title: Stimulated Ca2+ influx raises mitochondrial free Ca2+ to supramicromolar levels in a pancreatic beta-cell line. Possible role in glucose and agonist-induced insulin secretion.
abstract: The effects of stimulated Ca2+ influx on cytosolic ([Ca2+]c) or intramitochondrial free Ca2+ ([Ca2+]m) were examined in the new pancreatic beta-cell line, INS-1. [Ca2+]c was monitored by video imaging of single fura-2-loaded INS-1 cells, or in populations of cells transfected with non-targeted (cytosolic) aequorin. [Ca2+]m was measured after transfection with aequorin targeted to the mitochondria by fusion of the gene in frame with the signal peptide of cytochrome c oxidase subunit VIII. Two physiological stimuli of native beta-cells, glucose and ATP, raised [Ca2+]c in INS-1 cells largely by stimulating Ca2+ influx. Thus, glucose (20 mM) induced repetitive transient increases in [Ca2+]c (0.42 min-1, mean amplitude 229 nM above 102 nM basal). These transients were largely due to periodic stimulation of Ca2+ influx through voltage-sensitive Ca2+ channels, since they could be rapidly and reversibly blocked by chelation of external Ca2+, by addition of the hyperpolarizing agent diazoxide, or with the Ca2+ channel blocker SR 7037. ATP, by contrast, caused single transient [Ca2+]c increases, to about 300 nM above basal levels, which could be inhibited by > 90% upon external Ca2+ chelation. Challenge of aequorin-transfected cells with ATP increased [Ca2+]m to 4 microM or above, an effect blocked by EGTA. Furthermore, plasma membrane depolarization with high K+, used as a glucose surrogate to mimic, in a synchronized fashion, the influx-induced Ca2+ transients observed at the single-cell level, also increased [Ca2+]m to > 4 microM. Similar increases in [Ca2+]m were also measured in other aequorin-transfected insulin-secreting cells, RINm5F, during mobilization of internal Ca2+ with carbachol. In contrast, glucose-induced changes in [Ca2+]m were below the level of detection in INS-1 cell populations, consistent with the asynchrony of the [Ca2+]c transients induced by this nutrient at the single-cell level, and the consequent small average [Ca2+]c rise. These data are in line with the view that stimulated Ca2+ influx into excitable cells raises [Ca2+]m as efficiently as internal Ca2+ mobilization in nonexcitable cells. In the case of INS-1 and pancreatic beta-cells, this may be important both to enhance oxidative metabolism, hence fueling the secretory process, and also to maintain the production of metabolic signaling molecules.
authors.affiliation: Department of Medicine, University of Geneva, Switzerland.
authors.forename: G A
authors.initials: GA
authors.lastname: Rutter
authors.forename: J M
authors.initials: JM
authors.lastname: Theler
authors.forename: M
authors.initials: M
authors.lastname: Murgia
authors.forename: C B
authors.initials: CB
authors.lastname: Wollheim
authors.forename: T
authors.initials: T
authors.lastname: Pozzan
authors.forename: R
authors.initials: R
authors.lastname: Rizzuto
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000331:Aequorin / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D003981:Diazoxide / Q000494:pharmacology; D004164:Diphosphonates / Q000494:pharmacology; D016257:Fura-2; D005947:Glucose / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004164:Diphosphonates; D007328:Insulin; D011994:Recombinant Proteins; C061154:belfosdil; D000331:Aequorin; D000255:Adenosine Triphosphate; D005947:Glucose; D003981:Diazoxide; D011188:Potassium; D002118:Calcium; D016257:Fura-2