Jj. Evans et al., OXYTOCIN RECEPTOR-MEDIATED ACTIVATION OF PHOSPHOINOSITIDASE-C AND ELEVATION OF CYTOSOLIC CALCIUM IN THE GONADOTROPE-DERIVED ALPHA-T3-1 CELL-LINE, Endocrinology, 138(5), 1997, pp. 2049-2055
Gonadotropes synthesize and secrete LH and FSH under the control of Gn
RH, 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 i
n 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 ea
ch stimulated accumulation of [H-3]inositol phosphates in cells prelab
eled with [H-3]inositol, indicating activation of PIG. The rank order
of potency (oxytocin > vasotocin > vasopressin) and sensitivity to inh
ibition 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) increa
ses in the cytosolic Ca2+. The spike response to oxytocin and GnRH wer
e both retained in Ca2+-free medium, reflecting mobilization of intrac
ellular Ca2+, and were comparably reduced by thapsigargin, implying mo
bilization of Ca2+ from a shared thapsigargin-sensitive intracellular
pool. Brief stimulation with oxytocin, vasopressin, or vasotocin preve
nted subsequent Ca2+ responses to oxytocin, but not to GnRH, suggestin
g that the oxytocin receptor undergoes rapid homologous desensitizatio
n and reinforcing the interpretation that the nonapeptides act via the
same receptor type. Oxytocin did not increase Ca2+ in cells stimulate
d with Gr;RK, whereas GnRH caused a spike Ca2+ increase even in the pr
esence of oxytocin, implying that different mechanisms of desensitizat
ion (Ca2+ pool depletion and receptor uncoupling) are operating for tw
o distinct PIG-coupled receptors in these cells. The demonstration tie
r, that oxytocin acts directly via PIG-linked, oxytocin-selective rece
ptors to increase cytosolic Ca2+ in a gonadotrope-derived cell line is
consistent with the possibility that oxytocin has a comparable effect
on nonimmortalized gonadotropes.