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

Abstract

Addition of ATP to the incubation medium of freshly isolated rat hepatocytes causes a marked inhibition of the efflux of CaZ+ from the cells, and its accumulation in intracellular compartments. After an initial rise in cytosolic free Ca2 + concentration, as indicated by the activation of phosphorylase, Ca2 + is preferentially sequestered in the mitochondria, without any apparent contribution by the endoplasmic reticulum. Impairment of mitochondrial Ca2 + homeostasis by pyridine nucleotide oxidation associated with tert-butyl hydroperoxide metabolism, prevents the ATP-dependent cellular Ca2 + accumulation and causes a release of Ca2+ from the hepatocytes into the medium. Conversely, maintenance of the mitochondrial pyridine nucleotides in a more reduced state, e. g. in presence of 3-hydroxybutyrate in the medium, prevents this hydroperoxide-induced release of intracellular Ca2Under conditions of impaired mitochondrial CaZ + sequestration, there appears to be a redistribution of a minor fraction of the intracellular Ca2 + from the mitochondria to the endoplasmic reticulum. Our results provide additional evidence for the critical involvement of the plasma membrane Ca2 +-extruding system in the physiological regulation of the cytosolic free Ca2 + concentration in hepatocytes, and suggest that the mitochondria play amore important role than the endoplasmic reticulum in the regulation of the cytosolic free Ca2+ level when the plasma membrane Ca" pump is inhibited.In hepatocytes, the cytosolic free Ca2 + concentration is maintained at a level three to four orders of magnitude lower than that in the extracellular fluid by the concerted operation of compartmentation processes [l] and binding to cellular structures [2]. Active transport of Ca2+ occurs by more or less well-characterized translocases located in the mitochondria, endoplasmic reticulum and plasma membrane ; the mitochondria have been demonstrated to represent the predominant site for intracellular Ca2 + sequestration [3].Mitochondria1 CaZ ' homeostasis is regulated by a kinetic cycling process involving both uptake and release of the ion. Ca2 + uptake is purely electrophoretic, driven by the electrical component of the proton-motive force [4], and specifically inhibited by ruthenium red [ 5 ] . The route by which Ca2' is released has been shown to be distinct from the uptake uniporter and, in liver, appears to involve a Ca2+/2H+ antiporter [6]. Reports from several laboratories suggest that this latter mechanism for Caz+ release may be regulated by the intramitochondrial pyridine nucleotide redox level [7 -91. Liver microsomes have been found to actively sequester CaZ+ by a process linked to ATP hydrolysis [lo, 1 I]. Although this mechanism seems to be quantitatively less important than mitochondrial Ca2+ sequestration, liver microsomes have been demonstrated to contribute to the regulation of the free Ca2+ concentration in the medium of an incubation system containing isolated liver microsomes and mitochondria [I 21.Because of the continuous influx of Ca2...

Stimulation of glucose production from glycogen by glucagon, noradrenaline and non-degradable adenosine analogues is counteracted by adenosine and ATP in cultured rat hepatocytes

“…The data presented in this study (1) show that in cultured hepatocytes, adenosine and ATP can both increase and decrease glucose production; (2) indicate that in the culture system a purinergic increase in glycogen breakdown is mediated via the Pi receptor (adenosine and ATP-and p[CH2IppA-derived adenosine) and the P2 receptor (ATP); (3) suggest that the inhibitory signal arises from uptake of adenosine into the cell and (4) strengthen the view that adenosine and ATP can exert metabolic effects which are not mediated via extracellular receptor activation in isolated cell systems.…”

“…It is well documented that both purines activate glycogen phosphorylase [1][2][3][4] and increase glucose production in perfused livers from fed rats, an action comparable with the effects of noradrenaline and glucagon and of hepatic nerve stimulation [5][6][7]. The effects of adenosine are thought to be mediated by an increase in cyclic AMP concentration via P1 receptors [3,8,9], whereas the signal chain initiated by ATP appears to involve the breakdown of phosphatidylinositol to InsP3 and mobilization of intracellular Ca2+ via P2y receptors in a cyclic AMP-independent manner [10][11][12][13][14][15].…”

Calcium and long‐term transmission damage following anoxia in dentate gyrus and CA1 regions of the rat hippocampal slice.

“…Thus, by a process of elimination the data argue that inhibition of the Ca2+-ATPase-driven Ca2+-extrusion pump is the basis for the anoxic Ca2+ accumulation. Studies on synaptosomes (Snelling & Nicholls, 1985) and liver cells (Bellomo, Nicotera & Orrenius, 1984) have indicated that inhibition of this efflux system does lead to significant accumulation of tissue Ca2+.…”

P2-purinergic control of liver glycogenolysis

“…Since in a number of tissues ATP is a co-transmitter in the sympathetic nerve terminals (Burnstock, 1981), the question arises as to the possibility of a Ca2+-mediated purinergic control of liver glycogenolysis. At least at high concentrations, exogenous ATP reproduces several effects of the Ca2+-dependent glycogenolytic hormones, such as glucose production at 0.1 mM (De Witt & Putney, 1983), Ca2+ redistribution at 0.3-0.5 mM (Krell et al, 1983) and 0.8 mm (Bellomo et al, 1984), phosphorylase activation at 0.8 mM (Bellomo et al, 1984), hydrolysis of phosphatidylinositol 4,5-bisphosphate at 0.1 mM (Creba et al, 1983) and increased inositol trisphosphate at 0.1 mM (Charest et al, 1985). In the present paper, we show that ATP acts at much lower concentrations and present strong evidence for a common mode of action for ATP and the Ca2+-dependent glycogenolytic hormones.…”