Calcium Uptake in Isolated Hepatic Plasma‐Membrane Vesicles

Abstract

A liver plasma-membrane fraction capable of CaZ+ uptake was isolated. The fraction exhibited high Na', K + -ATPase, low glucose-6 phosphatase activity, and transported alanine in a Na+-dependent fashion. The uptake of Ca2+ was ATP-dependent; UTP, GTP, or CTP did not substitute for ATP. The presence of oxalate did not significantly alter the rate of uptake. The pH optimum of the reaction was basic (no uptake was visible at pH 6.8). These properties are at variance with those of the endoplasmic reticulum Ca2+ uptake system, which is oxalate-dependent, and has an acid pH optimum.The ATP-dependent Ca2+ uptake has a Km(Ca2+) of 1.4 x lop8 M and a Vmax of transport of 30 nmol x mg protein-' x min-l. No conclusive results were obtained on the calmodulin-sensitivity of the process: addition of calmodulin to the vesicles did not stimulate uptake, and the anti-calmodulin drug trifluoperazine had no inhibitory effect. However, another anti-calmodulin drug (R24571) had a limited, but statistically significant, inhibitory action. A partial release of the accumulated Ca2+ from the vesicles could be induced by the addition of Na', and incubation of the vesicles in a high Na+ medium (as compared to high K + medium) resulted in lower (about 25 %) calcium uptake. Partial release of the accumulated Ca2+ could be induced also by the addition of H'. The releasing effect of H', taken together with the absence of CaZ . The distribution of Ca2+ among the various cell phases is regulated at different sites. Mitochondria and the endoplasmic reticulum are of special importance [4,5], but it is also likely that calmodulin, which is present in the cytosol in micromolar concentration, influences the cytosolic level of free Ca2+. It is obvious, however, that the plasma membrane plays the ultimate role in the balancing of extracellular and intracellular Ca2+. This is so because most plasma membranes are somewhat permeable to Caz+. Since the Ca2+ gradient between the extracellular and intracellular compartements is in favour of influx, the CaZ+ that has entered the cell will eventually have to be ejected by energy-linked mechanisms. Indeed, perfusion of liver with 45Ca2 + In ' the presence of inhibitors of glycolysis and oxidative phosphorylation results in increased cellular Ca2+ [6]. Energy-dependent Caz + extrusion has now been demonstrated in many plasma membranes (for a review, see [7]), and a high-affinity Caz+-stimulated ATPase, possibly involved in CaZ + extrusion, has been recently isolated from those of liver [8]. The isolated liver enzyme is apparently regulated by an activator different from calmodulin [8].The problem of the extrusion of Ca2+ from liver has been approached in this work by studying the properties of the Ca2+ transport process in isolated plasma membrane vesicles. Extending previous work in this Laboratory [9] the results obtained have shown that an ATP-linked Ca2+ transport mechanism indeed exists. A Na+/Ca2+ exchange system, Abbreviations. Hepes = 4-(2-hydroxyethyl)-l-piperazineethanesul-

Hepatic adenosine triphosphate-dependent Ca2+ transport is mediated by distinct carriers on rat basolateral and canalicular membranes.

“…Although the values for the basolateral and canalicular Km (1.4 and 4.8. 10-8 M, respectively) agree closely with several previously reported values for the Km for ATP-dependent Ca2+ uptake (2,3,15) or (Ca2+-Mg2+)-ATPase activity (5,15) in mixed liver plasma membrane fractions, they are approximately an order of magnitude below current estimates of the cytosolic free Ca2+ concentration. As it is unlikely that hepatocyte Ca2+ transport is maximally driven at physiological concentrations of free Ca2+, this disparity might be explained by the absence of regulatory influences normally present in the intact cell.…”

ATP‐dependent Ca2+ transport in vesicles isolated from the bile canalicular region of the hepatocyte plasma membrane

“…4 5~~2 + uptake was measured with the Millipore filtration technique using filters having 0.22 pM pore diameter, essentially as described by Kraus-Friedman et al [12]. The incubation medium contained 100 mM KCl, 5 mM MgC12, 1 mM NaN3, 20 pM digitoxygenin, 20 mM Tris/Cl, pH 8, and 20 pM free Ca2+ (buffered with EGTA) containing 45Ca2+ (70 000 cpm/nmol) in a total volume of 0.5 ml.…”

Alterations in intracellular calcium compartmentation following inhibition of calcium efflux from isolated hepatocytes

“…This is achieved by the operation of a high-affinity Ca2 + translocase located in the hepatocyte plasma membrane [13], the activity of which does not appear to be calmodulin-regulated [14]. In recent studies we have found that both the Ca2 +-stimulated ATPase activity ofthe rat liver plasma membrane fraction and Ca2+ transport by inverted plasma membrane vesicles are critically dependent on free sulfhydryl groups [15].…”