Studies of transepithelial Cl - transport in cultured cauda epididymal cells of rats by the short-circuit current method

Abstract

1. Monolayer cultures of cauda epididymides from male Sprague-Dawley rats (210-230 g) were studied by the short-circuit current (I(SC)) technique to characterize the properties of the transepithelial chloride transport. In HCO 3 --free, HEPES-buffered solution, adrenaline (0.23 μM) added to the basolateral side led to an increase in I(SC) and transepithelial conductance (g(t)). 2. Decreasing apical chloride concentration ([Cl -](a)) progressively from 126.7 to 0 mM by substituting chloride with gluconate increased the I(SC) response to adrenaline (ΔI(SC)) in a linear fashion with a slope of 1.6 x 10 -3 μequiv h -1 cm -2 per millimolar change in [Cl -](a). Pretreating the tissue with a chloride channel blocker diphenylamine-2-carboxylate (DPC) on the apical side significantly reduced the slope to -4.9 x 10 -4,μequiv h -1 cm -2 per millimolar change in [Cl -](a). 3. By substituting apical chloride with various anions and measuring the change in I(SC) upon adrenaline stimulation, the selectivity sequence of the apical anion conductance was found to be NO 3 - ~ Br - > Cl - > I - > gluconate > isethionate. 4. When the monolayers were bathed with Krebs-Henseleit solution containing 25 mM HCO 3 - and 5% CO 2, the ΔI(SC) at each [Cl -](a) as well as the dependence of ΔI(SC) on [Cl -]a (slope = 3.3 x 10 -3 μequiv h -1 cm -2 per millimolar change in [Cl -](a)) were significantly greater than the HCO 3 --free counterpart. Addition of 0.1 mM acetazolamide or 0.5 mM SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) to the basolateral side significantly reduced the effects of HCO 3 - and CO 2. 5. When the tissues were bathed on both sides with HCO 3 --free, HEPES-buffered solution and were clamped at various transepithelial potential differences (PD(t)) from +30 mV (lumen positive) to -30 mV (lumen negative), the relationship between the clamping current response to adrenaline (ΔI(CL)) and the PD(t) applied was linear. Zero clamping current response was found at -6 mV. Decreasing [Cl -](a) to 0 mM reduced the dependence of ΔI(CL) on PD(t) and ΔI(CL) was positive at all PD(t) tested. The response of the transepithelial conductance to adrenaline (Δg(t)) did not depend on the PD(t) applied but was reduced with decreasing apical chloride concentration. 6. The results showed that the apical membrane of epididymal cells possessed an anion conductance which could be blocked by DPC, being selective against isethionate and gluconate, but was permeable to Cl -, Br - as well as NO 3 -. HCO 3 - enhanced transepithelial Cl - transport by replenishing intracellular Cl - through a SITS-sensitive HCO 3 -Cl - exchange at the basolateral membrane. Depending on the PD(t) and the [Cl -](a), the transport of chloride could be secretory (from blood to lumen) or absorptive (from lumen to blood). The transepithelial conductance response to adrenaline did not depend on the PD(t) applied but was reduced with decreasing [Cl -](a). It is suggested that the transepithelial Cl - transport might be important in maintaining the electrolyte composition in the epididymal fluid.