Metabolic Regulation of Ca2+ Handling in Permeabilized Insulinoma Cells

Abstract

Signal generation in the pancreaticscell requires metabolism of the stimulatory fuel and is accompanied by increases in oxygen consumption and intracellular free Ca2+ (Hedeskov, 1980; Matschinskyet al., 1983; Meglasson and Matschinsky, 1986; Prentki and Matschinsky, 1987). We hypothesized that fuel phosphorylation decreased the cytosolic MgATP/MgADP ratio sufficiently to stimulate O2consumption, and simultaneously reduced the activity of Ca2+ -ATPase with resulting increases in free-Ca2+ levels. To explore this hypothesis, we varied the MgATP/MgADP ratio in permeabilized RINm5F insulinoma cells that maintain a low-medium Ca2+ concentration in the presence of MgATP. Either of the following was added: (1) creatine phosphokinase plus various fixed ratios of creatine/creatine phosphate, or (2) a cell-free extract of rat skeletal muscle that exhibits spontaneous oscillatory behavior of glycolysis, and linked oscillations in the MgATP/MgADP ratio when provided with glucose and a hexokinase (Tornheim, 1979). We found that the free-Ca2+ level maintained by the permeabilized cells varied inversely with the MgATP/MgADP ratio, regardless of the mechanism used to vary MgATP/MgADP. In addition, free Ca2+ was decreased by increasing levels of orthophosphate (Pi). Ca2+ levels oscillated in phase with glycolytic oscillations and correlated closely with the MgATP/MgADP ratio. Ca2+ oscillations were evoked by increasing glucose levels from 2.5 to 10 or 20 mM in the presence of glucokinase, whereas oscillations occurred at 2.5 mM glucose in the presence of hexokinase and were unaffected by increasing the glucose concentration. These results provide the first demonstration of a link between metabolite changes and free-Ca2+ levels, and suggest a mechanism by which fuel metabolism might be coupled to activation of the Ca2+ messenger system in pancreaticβcells.