Electrophysiological studies of anion secretion in cultured human epididymal cells

Abstract

1. Primary monolayer cultures from adult human epididymis were grown on Petri dishes and previous supports. The epithelia so formed were used for whole-cell patch clamp recording and short-circuit current (ISC) measurement. 2. After 50 days of culture, the cells formed a tight epithelium with transepithelial potential of 5.5 +/- 1.3 mV (mean +/- S.E.M.., n = 16), apical side negative, and a basal ISC of 6.9 +/- 0.9 microA cm-2 (mean +/- S.E.M., n = 16). 3. Adrenaline, when added to the basolateral side, at a concentration of 0.23 mumol l-1 increased the ISC by 3.0 +/- 1.2 microA cm-2 (mean +/- S.E.M., n = 4). This increase was blockable by diphenylamine-2-carboxylate (DPC, 1 mmol l-1). Forskolin (10 mumol l-1) also evoked a similar response to adrenaline. 4. In whole-cell patch clamp experiment, the resting membrane potential of the cells after dialysis with pipette solution containing 135 mmol l-1 KCl was found to be -30 +/- 14 mV (mean +/- S.E.M., n = 15). 5. About 90% of the cells successfully forming patches responded to 1 mumol l-1 adrenaline by an increase in inward current at -70 mV holding potential (delta I = -1600 +/- 900 pA, mean +/- S.E.M., n = 15). This increase in current was accompanied by a shift in reversal potential to -2 +/- 1 mV (mean +/- S.E.M., n = 15). 6. The adrenaline-induced inward current was found to be blockable by the Cl- channel blocker, DPC (0.25 mmol l-1). Ion substitution experiments showed that the adrenaline-evoked current was carried mainly by Cl-. 7. The effect of adrenaline on the whole-cell current was found to be mimicked by forskolin and could be abolished by including GDP beta S or a protein kinase A inhibitor in the pipette solution. Propranolol, but not phentolamine, completely abolished the effect of adrenaline. 8. Inclusion of 20 mmol l-1 EGTA or 2 mmol l-1 BAPTA + 100 mumol l-1 TMB-8 (to inhibit intracellular Ca2+ release) in the pipette did not seem to have any marked effect on adrenaline-evoked whole-cell current. Lowering the pipette Ca2+ concentration to 1 nmol l-1 or raising it to 10 mumol l-1 had no effect on the whole-cell current response to adrenaline. 9. This study shows that adrenaline stimulates Cl- secretion in cultured human epididymal cells.(ABSTRACT TRUNCATED AT 400 WORDS)