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 V₁ receptor [11], we investigated AVP activation of calcium signaling pathways in cultured hippocampal neurons. Results of this investigation demonstrate that exposure of cultured hippocampal neurons prelabeled with [³H]myo-inositol to vasopressin induced a significant accumulation of [³H]inositol-1-phosphate ([³H]IP₁). The selective V₁ vasopressin receptor agonist, [Phe², Orn²]vasotocin, induced a s significant accumulation of [³H]IP₁ whereas a selective V₂ vasopressin receptor agonist, [deamino¹, d-Arg⁸]-vasopressin, did not. Moreover, V₁ agonist-induced accumulation of [³H]IP₁ was blocked by the selective V₁ vasopressin receptor antagonist d(CH₂)₅[Tyr(Me)²]-vasopressin. V₁ agonist-induced accumulation of [³H]IP₁ was concentration dependent and exhibited a steep inverted U-shaped curve that included both stimulation and inhibition of [³H]IP₁ accumulation. Time course analysis of V₁ agonist-induced accumulation of [³H]IP₁ revealed significant increase by 20 min which continued to be significantly elevated for 60 min. Investigation of the effect of closely related peptides on [³H]IP₁ accumulation indicated that the vasopressin metabolite peptide AVP_4-9 and oxytocin significantly increased [³H]IP₁ accumulation whereas the vasopressin metabolite peptide AVP_4-8 did not. AVP_4-9 and oxytocin induced [³H]IP₁ accumulation were blocked by the V₁ vasopressin receptor antagonist d(CH₂)₅[Tyr(Me)²]-vasopressin. V₁ receptor activation was associated with a pronounced rise in intracellular calcium. Results of calcium fluorometry studies indicated that V₁ agonist exposure induced a marked and sustained rise in intracellular calcium that exhibited oscillations. Interestingly, absence of calcium in the extracellular medium abolished both the rise in intracellular calcium and the appearance of oscillations. The loss of the intracellular calcium signal is not due to a failure to induce PIP₂ hydrolysis since activation of the phosphatidylinositol pathway occurred in the absence of extracellular calcium. V₁ agonist (250 nM) induced a highly significant increase in ⁴⁵Ca²⁺ uptake from the extracellular medium within 5 sec of exposure. ⁴⁵Ca²⁺ uptake remained significantly greater than basal for 300 sec. The increase in ⁴⁵Ca²⁺ uptake was followed by a significant inhibition of uptake by 20 min of exposure. These results indicate that in cultured hippocampal neurons, V₁ vasopressin receptor activation leads to activation of the phosphatidylinositol signaling pathway, uptake of calcium from the extracellular medium and induction of complex intracellular calcium signals. These data provide the first step in delineating the biochemical mechanism that underlies vasopressin-induced neutrophism.