Na-K-ATPase in rat cerebellar granule cells is redox sensitive

Abstract

Redox-induced regulation of the Na-K-ATPase was studied in dispersed rat cerebellar granule cells. Intracellular thiol redox state was modulated using glutathione (GSH)-conjugating agents and membrane-permeable ethyl ester of GSH (et-GSH) and Na-K-ATPase transport and hydrolytic activity monitored as a function of intracellular reduced thiol concentration. Depletion of cytosolic and mitochondrial GSH pools caused an increase in free radical production in mitochondria and rapid ATP deprivation with a subsequent decrease in transport but not hydrolytic activity of the Na-K-ATPase. Selective conjugation of cytosolic GSH did not affect free radical production and Na-K-ATPase function. Unexpectedly, overloading of cerebellar granule cells with GSH triggered global free radical burst originating most probably from GSH autooxidation. The latter was not followed by ATP depletion but resulted in suppression of active K(+) influx and a modest increase in mortality. Suppression of transport activity of the Na-K-ATPase was observed in granule cells exposed to both permeable et-GSH and impermeable GSH, with inhibitory effects of external and cytosolic GSH being additive. The obtained data indicate that redox state is a potent regulator of the Na-K-ATPase function. Shifts from an "optimal redox potential range" to higher or lower levels cause suppression of the Na-K pump activity.

S-Glutathionylation of the Na,K-ATPase Catalytic α Subunit Is a Determinant of the Enzyme Redox Sensitivity

“…Na,K-ATPase may be S-glutathionylated in a reaction of thiol-disulfide exchange within the physiological concentration range of GSH and GSSG of 1-10 mM and 50 -500 M, respectively (Figs. 3 and 5E) (9,31,32). Thiol-disulfide exchange reaction with glutathione is comparatively rare, and the few other proteins where it is also physiologically relevant are c-Jun (33) and aldose reductase (34).…”

“…Binding of glutathione to a single reduced cysteine residue in the regulatory ␤ subunit caused a modest decrease in the Na,K-ATPase activity in cardiomyocytes (16). These minor effects of S-glutathionylation on the enzyme function did not explain robust redox-induced responses of the enzyme (9,10,12). S-Glutathionylation for any of the 23 evolutionarily conserved cysteine residues of the catalytic ␣ subunit has never been reported.…”

“…However, accumulating evidence suggests that redox-induced responses of the Na,K-ATPase cannot be explained by irreversible oxidation of -SH groups alone. Our earlier findings indicated that both loading of cerebellar granule cells with reduced glutathione (GSH) and GSH depletion were equally efficient in blocking Na,K-ATPase (9). Maximal activity of the enzyme in freshly isolated cerebellar neurons was only observed within a "physiological" range of pO 2 and redox state characteristic for neonatal rat cerebellum.…”

“…Maximal activity of the enzyme in freshly isolated cerebellar neurons was only observed within a "physiological" range of pO 2 and redox state characteristic for neonatal rat cerebellum. Hypoxic and hyperoxic conditions and oxidative and reductive stress were associated with a decrease in Na,K-ATPase activity (9,10). Alterations of NO production in response to hypoxia or ischemia have been shown to play a decisive role in oxygen-induced inhibition of Na,K-ATPase (10 -13).…”

“…The activity of Na,K-ATPase (␣1␤ and ␣2␤ isozymes) from the rat heart was assessed either in the crude homogenate prepared from ventricular tissue (13) or in the sarcolemmal membrane fraction (20). Activity of the duck, rabbit, and rat Na,K-ATPase was measured as ouabain-sensitive (1 mM) ATP cleavage in the medium containing (in mM) 130 NaCl, 20 KCl, 3 MgCl 2 , 3 ATP, and 30 imidazole, pH 7.4, 37°C, when not stated otherwise (9,13,21).…”