VASOPRESSIN-INDUCED CALCIUM SIGNALING IN CULTURED HIPPOCAMPAL NEURONS

We recently demonstrated that the neural peptide vasopressin (AVP) can act as a neurotrophic factor for hippocampal nerve cells in culture. Because the neurotrophic effect of vasopressin is mediated by the V-1 receptor [11], we investigated AVP activation of calcium signaling pat hways in cultured hippocampal neurons. Results of this investigation d emonstrate that exposure of cultured hippocampal neurons prelabeled wi th [H-3]myo-inositol to vasopressin induced a significant accumulation of [H-3]inositol-1-phosphate ([H-3]IP1). The selective V1 vasopressin receptor agonist, [Phe(2), Orn(2)]vasotocin, induced a significant ac cumulation of [H-3]IP1 whereas a selective V-1 vasopressin receptor ag onist, [deamino(1), D-Arg(8)]-vasopressin, did not. Moreover, V-1 agon ist-induced accumulation of [H-3]IP1 was blocked by the selective V-1 vasopressin receptor antagonist d(CH2)(5)[Tyr(Me)(2)]-vasopressin. V-1 agonist-induced accumulation of [H-3]IP1 was concentration dependent and exhibited a steep inverted U-shaped curve that included both stimu lation and inhibition of [H-3]IP1 accumulation. Time course analysis o f V-1 agonist-induced accumulation of [H-3]IP1 revealed significant in crease by 20 min which continued to be significantly elevated for 60 m in. Investigation of the effect of closely related peptides on [H-3]IP 1 accumulation indicated that the vasopressin metabolite peptide AVP(4 -9) and oxytocin significantly increased [H-3]IP1 accumulation whereas the vasopressin metabolite peptide AVP(4-8) did not. AVP(4-9) and oxy tocin induced [H-3]IP1 accumulation were blocked by the V-1 vasopressi n receptor antagonist d(CH2)(5)[Tyr(Me)2]vasopressin. V-1 receptor act ivation was associated with a pronounced rise in intracellular calcium . Results of calcium fluorometry studies indicated that V-1 agonist ex posure induced a marked and sustained rise in intracellular calcium th at exhibited oscillations. Interestingly, absence of calcium in the ex tracellular medium abolished both the rise in intracellular calcium an d the appearance of oscillations. The loss of the intracellular calciu m signal is not due to a failure to induce PIP2 hydrolysis since activ ation of the phosphatidylinositol pathway occurred in the absence of e xtracellular calcium. V-1 agonist (250 nM) induced a highly significan t increase in Ca-45(2+) uptake from the extracellular medium within 5 sec of exposure. Ca-45(2+) uptake remained significantly greater than basal for 300 sec. The increase in Ca-45(2+) uptake was followed by a significant inhibition of uptake by 20 min of exposure. These results indicate that in cultured hippocampal neurons, V-1 vasopressin recepto r activation leads to activation of the phosphatidylinositol signaling pathway, uptake of calcium from the extracellular medium and inductio n of complex intracellular calcium signals. These data provide the fir st step in delineating the biochemical mechanism that underlies vasopr essin-induced neurotrophism.