Secretagogues modulate the calcium concentration in the endoplasmic reticulum of insulin-secreting cells -: Studies in aequorin-expressing intact and permeabilized INS-1 cells

The precise regulation of the Ca2+ concentration in the endoplasmic reticulum ([Ca2+](er)) is important for protein processing and signal transduction, In the pancreatic beta-cell, dysregulation of [Ca2+](er) may cause impaired insulin secretion. The Ca2+-sensitive photoprotein aequorin mutated to lower its Ca2+ affinity was stably expressed in the endoplasmic reticulum (ER) of rat insulinoma INS-1 cells. The steady state [Ca2+](er) was 267 +/- 9 mu M. Both the Ca2+-ATPase inhibitor cyclopiazonic acid and 4-chloro-m-cresol, an activator of ryanodine receptors, caused an almost complete emptying of ER Ca2+. The inositol 1,4,5-trisphosphate generating agonists, carbachol, and ATP, reduced [Ca2+](er) by 20-25%, Insulin secretagogues that raise cytosolic [Ca2+] by membrane depolarization increased [Ca2+](er) in the potency order K+ much greater than glucose > leucine, paralleling their actions in the cytosolic compartment, Glucose, which augmented [Ca2+](er) by about 25%, potentiated the Ca2+-mobilizing effect of carbachol, explaining the corresponding observation in cytosolic [Ca2+]. The filling of ER Ca2+ by glucose is not directly mediated by ATP production as shown by the continuous monitoring of cytosolic ATP in luciferase expressing cells. Both glucose and K+ increase [Ca2+](er), but only the former generated whereas the latter consumed ATP, Nonetheless, drastic lowering of cellular ATP with a mitochondrial uncoupler resulted in a marked decrease in [Ca2+](er), emphasizing the requirement for mitochondrially derived ATP above a critical threshold concentration. Using alpha-toxin permeabilized cells in the presence of ATP, glucose 6-phosphate did not change [Ca2+](er), invalidating the hypothesis that glucose acts through this metabolite. Therefore, insulin secretagogues that primarily stimulate Ca2+ influx, elevate [Ca2+](er) to ensure beta-cell homeostasis.