title: Secretagogues modulate the calcium concentration in the endoplasmic reticulum of insulin-secreting cells. Studies in aequorin-expressing intact and permeabilized ins-1 cells.
abstract: 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 microM. 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+ >> 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.
authors.affiliation: Division of Clinical Biochemistry and Experimental Diabetology, Department of Internal Medicine, University Medical Center, CH-1211 Geneva 4, Switzerland.
authors.forename: P
authors.initials: P
authors.lastname: Maechler
authors.forename: E D
authors.initials: ED
authors.lastname: Kennedy
authors.forename: E
authors.initials: E
authors.lastname: Sebö
authors.forename: A
authors.initials: A
authors.lastname: Valeva
authors.forename: T
authors.initials: T
authors.lastname: Pozzan
authors.forename: C B
authors.initials: CB
authors.lastname: Wollheim
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000331:Aequorin / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D017136:Ion Transport; D007700:Kinetics; D007930:Leucine / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats
chemical_list: D007328:Insulin; D000331:Aequorin; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D002217:Carbachol; D007930:Leucine; D005947:Glucose; D002118:Calcium