17β-estradiol potentiates the cardiac cystic fibrosis transmembrane conductance regulator chloride current in guinea-pig ventricular myocytes

There is a well-characterized membrane chloride current (I-CI,I-cAMP) in the heart that can be activated by beta-adrenergic agonists and is due to expression of the cardiac isoform of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR). We have investigated whether 17 beta-estradiol (E2) modulates I-CI/cAMP in single ventricular myocytes. Under whole-cell tight-seal voltage-clamp conditions, I-CI,I-cAMP was evoked by exposing cells to 20 nM isoprenaline. On the addition of 30 mu m 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 mu m and a Hill coefficient of 1.63. The application of membrane-impermeant E2 conjugated to bovine serum albumin (E2-BSA) potentiated isoprenaline-evoked I-CI/cAMP by approximately the same degree as that for the equivalent level of free E2. Cell surface binding was observed with confocal microscopy by using BSA-FITC tagged E2. This binding was inhibited by nonlabeled, nonconjugate E2, the specific E2 antagonist ICI 182,780, and incubation of E2coBSA with a specific anti-E2 antibody (E2885). ICI 182,780 (100 mu m) significantly reduced the increase in I-CI,I-cAMP evoked by 10 mu m E2 to 1.46 +/- 0.10 (p < 0.02). The preincubation of myocytes with the NOS inhibitor N-omega-nitro-arginine (L-NNA, 1 mm) reduced the potentiation of I-CI,I-cAMP by 30 mu m E2, to 1.93 +/- 0.06 (p < 0.02), and for 10 mu M E2, to 1.32 +/- 0.05 (p < 0.002). E2 also increased I-CI,I-cAMP evoked by bath application of 0.5 mu M Forskolin. These experiments demonstrate that, under our experimental conditions, E2 dramatically increases I-CI,I-cAMP in ventricular myocytes by mechanisms involving a contribution by NOS, but that can be only partially accounted for through binding to classical plasma membrane estrogen receptor sites. This potentiation of I-CI,I-cAMP by E2 may play a significant role in the observed clinical actions of E2 on the incidence of cardiac arrhythmias and hypertrophy.