Whole cell sodium conductance of principal cells freshly isolated from rat cortical collecting duct

Abstract

Cortical collecting duct fragments were manually dissected from 6-wk-old Sprague-Dawley rats. The fragments were enzymatically digested (collagenase A) into single cells, washed, and resuspended in serum-free RPMI 1640. Individual cells were examined electrophysiologically using the whole cell patch-clamp technique. Two morphologically distinct cell types were present in the cell suspension. Small round cells that had a capacitance of 7 pF and larger oval cells with a capacitance of 29 pF were consistently observed. Whole cell electrophysiological examination revealed that the small round cells had virtually no plasma membrane ionic conductance, whereas both inward and outward currents were observed in the larger oval-type cells. Also, superfusion of 250 pM arginine vasopressin specifically increased the inward conductance of only the larger cells. The effect could be completely inhibited by 2 microM amiloride or 100 mumol of the Rp diastereomer of 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (a specific adenosine 3',5'-cyclic monophosphate inhibitor). These findings are consistent with the hypothesis that the larger cells are principal cells and the smaller cells are intercalated cells and directly demonstrate that an amiloride-sensitive whole cell conductance is readily observable in freshly isolated cortical collecting duct cells. Thus the whole cell configuration of the patch-clamp technique appears to be well suited for assessing cellular mechanisms that regulate the ionic conductances of cortical collecting duct cells.