Abstract
There is a well-characterized membrane chloride current (ICl,cAMP) in the heart, which can be activated by β-adrenergic agonists and arises due to expression of the cardiac isoform of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR). We have investigated whether 17β-estradiol (E2) modulates ICl,cAMP in single ventricular myocytes. Under whole-cell tight-seal voltage-clamp conditions, ICl,cAMP was evoked by exposing cells to 20 nM isoprenaline. On addition of 30 µM E2, membrane slope-conductance, measured at potentials near 0 mV, increased over that induced by isoprenaline alone by 2.46 ± 0.16 (p < 0.001). The effects of E2 were concentration-dependent and described by a Hill Plot with an EC50 of 8.2 µM and a Hill coefficient of 1.63. Application of membrane-impermeant E2 conjugated to bovine serum albumin (E2-BSA), potentiated ICl,cAMP evoked by isoprenaline by approximately the same degree as that for the equivalent level of free E2. Cell surface binding was observed with confocal microscopy using BSA-FITC tagged E2. This binding was inhibited by non-labeled, non-conjugate E2, the specific E2 antagonist ICI 182,780, and by incubation of E2coBSA with a specific anti-E2 antibody (E2885). ICI 182,780 (100 µM) reduced significantly the increase in ICl,cAMP evoked by 10 µM E2 to 1.46 ± 0.10 (p < 0.02). Preincubation of myocytes with the NOS inhibitor N-ω-nitro-arginine (L-NNA, 1 mM) reduced the potentiation of ICl,cAMP by 30 µM E2 to 1.93 ± 0.06 (p < 0.02) and to 1.32 ± 0.05 for 10 µM E2 (p < 0.002). E2 also increased ICl,cAMP evoked by bath application of 0.5 µM Forskolin. These experiments demonstrate that, under our experimental conditions, E2 dramatically increases ICl,cAMP in ventricular myocytes, by mechanisms which involve a contribution by NOS, but can only be partially accounted for through binding to classical plasma membrane estrogen receptor sites. This potentiation of ICl,cAMP by E2, may play a significant role in the observed clinical actions of E2 on the incidence of cardiac arrhythmias and hypertrophy.
