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Oxytocin Receptor-Mediated Activation of Phosphoinositidase C and Elevation of Cytosolic Calcium in the Gonadotrope-Derived T3–1 Cell Line¹

Department of Obstetrics and Gynaecology, Christchurch School of Medicine (J.J.E.), Christchurch, New Zealand; and Department of Medicine, University of Bristol (W.F.-O., C.A.McA.), Bristol, BS2 8HW, United Kingdom

Address all correspondence and requests for reprints to: J. J. Evans, University Department of Obstetrics and Gynaecology, Christchurch Women’s Hospital, Private Bag 4711, Christchurch, New Zealand. E-mail: jevans{at}chmeds.ac.nz

Gonadotropes synthesize and secrete LH and FSH under the control of GnRH, which acts via phosphoinositidase C (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 T3–1 cells, a gonadotrope-derived cell line. Oxytocin, vasopressin, and vasotocin each stimulated accumulation of [³H]inositol phosphates in cells prelabeled with [³H]inositol, indicating activation of PIC. 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 GnRH, 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 PIC-coupled receptors in these cells. The demonstration that oxytocin acts directly via PIC-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.

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