Modulation of cardiac Na(+)-K+ pump current: role of protein and nonprotein sulfhydryl redox status

Abstract

Oxidant stress alters protein structure and function, possibly through the modification of the redox status of regulatory protein sulfhydryl groups. We used the sulfhydryl-blocking reagent p-chloromercuriphenylsulfonic acid (pCMPSA), applied selectively and independently to either the intracellular or extracellular environment, to study the relationship between blocking protein sulfhydryl groups and Na(+)-K+ pump current (i.p.). In guinea pig ventricular myocytes voltage clamped at -30 mV, extracellular pCMPSA (50, 100, and 400 microM) caused a concentration-dependent reduction in holding current. The selective intracellular administration of pCMPSA (100 microM) induced a similar inhibition of i.p., albeit over a longer time course. The inhibition of ip resulting from either the intracellular or extracellular application of pCMPSA (100 microM) was reversed, in part, by the extracellular application of dithiothreitol (3 mM). An intracellular oxidant stress was also imposed by using diethyl maleate to deplete the intracellular nonprotein sulfhydryl content [represented by reduced glutathione (GSH)]. In myocytes isolated from diethyl maleate-treated guinea pigs *860 mg/kg i.p., 30 min before study), intracellular GSH was depleted by 93% and i.p. was depressed by 38% at all membrane potentials tested. We propose that Na(+)-K+ pump function may be related to protein and nonprotein sulfhydryl status. Protein sulfhydryl oxidation and glutathione depletion may account, in part, for a depression in Na(+)-K+ pump activity during reperfusion-induced oxidant stress.

Detection, Quantitation, Purification, and Identification of Cardiac Proteins S-Thiolated during Ischemia and Reperfusion

“…Protein thiols have been implicated as targets of this oxidative attack (2), and it is likely that injury involves terminal or irreversible oxidation of these reactive groups. Less severe oxidative damage occurs during reperfusion after shorter sublethal periods of ischemia and reperfusion (ischemic preconditioning), and this initiates a stress-adaptive signaling response (3,4).…”

Mitochondrial Ca2+uptake, efflux, and sarcolemmal damage in Ca2+-overloaded cultured rat cardiomyocytes

“…Experiments with pCMPSA also showed that at least some of the functionally important SH groups of Na ϩ -K ϩ -ATPase protein, which spans the cell membrane, are exposed to the extracellular environment. SH group location on the extracellular face of Na ϩ -K ϩ -ATPase has been demonstrated by recording the effect of selective application of pCMPSA on Na ϩ -K ϩ pump current in guinea pig ventricular myocytes (17), and this location also explains the rapid effect of pCMB (Fig. 1).…”

Regulation of the cardiac Na+ pump by palmitoylation of its catalytic and regulatory subunits

“…For example, superoxide generators reduce pump current in voltage-clamped rabbit ventricular myocytes by approximately 50% at all membrane potentials within 10 min [33]. Pump activity is also significantly reduced by intracellular application of thiolmodifying reagents, or depletion of cellular glutathione [34], [2] transmembrane region is in blue, the N-domain is in cyan, the P-domain is in yellow, and the A-domain is in purple.…”