On the Mechanisms of ATP-Induced and Succinate-Induced Redistribution of Cations in Isolated Rat Liver Cells

Abstract

(Received Septcmber 1 Deccmbcr 7, 19x2) -EJH 59591. The ability of external ATP to induce calcium uptake in isolated rat liver cells was further characterized. Stimulation of calcium uptake was specific for ATP, other nucleotides or ATP metabolites had no comparable effect. ATP was dephosphorylated while stimulating calcium uptake, but there was no stoichiometry between ATP hydrolysis and calcium uptake nor did dephosphorylation depend on calcium concentration. ATP acted from outside and was dephosphorylated by an ccto-ATPase of the cells.2. In addition to its direct action, ATP enhanccd succinate-dependent calcium uptake in a coopcrativc fashion. This is best explained by different sites of action. ATP increases cell membrane permeability while succinatc stimulates uptake into mitochondria.3. ATP was able to lower Na' and K t gradients and the pH gradient between cells and incubation medium. Increasing calcium concentration countcracted this effect though calcium uptake was then stimulated.4. Succinate alone did not affect monovalent cation gradients but raised the pH gradient. It partially coiinteractcd the ATP effects on these gradients.5. Since catecholamine-like actions of ATP may be mediated by an increase in cytoplasmic calcium concentration, the action of extracellular ATP can be taken as a model t o study the role of calcium as a transmitter of hormone actions. From interdependence between ATP-stimulated and succinatc-stimulated calcium uptake, conclusions can be drawn on the resulting cytoplasmic calcium concentration and its effect on plasma membrane permeability.In a previous study, isolated liver cells proved to be a useful model to study the delicately poised equilibrium of calcium distribution across the various cellular compartments [l]. This was especially true for disclosing the role of cellular mitochondria in this interplay. Respiratory substrates, pH, inorganic phosphate, and the redox grade of pyridine nucleotides were found important in triggering calcium movements at the mitochondria1 site, whereas external ATP and La3+ induced calcium uptake at the plasma membrane site [I. 21.In this report, thc action of added ATP is further characterizcd. The responses of functional parameters such as cation distribution and the pH gradient between cells and incubation medium are compared with those seen after succinate addition. Relations between the different responses and the probablc changes in cytoplasmic calcium concentrations are discussed. This is of interest because external ATP was previously reported to exert a catecholamine-like action on the plasma membrane permeability [3, 41 and to prcvent the stimulatory effect of insulin on hexose transport and oxidation [S -71. As r-adrenergic stimulation generally results in changes in both calcium and potassium distribution (for review see [8]) the study of ATP action might shed light on the relcvance of various cellular parameters in a state similar to that during a-adrenergic action. MATERIALS AND METHODS Radiochmicals.

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

“…The results presented in this report confirm previous observations that extracellular ATP induces Ca2 + accumulation in isolated hepatocytes [20,33], and demonstrate that this effect is not due to free organelles contaminating the cell suspension or to a nonspecific increase in membrane permeability. Two lines of evidence strongly support the hypothesis that inhibition of Ca2 + efflux is the biochemical mechanism underlying ATPinduced cellular Ca2+ accumulation.…”

“…At present, the molecular mechanism by which extracellular ATP inhibits plasma membrane Ca2 + extrusion remains unknown, although it appears that this effect could be part of a more general inhibition of plasma membrane transport ATPases, resulting in the intracellular accumulation of both Ca2 + and Na' [20] (this work).…”

“…However, Krell and associates [20] have reported that incubation of hepatocytes at 37 "C in presence of exogeneously added ATP, is also associated with an increase in cell Ca2+, and attributed this effect to enhanced negative surface charge density of the plasma membrane resulting in a change in its permeability to CaZ+. In the present study we have used this approach in an attempt to obtain further information about the events involved in cellular Ca2+ accumulation, and the factors that control Ca2+ compartmentation in the intact cell.…”

Actions of extracellular UTP and ATP in perfused rat liver

“…A different subacinar localization of the UTP and ATP-hydrolyzing activities could therefore explain the higher effectivity of UTP and the different response pattern, because different hepatocyte populations may be involved in the response to extracellular UTP and ATP, respectively. Extracellular ATP is known to be hydrolyzed by ATPases and the nucleotide pyrophosphatase in rat liver plasma membrane [4,29,301, and the latter enzyme has a similar affinity for UTP and ATP [30,31]. In our experiments ATP in concentrations up to 100 pM was completely hydrolyzed during a single liver passage and no ADP was found in effluent (not shown).…”

“…In isolated rat hepatocytes or isolated perfused rat liver, ATP causes a K t uptake [2, 31 or release [4], stimulation of oxoglutarate dehydrogenase flux [5] and of glycogenolysis , and an increase in portal pressure [6]. Further, ATP decreases the bile flow [8].…”