Ovine male genital duct epithelial cells differentiate in vitro and express functional CFTR and ENaC

To investigate the biology of the male genital duct epithelium, we have est ablished cell cultures from the ovine vas deferens and epididymis epitheliu m. These cells develop tight junctions, high transepithelial electrical res istance, and a lumen-negative transepithelial potential difference as a sig n of active transepithelial ion transport. In epididymis cultures the equiv alent short-circuit current (I-sc) averaged 20.8 +/- 0.7 mu A/cm(2) (n = 15 0) and was partially inhibited by apical application of amiloride with an i nhibitor concentration of 0.64 mu M. In vas deferens cultures, I-SC average d 14.4 +/- 1.1 mu A/cm(2) (n = 18) and was also inhibited by apical applica tion of amiloride with a half-maximal inhibitor concentration (K-i) of 0.68 mu M. The remaining amiloride-insensitive I-SC component in epididymis and vas deferens cells was partially inhibited by apical application of the Cl - channel blocker diphenylamine-2-carboxylic acid (1 mM). It was largely de pendent on extracellular Cl- and, to a lesser extent, on extracellular HCO; . It was further stimulated by basolateral application of forskolin (10(-5) M), which increased I-SC by 3.1 +/- 0.3 mu A/cm(2) (n = 65) in epididymis and 0.9 +/- 0.1 mu A/cm(2) (n = 11) in vas deferens. These findings suggest that cultured ovine vas deferens and epididymis cells absorb Na+ via amilo ride-sensitive epithelial Na+ channels (ENaC) and secrete Cl- and HCO, via apical cystic fibrosis transmembrane conductance regulator (CFTR) Cl- chann els. This interpretation is supported by RT-PCR data showing that vas defer ens and epididymis cells express CFTR and ENaC mRNA.