Astrocytes synthesize a series of regulatory peptides called endozepines, which act as endogenous ligands of benzodiazepine receptors. We have recently shown that one of these endozepines, the triakontatetraneuropeptide TTN, stimulates DNA synthesis in astroglial cells. The purpose of the present study was to determine the mechanism of action of TTN on cultured rat astrocytes. Binding of the peripheral-type benzodiazepine receptor ligand [3H]Ro5-4864 to intact astrocytes was displaced by TTN, whereas its C-terminal fragment (TTN[17-34], the octadecaneuropeptide ODN) did not compete for [3H]Ro5-4864 binding. Microfluorimetric measurement of cytosolic calcium concentrations ([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 in cultured astrocytes. Simultaneous administration of TTN (10(-8) M) and Ro5-4864 (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 channel blockers omega-conotoxin (10(-6) M) and mibefradil (10(-6) M), but was significantly 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-type benzodiazepine receptors, provokes chloride efflux, which in turn induces calcium influx via L-type calcium channels in rat astrocytes.
Animals Animals, Newborn