Small conductance potassium channels drive ATP-activated chloride secretion in the oviduct

Abstract

The molecular mechanisms controlling fluid secretion within the oviduct have yet to be determined. As in other epithelia, both secretory and absorptive pathways are likely to work in tandem to drive appropriate ionic movement to support fluid movement across the oviduct epithelium. This study explored the role of potassium channels in basolateral extracellular ATP (ATPe)-stimulated ion transport in bovine oviduct epithelium using the Ussing chamber short-circuit current ( ISC) technique. Basal ISCin bovine oviduct epithelium comprises both chloride secretion and sodium absorption and was inhibited by treatment with basolateral K+channel inhibitors tetrapentlyammonium chloride (TPeA) or BaCl2. Similarly, ATP-stimulated chloride secretion was significantly attenuated by pretreatment with BaCl2,tetraethylammonium (TEA), tolbutamide, and TPeA. Basolateral K+current, isolated using nystatin-perforation technique, was rapidly activated by ATPe, and pretreatment of monolayers with thapsigargin or TPeA abolished this ATP-stimulated K+current. To further investigate the type of K+channel involved in the ATP response in the bovine oviduct, a number of specific Ca2+-activated K+channel inhibitors were tested on the ATP-induced Δ ISCin intact monolayers. Charbydotoxin, (high conductance and intermediate conductance inhibitor), or paxilline, (high conductance inhibitor) did not significantly alter the ATPeresponse. However, pretreatment with the small conductance inhibitor apamin resulted in a 60% reduction in the response to ATPe. The presence of small conductance family member KCNN3 was confirmed by RT-PCR and immunohistochemistry. Measurements of intracellular calcium using Fura-2 spectrofluorescence imaging revealed the ability of ATPeto increase intracellular calcium in a phospholipase C-inositol 1,4,5-trisphosphate pathway-sensitive manner. In conclusion, these results provide strong evidence that purinergic activation of a calcium-dependent, apamin-sensitive potassium conductance is essential to promote chloride secretion and thus fluid formation in the oviduct.