17β‐estradiol prevents phosphorylation of serine 707 of the myocardial sodium/proton exchanger (NHE1 isoform) in perfused rat hearts: a postulated mechanism of estrogen inhibition of NHE1 activity.

Abstract

The mammalian myocardial Na/H exchanger (NHE1 isoform) mediates net Na accumulation during ischemia, which increases the driving force for cytotoxic calcium entry via Na/Ca exchange (NCX), to promote ischemia/reperfusion (IR) injury. Inhibition of NHE1 with pharmacological inhibitors, or mildly hypertonic perfusion protects the heart from ischemic damage. In addition, estrogen (17β-estradiol (E2)) appears to be cardioprotective, perhaps due to inhibition of NHE1 and/or NCX activity (Anderson et al, AJP 2005). The exact mechanism of NHE1 inhibition by E2 is not known, though we have hypothesized that the effect is mediated by stimulation of NO synthesis, leading to increased p38 MAP kinase activity, down regulation of ERK1/2 kinase and subsequent dephosphorylation of NHE1. In this investigation, we immunoprecipitated NHE1 from Langendorff perfused hearts of healthy adult Sprague-Dawley rats (female, male, and male + 1 nM E2), and analyzed phosphorylation of NHE1 using an LTQ-FT mass spectrometer (MS) and LC MS/MS. MS coverage of NHE1 included 14%, 17%, and 20%, respectively. Three sites were identified as phosphorylated on NHE1 in the male rat heart: serines 697, 707, and 790. E2 treatment abolished phosphorylation of s707 (s703 in human) in the male rat heart. In the female rat heart, s790 was phosphorylated, while s707 and s697 were not. Human s703 is an important phosphorylation site for activation of NHE1 by p90RSK, and phosphorylation of NHE1 by p90RSK downstream of ERK1/2 is associated with IR-stimulated NHE1 activity in perfused rat hearts (Moor et al, JBC 2001). Thus dephosphorylation of s707 by E2 may confer cardioprotective inhibition of NHE1 during IR.