Postreceptor signalling of growth hormone and prolactin and their effects in the differentiated insulin-secreting cell line, INS-1.
Signal transduction of two mitogens for pancreatic beta-cells, GH and PRL, was investigated using the differentiated insulin-secreting cell line, INS-1. Addition of human GH (hGH) or ovine PRL in a serum-substitute medium increased growth, insulin content, and nutrient metabolism evaluated by tetrazolium salt reduction. hGH, bovine GH (bGH), and PRL also stimulated [3H]thymidine incorporation in a dose-dependent manner (1 pM - 1 nM). hGH induced cytosolic Ca2+ ([Ca2+]i) rises, which were transient, dependent on the presence of extracellular Ca2+, blocked by verapamil, calciseptine, and the hyperpolarizing agent diazoxide, suggesting that hGH stimulates Ca(2+)-influx through L-type Ca(2+)-channels. Similar effects on [Ca2+]i were observed with bGH or PRL. hGH caused membrane depolarization in a small proportion of the cells ( < 25%) as detected by cell-attached patch-clamp analysis. However, hGH failed to stimulate acute insulin secretion. hGH, bGH, and PRL promoted tyrosine phosphorylation of JAK2 tyrosine kinase. Verapamil inhibited neither [3H]thymidine incorporation nor JAK2 phosphorylation stimulated by hGH, whereas a tyrosine kinase inhibitor, lavendustin A, blocked the mitogenic effect. Involvement of cAMP is suggested because Rp-cyclic adenosine-3', 5'-monophosphorothioate, a competitive inhibitor of protein kinase A, abolished hGH-induced [Ca2+]i rises and DNA synthesis. cAMP appears to play a permissive role, although hGH failed to raise cellular cAMP levels. These results support the idea that activation of JAK2 is a major signaling event, whereas the [CA2+]i rise is not a prerequisite, for the mitogenic effects of GH and PRL in insulin-secreting cells.
Sekine
N
N
Division de Biochimie Clinique, University of Geneva, Switzerland.
Ullrich
S
S
Regazzi
R
R
Pralong
W F
WF
Wollheim
C B
CB
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D003470:Culture Media; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004247:DNA / Q000096:biosynthesis; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D013006:Growth Hormone / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D053614:Janus Kinase 2; D010636:Phenols / Q000494:pharmacology; D019000:Phosphotyrosine / Q000378:metabolism; D011388:Prolactin / Q000378:metabolism / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D012756:Sheep; D015398:Signal Transduction
D003470:Culture Media; D004791:Enzyme Inhibitors; D007328:Insulin; D010636:Phenols; D011518:Proto-Oncogene Proteins; D019000:Phosphotyrosine; C062898:lavendustin A; D011388:Prolactin; D013006:Growth Hormone; D004247:DNA; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium