Evidence for direct regulation of myocardial Na+/H+ exchanger isoform 1 phosphorylation and activity by 90-kDa ribosomal S6 kinase (RSK): effects of the novel and specific RSK inhibitor fmk on responses to alpha1-adrenergic stimulation

Multiple stimuli of physiological and pathophysiological significance, including alpha1-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 alpha1-adrenergic stimulation, in ventricular myocytes isolated from the adult rat heart. Initial experiments confirmed that pretreatment of myocytes with fmk before exposure to the alpha1-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 alpha1-adrenergic stimulation in adult myocardium.