β ₃ Adrenergic Stimulation of the Cardiac Na ⁺ -K ⁺ Pump by Reversal of an Inhibitory Oxidative Modification

Abstract

Background-Inhibition of L-type Ca 2ϩ current contributes to negative inotropy of ␤ 3 adrenergic receptor (␤ 3 AR) activation, but effects on other determinants of excitation-contraction coupling are not known. Of these, the Na ϩ -K ϩ pump is of particular interest because of adverse effects attributed to high cardiac myocyte Na ϩ levels and upregulation of the ␤ 3 AR in heart failure. Methods and Results-We voltage clamped rabbit ventricular myocytes and identified electrogenic Na ϩ -K ϩ pump current (I p ) as the shift in holding current induced by ouabain. The synthetic ␤ 3 AR agonists BRL37344 and CL316,243 and the natural agonist norepinephrine increased I p . Pump stimulation was insensitive to the ␤ 1 /␤ 2 AR antagonist nadolol and the protein kinase A inhibitor H-89 but sensitive to the ␤ 3 AR antagonist L-748,337. Blockade of nitric oxide synthase abolished pump stimulation and an increase in fluorescence of myocytes loaded with a nitric oxide-sensitive dye. Exposure of myocytes to ␤ 3 AR agonists decreased ␤ 1 Na ϩ -K ϩ pump subunit glutathionylation, an oxidative modification that causes pump inhibition. The in vivo relevance of this was indicated by an increase in myocardial ␤ 1 pump subunit glutathionylation with elimination of ␤ 3 AR-mediated signaling in ␤ 3 AR Ϫ/Ϫ mice. The in vivo effect of BRL37344 on contractility of the nonfailing and failing heart in sheep was consistent with a beneficial effect of Na ϩ -K ϩ pump stimulation in heart failure. Conclusions-The ␤ 3 AR mediates decreased ␤ 1 subunit glutathionylation and Na ϩ -K ϩ pump stimulation in the heart. Upregulation of the receptor in heart failure may be a beneficial mechanism that facilitates the export of excess Na ϩ . (Circulation. 2010;122:2699-2708.)Key Words: heart failure Ⅲ molecular biology Ⅲ myocardium Ⅲ nitric oxide synthase Ⅲ receptors, adrenergic T he ␤ 3 adrenergic receptor (␤ 3 AR) differs from ␤ 1 /␤ 2 ARs in its molecular structure, genetic coding, 1,2 and affinities for adrenergic ligands. 3 Cellular responses to receptor activation also differ. In cardiac tissue, ␤ 3 AR activation decreases contractility, contrasting with the increase mediated by the ␤ 1 AR. 4 Consistent with this, ␤ 3 AR activation is associated with a decrease in the amplitude of the cytosolic Ca 2ϩ transient that initiates contraction. 5-7 Inhibition of sarcolemmal L-type Ca 2ϩ current 5,7 that triggers release of sarcoplasmic reticulum Ca 2ϩ is likely to contribute to this. However, effects of ␤ 3 AR activation on other proteins that are important for excitation-contraction coupling remain to be determined. 7 The membrane Na ϩ -K ϩ pump is of particular interest because it maintains the transmembrane electrochemical gradient for Na ϩ that provides the electrochemical energy for the Na ϩ -Ca 2ϩ exchanger, quantitatively the most important determinant of total cell Ca 2ϩ and hence sarcoplasmic reticulum Ca 2ϩ that is triggered for release during systole. A modest increase in the intracellular Na ϩ concentration, and hence cell Ca 2ϩ , increases con...

β3 Adrenergic Stimulation Restores Nitric Oxide/Redox Balance and Enhances Endothelial Function in Hyperglycemia

“…This increase in pump current was abolished by L‐ N G ‐nitroarginine methyl ester, showing that stimulation was mediated by eNOS‐derived NO production enhanced by ex vivo supplementation of BH 4 . In previous reports we have shown that NO generation from eNOS stimulates the Na + ‐K + pump,10, 38 and conditions that cause eNOS uncoupling inhibit the Na + ‐K + pump 24, 3940 where changes in redox milieu are sensed by eNOS and transduced to regulation of the Na + ‐K + pump function via redox‐dependent mechanisms.…”

“…To detect glutathionylation of eNOS and β 1 Na + ‐K + pump subunit in co‐immunoprecipitation experiments, an antibody against glutathionylated protein (anti‐glutathione antibody) was used to detect glutathionylation 10. Aorta was homogenized in ice‐cold lysis buffer containing 150 mmol/L NaCl, 50 mmol/L Tris‐HCl (pH 8.0), 1% Triton X‐100, 2 mmol/L EDTA, and protease inhibitor (Complete EGTA‐free, Roche Diagnostics), followed by centrifugation at 16 000 g for 20 minutes.…”

“…To detect glutathionylation of β 1 Na + ‐K + subunit in cardiac myocytes, freshly isolated myocytes were loaded with biotinylated glutathione ester (500 μmol/L) for 1 hour at room temperature. The biotin‐tag was used to precipitate glutathionylated proteins as previously described 10. Western blot chemiluminescence was read by a LAS‐4000 image reader and quantified by densitometry using Multi Gauge 3.1 software (Fujifilm Life Science, Tokyo, Japan).…”

“…β 3 ARs are expressed in endothelial cells8 and enriched in the caveolae9 and their stimulation is coupled to eNOS activation and NO release 10, 11. We have previously shown that β 3 AR stimulation reverses oxidative inhibition of the cardiac Na + ‐K + pump,10, 11 a biomolecule also enriched in the caveolae 12.…”

“…We have previously shown that β 3 AR stimulation reverses oxidative inhibition of the cardiac Na + ‐K + pump,10, 11 a biomolecule also enriched in the caveolae 12. This effect was mediated by a decrease in glutathionylation of the pump's β 1 subunit 10. Glutathionylation, which involves formation of a reversible disulphide bond between glutathione and reactive cysteines on proteins with significant structural and functional consequences, is increasingly recognized in physiological and pathophysiological cell signaling 13.…”