Abstract
Multiple stimuli of physiological and pathophysiological significance, including α1-adrenoceptor agonists, stimulate the cardiac sarcolemmal Na+/H+ exchanger isoform 1 (NHE1) through activation of the mitogen-activated or extracellular signal-regulated kinase (ERK) kinase (MEK) ERK-90-kDa ribosomal S6 kinase (RSK) signaling cascade. However, the individual contributions of ERK and RSK, which can each phosphorylate the NHE1 regulatory domain, to such stimulation are unknown. In the present study, we have used the novel RSK inhibitor fmk to determine the role of RSK as a direct regulator of NHE1 phosphorylation and activity in response to α1-adrenergic stimulation, in ventricular myocytes isolated from the adult rat heart. Initial experiments confirmed that pretreatment of myocytes with fmk before exposure to the α1-adrenoceptor agonist phenylephrine inhibited RSK C-terminal kinase activity and thereby RSK N-terminal kinase activation, without affecting MEK or ERK activation. Pretreatment of myocytes with fmk also inhibited phenylephrine-induced increases in NHE1 phosphorylation and the rate of NHE1-mediated H+ efflux under conditions of intracellular acidosis. These findings reveal, for the first time to our knowledge, that RSK is the principal regulator of NHE1 phosphorylation and activity after α1-adrenergic stimulation in adult myocardium.
Footnotes
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The posts of F.C. and A.K.S. were funded by grants from the Medical Research Council (Co-operative Group Component Grant G0300052) and the British Heart Foundation (Intermediate Research Fellowship FS/04/053), respectively.
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F.C. and A.K.S. contributed equally to this work.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.106.029900.
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ABBREVIATIONS: RSK, 90-kDa ribosomal S6 kinase; CTK, C-terminal kinase; NHE1, Na+/H+ exchanger isoform 1; ARVM, adult rat ventricular myocyte; NHE, Na+/H+ exchanger; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase or extracellular signal-regulated kinase kinase; GST, glutathione transferase; aa, amino acid; BCECF, 2′,7′-bis-(carboxyethyl)-5(6′)-carboxyfluorescein; HA, hemagglutinin; Adv, adenovirus, m, mouse; h, human; PFU, plaque-forming unit; DMSO, dimethyl sulfoxide; pHi, intracellular pH; PAGE, polyacrylamide gel electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid; NTK, N-terminal kinase; PDK, phosphoinositide-dependent kinase; IB, immunoblot/immunoblotting; PHEN, phenylephrine; CONT, control.
- Received August 11, 2006.
- Accepted December 1, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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