Hormonal effects on calcium homeostasis in isolated hepatocytes.

Abstract

A new method is described to determine the cytosolic free Ca”-concentration in isolated hepatocytes. Calcium concentrations in the medium were monitored spectrophotometrically using arsenazo 111. Digitonin ( 5 pg/mg dry weight of cells) was added to make the plasma membrane permeable to Ca”, and the magnitude of the Ca“ change was measured for different initial external Ca2+ concentrations. The cytosolic free Ca2+ concentration was estimated by determining the Ca2+ null point when no net Ca2+ changes were observed upon addition of digitonin. Values for the Caz+ null point between 100 and 200 nm were obtained for normal hepatocytes from fed or fasted rats, depending upon the pH and Mg2+ concentration of the incubation medium. Rapid cell fractionation of calcium-loaded cells permitted determination of the intracellular calcium distribution under these conditions. In normal cells, the mitochondrial calcium content accounted for 60 to 70% of the total cell calcium content, with the remainder located mainly in the microsomes. This proportion was approximately constant over a 10-fold range of total cell calcium. Glucagon had no effect on the cytosolic free Ca2+. On the other hand, a-adrenergic stimulation increased the cytosolic free Ca2+ concentration 2to %fold; phenylephrine M) increased mean values from 0.19 ? 0.01 to 0.46 f 0.04 p~ within 2 min. The time course and dose-response relationship of cytosolic free Caz+ changes closely followed the increase of phosphorylase a. Rapid cell-fractionation studies showed that the calcium content of the mitochondrial fraction was decreased after norepinephrine addition to the intact cells, while that of the microsomal fraction was increased. The above effects were abolished by a-adrenergic antagonists but were little affected by /3-adrenergic antagonists. These data indicate that the mitochondrial calcium pool is highly labile and is influenced by an as yet unknown transducing signal which is regulated by interaction of a-adrenergic hormones with the plasma membrane receptor. Increased efflux of calcium from the mitochondria causes a rise of cytosolic free Ca2+ and regulates enzymes of carbohydrate metabolism possibly by enhanced binding of calcium to calmodulin.