Ca2+ depletion from granules inhibits exocytosis. A study with insulin-secreting cells.
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.
Department of Biomedical Sciences, Consiglio Nazionale delle Ricerche, Center of Biomembranes, University of Padova, Via G. Colombo 3, 35100 Padova Italy. scheenen@sci.kun.nl
W J
WJ
Scheenen
C B
CB
Wollheim
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T
Pozzan
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Fasolato
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002738:Chloroquine / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism*; D004553:Electric Conductivity; D005089:Exocytosis; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D008985:Monensin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D007328:Insulin; D007476:Ionophores; D002738:Chloroquine; D008985:Monensin; D002118:Calcium