Abstract
The secretory compartment is characterized by low luminal pH and high Ca2+ content. Previous studies in several cell types have shown that the size of the acidic Ca2+ pool, of which secretory granules represent a major portion, could be estimated by applying first a Ca2+ ionophore followed by agents that collapse acidic pH gradients. In the present study we have employed this protocol in the insulin-secreting cell line Ins-1 to determine whether the Ca2+ trapped in the secretory granules plays a role in exocytosis. The results demonstrate that a high proportion of ionophore-mobilizable Ca2+ in Ins-1 cells resides in the acidic compartment. The latter pool, however, does not significantly contribute to the [Ca2+]i changes elicited by thapsigargin and the inositol 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 reduces secretion. It is suggested that a high Ca2+ concentration in the secretory compartment is needed to sustain optimal exocytosis.
Highlights
The secretory compartment is characterized by low luminal pH and high Ca2؉ content
Previous studies in several cell types have shown that the size of the acidic Ca2؉ pool, of which secretory granules represent a major portion, could be estimated by applying first a Ca2؉ ionophore followed by agents that collapse acidic pH gradients
The results demonstrate that a high proportion of ionophore-mobilizable Ca2؉ in Ins-1 cells resides in the acidic compartment
Summary
(Received for publication, December 24, 1997, and in revised form, May 13, 1998). Wim J. Another line of evidence suggesting that intragranular Ca2ϩ is implicated in secretion comes from the recent identification of an acidic Ca2ϩ-binding protein, granule lattice Protein 1 (Grl1p), in dense core secretory granules of Tetrahymena thermophila that appears essential for regulated secretion [20] Another common feature between secretory vesicles and granules is their low luminal pH. The aim of the present study was to establish whether the Ca2ϩ stored within the acidic pool is important in the late steps of exocytosis For this purpose we employed as a model system the -cell line Ins-1, an insulin-secreting cell line established from a rat insulinoma that, among different lines, best retains the phenotype of -cells [27,28,29]. A low pH in the granules is not required for the late steps of secretion, the level of intragranular Ca2ϩ significantly affects the secretory profile
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