Oxytocin receptor-mediated activation of phosphoinositidase C and elevation of cytosolic calcium in the gonadotrope-derived alpha T3-1 cell line

Gonadotropes synthesize and secrete LH and FSH under the control of GnRH, which acts via phosphoinositidase G (PIC)-linked G protein coupled receptors. Additionally, gonadotropin released from the pituitary is influenced by oxytocin, a peptide that has been shown to play a role in generation of the preovulatory LH surge. Although oxytocin receptors are present in the pituitary, studies have identified their presence on lactotropes but not on gonadotropes, raising the question of which cells act as the direct target of oxytocin in gonadotrope regulation. In this study, we examined effects of oxytocin on alpha T3-1 cells, a gonadotrope-derived cell line. Oxytocin, vasopressin, and vasotocin each stimulated accumulation of [H-3]inositol phosphates in cells prelabeled with [H-3]inositol, indicating activation of PIG. The rank order of potency (oxytocin > vasotocin > vasopressin) and sensitivity to inhibition by oxytocin and vasopressin receptor antagonists, revealed the effect to be mediated by oxytocin-selective receptors. Like other PIC activators, these nonapeptides caused biphasic (spike-plateau) increases in the cytosolic Ca2+. The spike response to oxytocin and GnRH were both retained in Ca2+-free medium, reflecting mobilization of intracellular Ca2+, and were comparably reduced by thapsigargin, implying mobilization of Ca2+ from a shared thapsigargin-sensitive intracellular pool. Brief stimulation with oxytocin, vasopressin, or vasotocin prevented subsequent Ca2+ responses to oxytocin, but not to GnRH, suggesting that the oxytocin receptor undergoes rapid homologous desensitization and reinforcing the interpretation that the nonapeptides act via the same receptor type. Oxytocin did not increase Ca2+ in cells stimulated with Gr;RK, whereas GnRH caused a spike Ca2+ increase even in the presence of oxytocin, implying that different mechanisms of desensitization (Ca2+ pool depletion and receptor uncoupling) are operating for two distinct PIG-coupled receptors in these cells. The demonstration tier, that oxytocin acts directly via PIG-linked, oxytocin-selective receptors to increase cytosolic Ca2+ in a gonadotrope-derived cell line is consistent with the possibility that oxytocin has a comparable effect on nonimmortalized gonadotropes.