Role of Protein Kinases in the Prolactin‐Induced Intracellular Calcium Rise in Chinese Hamster Ovary Cells Expressing the Prolactin Receptor

Abstract

There is still only limited understanding of the early steps of prolactin signal transduction in target cells. It has been shown that prolactin actions are associated with cell protein phosphorylation, Ca2+ increases, and so on. However, the link between the activation of kinases and calcium influx or intracellular Ca2+ mobilization has not yet been clearly established. Chinese hamster ovary (CHO) cells, stably transfected with the long form of rabbit mammary gland prolactin receptor (PRL-R) cDNA were used for PRL-R signal transduction studies. Spectrofluorimetric techniques were used to measure intracellular calcium ([Ca2+]i) in cell populations with Indo1 as a calcium fluorescent probe. We demonstrate that, although protein kinase C activation (PMA or DiC8) caused a calcium influx in CHO cells, prolactin-induced PKC activation was not responsible for the early effect of prolactin on [Ca2+]i. Activation of protein kinase A (PKA) or protein kinase G did not modify [Ca2+]i and inhibition of PKA pathway did not affect the prolactin response. In the same way, phosphatidylinositol-3 kinaseinhibition had no effect on the prolactin-induced Ca2+ increase. On the other hand, tyrosine kinase inhibitors (herbimycin A, lavendustin A, and genistein) completely blocked the effect of prolactin on [Ca2+]i (influx and release). W7, a calmodulin-antagonist, and a specific inhibitor of calmodulin kinases (KN-62), only blocked prolactin-induced Ca2+ influx but had no significant effect on Ca2+ release. Using pharmacological agents, we present new data concerning the involvement of protein phosphorylations in the early effects of prolactin on ionic channels in CHO cells expressing the long form of PRL-R. Our results suggest that, at least in the very early steps of prolactin signal transduction, serine-threonine phosphorylation does not participate in the prolactin-induced calcium increase. On the other hand, tyrosine phosphorylation is a crucial, very early step, since it controls K+ channel activation, calcium influx, and intracellular calcium mobilization. Calmodulin acts later, since its inhibition only blocks the prolactin-induced Ca2+ influx.

Expression of the long form of the prolactin receptor in magnocellular oxytocin neurons is associated with specific prolactin regulation of oxytocin neurons

“…Prolactin induces rapid changes in excitability of arcuate nucleus neurons (42) and ventromedial hypothalamic neurons (10,25,41) in vitro. The mechanism of prolactin action to induce these rapid effects is not fully understood, but it is not likely to involve activation of the , as well as a mobilization of intracellular Ca 2ϩ stores (52,66). In addition, prolactin induces a membrane hyperpolarization, through direct activation of Ca 2ϩ -dependent potassium channels (52).…”

Involvement of JAK2 and Src kinase tyrosine phosphorylation in human growth hormone-stimulated increases in cytosolic free Ca2+ and insulin secretion

“…This apparent discrepancy may be due to the fact that hGH mainly acts through the PRLR in rodent islets (4,5). Accordingly, the increase in [Ca 2ϩ ] i induced by activation of CHO cells transfected with PRLR requires activation of JAK2 and tyrosine phosphorylation (41,45). The JAK2-independent pattern of [Ca 2ϩ ] i increase, mediated through the GH receptor, suggests that rise in [Ca 2ϩ ] i may not be directly involved in the rat GH-stimulated proliferation in pancreatic ␤-cells (9).…”

“…Means Ϯ SE derived from 3 separate experiments for each are shown (*P Ͻ 0.05 for a chance difference by ANOVA). [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50], were cultured in RPMI 1640 tissue culture medium containing 10% (vol/vol) FBS, 100 IU/ml penicillin, and 100 g/ml streptomycin. The BRIN-BD11 ␤-cell line was established by electrofusion of RINm5F insulinoma cells with New England Deaconess Hospital (NEDH) rat pancreatic islet cells and shares the major features of normal pancreatic ␤-cells (35,36).…”

A Calcium/Calmodulin-dependent Activation of ERK1/2 Mediates JunD Phosphorylation and Induction of nur77 and20α-hsd Genes by Prostaglandin F2α in Ovarian Cells

“…5C, left panel). Since recent evidence revealed that activation of PKC by PMA promoted calcium influx in Chinese hamster ovarian (26) and glomerular mesangial (27) cells, we tested whether the induction of nur77 by PKC activation is through an increase in [Ca 2ϩ ] i . Cells were treated with either PMA alone or PMA plus BAPTA/AM.…”