Expression and activity of epithelial sodium channel in rat cardiac tissue

Abstract

Intracellular sodium concentration ([Na+]i) in heart tissue is tightly linked to inotropic and pathological states. We studied the expression and function of the epithelial sodium channel (ENaC) in rat cardiac myocytes. Using RT-PCR we detected α, β and γ ENaC subunits mRNAs in rat left ventricle. Similar results were obtained with neonatal rat cultured cardiac myocytes. Western Blot studies with ventricular total protein homogenates using anti α, β and γ ENaC antibodies demonstrated the presence of specific inmunoreactive bands of 96, 102 and 92 kDa, respectively, consistent with the bands seen in epithelial tissue. Inmunoprecipitation assays with anti-β ENaC co-precipitated α and γ ENaC; α and β ENaC were co-precipitated with anti-γ ENaC. Functional studies were performed using benzamil (specific ENaC blocker, 10−6M) in isolated adult rat ventricular myocytes loaded with the fluorescent Na+-sensitive dye, SBFI. [Na+]i was determined by in situ calibration using cation selective ionophores. Benzamil (10 min) significantly diminished [Na+]i in adult ventricular myocytes from 9.6±0.1 mM to 6.2±1.4 mM (P<0.05, n=12). [Na+]i did not change in vehicle controls (11.90 ± 0.02 mM, N.S.). Further inhibition of Na+-K+ ATPase with ouabain (1mM, 10 min) increased [Na+]i to 43±4 mM in control cardiomyocytes but only to 26±3 mM in benzamil-treated cells (P<0.05). The results are consistent with the expression of functional ENaC channels in rat heart. FONDECYT 1050690.