The free level of calcium (Ca2+)i is a primary intracellular secretory signal coupling the stimulus to secretion. Understanding this process may hold the key to unraveling the beta cell defects in type II diabetes where alterations in the glucose sensitivity and defective dynamics of insulin release are major abnormalities of that disease. The mechanisms by which Ca2+ regulates insulin release are unclear, but may involve calmodulin (CaM). CaM interacts with many intracellular enzymes and proteins which it regulates. Using the analogy with smooth muscle, CaM should couple Ca2+ mediated secretory signals to the generatiom of energy essenital for insulin release by regulating phosphirylation of the myosin light chains. This proposal will use an unique system, a hamster beta cell line which responds in a highly regulated manner to secretagogues including glucose to probe how Ca2+ regulates exocytosis. These studies will utilize static incubations and a perifusion system we have developed to correlate rapid secretory dynamics with biochemical changes in the cell. The (Ca2+)i will be measured during stimulation or inhibition of insulin secretion utilizing a new fluorescent probe, Quin-2. Ca2+CaM-mediated phosphorylations in HIT cells will be characterized quantitating CaM-mediated protein kinase activity in a cell free system and in vivo phosphorylation utilizing p32 and 2D gel systems. The major phosphoproteins identified during secretion will be further characterized by peptide mapping and phosphoamino acid analysis. The temporal relationship between P-light chain phosphorylation and the acute release of insulin will be examined using new systems developed for studies in smooth muscle. The anti-CaM drug, W13, will be used to block insulin release in a reversible manner analying the phosphorylation patterns before, during and after treatment of HIT cells with W13 and its inactive analogue. Polyclonal and monoclonal antibodies to CaM will be microinjected into the HIT cells using preloaded red cells to study the effect of CaM neutralization on insulin release, phosphorylation and (Ca2+)i levels. These studies should establish the molecular events linking Ca2+ signaling to insulin secretion.
