Search Results

You are looking at 1 - 1 of 1 items for

  • Author: J. L. Nicks x
  • Refine by Access: All content x
Clear All Modify Search
Restricted access

S. J. Hughes, A. Carpinelli, I. Niki, J. L. Nicks, and S. J. H. Ashcroft


We have studied the effects of vasopressin and tetradecanoyl phorbol acetate (TPA) on cytosolic free Ca2+ ([Ca2+]i) and insulin release in HIT-T15 β-cells. Saturable binding of [3H] [Arg8]-vasopressin to HIT cell microsomes indicated a single class of receptors with a dissociation constant (K d) 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. These data indicate that vasopressin-induced insulin release is associated with a large increase in [Ca2+]i via increased Ca2+ entry and that this signal is markedly reduced by protein kinase C activation. Thus, protein kinase C does not have a direct role in vasopressin-induced insulin release. Simultaneous addition of vasopressin and TPA promotes insulin release, possibly through increased sensitivity of the secretory system to Ca2+ on activation of protein kinase C by TPA.