administration of TTN (10(-8) M) and Ro 5-4864 (10(-5) M) induced an increase in [Ca2+](i) similar to that obtained with Ro5-4864 alone. In contrast, the effects of TTN (10-8 M) and ODN (10( -8) M) on [Ca2+](i) were strictly additive. Chelation of extracellular Ca2 by EGTA (6 mM) or blockage of Ca2+ channels with Ni2+ (2 mM) abrogated the stimulatory effect of TTN. The calcium influx evoked by TTN (10(-7) M) or by Ro5-4864 (10(-5) M) was not affected by the N- and T-type calcium channe l blockers co-conotoxin (10(-6) M) and mibefradil (10(-6) M), but was signi ficantly reduced by the L-type calcium channel blocker nifedipine (10(-7) M ). Patch-clamp studies showed that, at negative potentials, TTN (10(-7) M) induced a sustained depolarization. Reduction of the chloride concentration in the extracellular solution shifted the reversal potential from 0 mV to a positive potential. These data show that TTN, acting through peripheral-t ype benzodiazepine receptors, provokes chloride efflux, which in turn induc es calcium influx via L-type calcium channels in rat astrocytes. (C) 2001Wi ley-Liss, Inc.