Suppression of mitochondrial respiratory function after short-term anoxia

6 downloads 0 Views 2MB Size Report
ATP synthase. These results show that short-term anoxia suppresses mitochondrial function in hepatocytes and suggest that mitochondrial. Ca*+ content may be ...
Suppression of mitochondrial after short-term anoxia TAK YEE Department

AW, BO S. ANDERSSON, AND of Biochemistry, Emory University

Aw, TAK YEE, Bo S. ANDERSSON, AND DEAN P. JONES. Suppression of mitochondrial respiratory function after shortterm anoxia. Am. J. Physiol. 252 (Cell Physiol. 21): C362C368, 1987.-Exposure of rat hepatocytes to 30 min anoxia resulted in a substantial decrease in 0, consumption on reoxygenation. Measurement of the sequestered Ca*’ pool of mitochondria by selective release with the protonophore, carbonylcyanide-ptrifluoromethoxyphenylhydrazone (FCCP), and quantitation with the metallochromic indicator, arsenazo III, showed that anoxia caused a marked decrease in mitochondrial Ca*+. This loss could, in part, be due to decreased electrophoretic uptake resulting from a 20% decrease in the magnitude of the mitochondrial transmembranal potential. The decrease was associated with a decrease in ATP synthase activity as expected from the Ca*+ dependence of endogenous inhibitor binding to the ATP synthase. These results show that short-term anoxia suppresses mitochondrial function in hepatocytes and suggest that mitochondrial Ca*+ content may be important in this regulation. Regulation of the ATP synthase and other ion transport systems may provide a means to preserve ion distribution and protonmotive force and thereby prolong the period during which cells can tolerate anoxia. adenosine tonmotive

triphosphate; force

synthase;

oxygen; liver; ischemia;

pro-

OF AEROBIC CELLS to survive periods of anoxia depends on the retention of structural and metabolic conditions that allow recovery of normal function on reoxygenation. We recently observed that during 30 min anoxia, isolated hepatocytes respond to preserve the mitochondrial membrane potential (A$), the mitochondrial proton gradient ( ApH), and the distribution of many metabolic anions (1, 2, 4). Such preservation requires selective inhibition of the transmembranal flux of ions, which would otherwise lead to collapse of the membrane potential and ion gradients. Physiological regulatory systems for several mitochondrial ion transport systems have been proposed, e.g., the redox state of pyridine nucleotides modulates the Ca’+/ 2H+ antiporter (18), acyl CoA’s inhibit the ATP-ADP carrier (23), a low molecular weight protein inhibits the ATP synthase (24), and voltage gating, as well as Mg2+, regulates the K+-H+ antiporter (7, 19). Although modulation of several ion transport systems by such diverse mechanisms may be necessary to preserve the protonmotive force during anoxia, regulation of the ATP synthase is of primordial importance because this system accounts for such a large portion of the inward flux of THE ABILITY

C362

respiratory

DEAN P. JONES School of Medicine,

function

Atlanta,

Georgia 30322

ions. This enzyme system normally couples inwardly directed proton flux to the synthesis of ATP from ADP and inorganic phosphate. This system is reversible so that during anoxia it could contribute to either the collapse of All/ by continued ATP synthesis (if ATP hydrolysis continues elsewhere in the cell) or to ATP hydrolysis to maintain the A# (if net ion flux continues across the mitochondrial membrane during anoxia). Thus inhibition of the ATP synthase during anoxia may act to protect cells against functional loss. An endogenous inhibitor of ATP synthase has been purified (24) and has been shown to block the movement of protons across the membrane. Binding of this inhibitor to the synthase is inversely related to mitochondrial function (12,29) and is prevented by high concentrations of Ca2’ (32). Thus changes in mitochondrial Ca2+during anoxia could, in principle, regulate ATP synthase function through the modulation of the binding of the inhibitor. Calcium ion is normally maintained in the mitochondrial matrix at concentrations at least two orders of magnitude greater than in the cytosol (31). In liver, this balance is maintained by an electrophoretic uptake (8) in response to the substantial membrane potential (-140-160 mV, negative inside; 1, 32) and a protoncompensated electroneutral Ca2’ efflux (25). During anoxia, A$ decreases -2O%, which could decrease the electrophoretic uptake of Ca2’. Since ApH is largely retained during anoxia (1, 2), efflux of Ca”+ may be unaffected. Thus one may expect mitochondrial Ca2+ to decrease. Since the binding of the inhibitor to the synthase is inhibited by Ca2+, such a decrease in mitochondrial Ca2+ concentration could result in inhibition of both ATP synthesis and proton movement across the membrane. Several studies have shown that decreasing extracellular Ca2+ decreases respiratory functions in isolated hepatocytes (3, 11). Moreover, Denton and McCormack (9) have suggestedthat hormonal modulation of cytosolic Ca2’ could result in variations in mitochondrial Ca2’ that could provide an extrinsic control of respiratory functions. In their proposal, they suggest that increased cytosolic Ca2+ increases mitochondrial Ca2’ and stimulates oxidative metabolism. Although this remains controversial (16), Moreno-Sanchez (22) has recently shown that oxidative phosphorylation in rat liver mitochondria is regulated by extramitochondrial Ca2’ over the free Ca2+ concentration range thought to occur in the cytoplasm. Since large increases in mitochondrial Ca2’ are

0363-6143/87 $1.50 Copyright 0 1987 the American Physiological Society

MITOCHONDRIAL

FUNCTION

also known to inhibit mitochondrial function (22), the various studies suggest that both Ca2+ loss and Ca2+ loading can result in decreased oxidative phosphorylation. Takano et al. (28) recently showed that short-term anoxia in rabbit proximal tubules causes a substantial inhibition of nystatin-stimulated respiration. However, their results indicated that in renal proximal tubules, this loss is not associated with a change in cellular Ca2’. Because of the central importance of suppression of mitochondrial respiration after anoxia and the potential role for Ca” in mitochondrial function as discussed above, we have examined the effect of short-term anoxia on respiratory function and mitochondrial Ca2+ content in isolated rat hepatocytes. The results show that 30 min anoxia causes a dramatic suppression of respiratory function. This suppression is coincident with a decrease in mitochondrial Ca2+ content and a decrease in ATP synthase activity. The results suggest that over a limited range of mitochondrial Ca2+ content, Ca2+ contributes to the regulation of respiratory capacity and ATP synthase activity. The results are consistent with a model of regulation of mitochondrial function during anoxia in which normal respiratory functions are inhibited to preserve the mitochondrial transmembrane potential and ion gradients. Such regulation may provide a quiescent but viable state during which the cell is capable of recovery on reoxygenation. MATERIALS

AND

METHODS

Materials. Collagenase (type IV), arzenazo III, antimycin A, rotenone, digitonin, oligomycin, N-Zhydroxyethylpiperazine-N’-2-ethanesulfonic acid HEPES, and A23187 were obtained from Sigma Chemical (St. Louis, MO). The radioactive chemical, [ methyl-3H]TPMP was purchased from New England Nuclear (Boston, MA). Carbonylcyanide - p - trifluoromethoxyphenylhydrazone (FCCP) was obtained from Boehringer-Mannheim (Mannheim, FRG). Ultrapure argon (