Ionic mechanisms of Ca(2+)-dependent electrolyte transport across equine sweat gland epithelium

1. The ionic mechanism involved in Ca(2+)-stimulated electrolyte transport in cultured equine sweat gland epithelial cells was studied using the short-circuit current (ISC) technique. 2. Microscopy revealed that the cultured cells grown on Millipore filters formed polarized monolayers with tight junctions. Monolayers exhibited a mean transepithelial resistance of 333.9 +/- 40.4 omega cm2. 3. Ca(2+)-mobilizing agents, A23187 (1 microM) or thapsigargin (0.01-1 microM), stimulated ISC while forskolin exerted little effect on the ISC. 4. Replacement of external Cl- by gluconate significantly reduced the ISC by 63% when stimulated by 0.1 microM thapsigargin. Residual ISC could be abolished (> 99%) by elimination of HCO3- from the bathing solution. 5. Basolateral addition of bumetanide (0.1 mM), ouabain (0.01 mM) and acetazolamide (45 microM) and apical addition of methyl isobutyl amiloride (MIA, 1-100 microM) all had inhibitory effects on the thapsigargin-stimulated ISC to various extents. 6. Substantial current inhibition could be obtained using 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and diphenylamine-2-carboxylate (DPC) in a concentration-dependent manner. 7. The K+ channel blocker barium (5 mM) was effective on both sides of the epithelium with a much larger effect on the basolateral side. 8. The inhibitory effects of acetazolamide, amiloride, MIA, DIDS and DPC on the thapsigargin-stimulated ISC were also observed when a Cl(-)-free solution was used. 9. The results provide evidence for Ca(2+)-stimulated HCO3- as well as Cl- secretion by equine sweat gland epithelium.