CA2- A STUDY WITH INSULIN-SECRETING CELLS( DEPLETION FROM GRANULES INHIBITS EXOCYTOSIS )

The secretory compartment is characterized by low luminal pH and high Ca2+ content. Previous studies in several cell types have shown that t he size of the acidic Ca2+ pool, of which secretory granules represent a major portion, could be estimated by applying first a Ca2+ ionophor e followed by agents that collapse acidic pH gradients. In the present study we have employed this protocol in the insulin-secreting cell li ne Ins-1 to determine whether the Ca2+ trapped in the secretory granul es plays a role in exocytosis. The results demonstrate that a high pro portion of ionophore-mobilizable Ca2+ in Ins-1 cells resides in the ac idic compartment. The latter pool, however, does not significantly con tribute to the [Ca2+](i) changes elicited by thapsigargin and the inos itol trisphosphate-producing agonist carbachol. By monitoring membrane capacitance at the single cell level or by measuring insulin release in cell populations, we show that Ca2+ mobilization from nonacidic Ca2 + pools causes a profound and long lasting increase in depolarization- induced secretion, whereas breakdown of granule pH had no significant effect. In contrast, releasing Ca2+ from the acidic pool markedly redu ces secretion. It is suggested that a high Ca2+ concentration in the s ecretory compartment is needed to sustain optimal exocytosis.