VASOPRESSIN-INDUCED CALCIUM SIGNALING IN CULTURED HIPPOCAMPAL-NEURONS(VOL 661, PG 274, 1994)

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 V-1 vasopressi n receptor agonist, [Phe(2), Orn(2)]vasotocin, induced a significant a ccumulation of [H-3]IP1 whereas a selective V-2 vasopressin receptor a gonist, [deamino(1), D-Arg(8)]-vasopressin, did not. Moreover, V-1 ago nist-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 stim ulation and inhibition of [H-3]IP1 accumulation. Time course analysis of V-1 agonist-induced accumulation of [H-3]IP1 revealed significant i ncrease by 20 min which continued to be significantly elevated for 60 min. Investigation of the effect of closely related peptides on [H-3]I P1 accumulation indicated that the vasopressin metabolite peptide AVP( 4-9) and oxytocin significantly increased [H-3]IP1 accumulation wherea s the vasopressin metabolite peptide AVP(4-8) did not. AVP(4-9) and ox ytocin induced [H-3]IP1 accumulation were blocked by the V-1 vasopress in receptor antagonist d(CH2)(5)[Tyr(Me)(2)]-vasopressin. V-1 receptor activation was associated with a pronounced rise in intracellular cal cium. Results of calcium fluorometry studies indicated that V-1 agonis t exposure induced a marked and sustained rise in intracellular calciu m that exhibited oscillations. Interestingly, absence of calcium in th e extracellular medium abolished both the rise in intracellular calciu m and the appearance of oscillations. The loss of the intracellular ca lcium signal is not due to a failure to induce PIP2 hydrolysis since a ctivation of the phosphatidylinositol pathway occurred in the absence of extracellular calcium. V-1 agonist (250 nM) induced a highly signif icant increase in Ca-45(2+) uptake from the extracellular medium withi n 5 sec of exposure. Ca-45(2+) uptake remained significantly greater t han basal for 300 sec. The increase in Ca-45(2+) uptake was followed b y a significant inhibition of uptake by 20 min of exposure. These resu lts indicate that in cultured hippocampal neurons, V-1 vasopressin rec eptor activation reads to activation of the phosphatidylinositol signa ling pathway, uptake of calcium from the extracellular medium and indu ction of complex intracellular calcium signals. These data provide the first step in delineating the biochemical mechanism that underlies va sopressin-induced neurotrophism.