Synthesis, conformational analysis and biological activity of cyclic analogs of the octadecaneuropeptide ODN. Design of a potent endozepine antagonist.

The octadecaneuropeptide (ODN; QATVGDVNTDRPGLLDLK) and its C-terminal octapeptide (OP; RPGLLDLK), which exert anxiogenic activity, have been previously shown to increase intracellular calcium concentration ([Ca2+]i) in cultured rat astrocytes through activation of a metabotropic receptor positively coupled to phospholipase C. It has also been found that the [d-Leu5]OP analog possesses a weak antagonistic activity. The aim of the present study was to synthesize and characterize cyclic analogs of OP and [d-Leu5]OP. On-resin homodetic backbone cyclization of OP yielded an analog, cyclo1-8 OP, which was three times more potent and 1.4-times more efficacious than OP to increase [Ca2+]i in cultured rat astrocytes. Cyclo1-8 OP also mimicked the effect of both OP and ODN on polyphosphoinositide turnover. Conversely, the cyclo1-8 [d-Leu5]OP analog was totally devoid of agonistic activity but suppressed the effect of OP and ODN on [Ca2+]i and phosphoinositide metabolism in astrocytes. The structure of these cyclic analogs has been determined by two-dimensional 1H-NMR and molecular dynamics. Cyclo1-8 OP exhibited a single conformation characterized by a gamma turn comprising residues Pro2-Leu4 and a type III beta turn encompassing residues Leu5-Lys8. Cyclo1-8 [d-Leu5]OP was present as two equimolar conformers resulting from cis/trans isomerization of the Arg-Pro peptide bond. These pharmacological and structural data should prove useful for the rational design of non peptidic ODN analogs.

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, 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.