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.
Scheenen
W J
WJ
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
Wollheim
C B
CB
Pozzan
T
T
Fasolato
C
C
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