Stimulation of insulin release by vasopressin in the clonal beta-cell line, HIT-T15: the role of protein kinase C.
We have studied the effects of vasopressin and tetradecanoyl phorbol acetate (TPA) on cytosolic free Ca2+ ([Ca2+]i) and insulin release in HIT-T15 beta-cells. Saturable binding of [3H] [Arg8]-vasopressin to HIT cell microsomes indicated a single class of receptors with a dissociation constant (Kd) of 2.5 nM and a total number of binding sites (Bmax) equal to 120 fmol/mg protein. [Arg8]-vasopressin (0.1-100 nM) elicited dose-dependent insulin release from HIT cells by up to 25-fold. This increase was dependent on the presence of extracellular glucose and was blocked by omission of extracellular Ca2+ or addition of verapamil. The stimulation was biphasic; a rapid but short-lived large increase in release was followed by a smaller sustained rise. Vasopressin also evoked a marked, concentration-dependent increase in [Ca2+]i which was also biphasic; an initial spike was followed by a sustained elevation. This increase also required glucose and was blocked by the absence of extracellular Ca2+ or the addition of verapamil. Pretreatment of the cells with TPA overnight to deplete protein kinase C activity did not affect the [Ca2+]i or insulin responses to vasopressin. However, short-term exposure to TPA markedly reduced glucose-induced steady-state [Ca2+]i, despite potentiating glucose-stimulated insulin release sevenfold, and blocked the [Ca2+]i increase induced by vasopressin. These inhibitory effects of TPA were absent in protein kinase C-depleted cells and were prevented by staurosporine. TPA had no significant effect on vasopressin-induced insulin release. Vasopressin did not modify the activity of ATP-sensitive K+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)
Hughes
S J
SJ
Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford, U.K.
Carpinelli
A
A
Niki
I
I
Nicks
J L
JL
Ashcroft
S J
SJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation; D007328:Insulin / Q000378:metabolism*; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D015221:Potassium Channels / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D014667:Vasopressins / Q000502:physiology*
D007328:Insulin; D015221:Potassium Channels; D014667:Vasopressins; D000255:Adenosine Triphosphate; D001120:Arginine; D011493:Protein Kinase C; D002118:Calcium