Ion conductances of isolated cortical collecting duct cells.

Abstract

The study of ion conductances in the intact cortical collecting duct (CCD) with the patch-clamp method is rather difficult. An optimized method to isolate CCD cells from rat kidneys using an in vivo followed by an in vitro enzyme digestion is described. Individual CCD segments were collected after this digestion and incubated in EGTA-buffered medium. This procedure resulted in single cells or cell clusters. These freshly isolated CCD cells were studied with different modifications of the patch-clamp method. Membrane voltages measured in the cell-attached-nystatin configuration were -74 +/- 1 mV (n = 13) and -68 +/- 3 mV (n = 22) in cells isolated from normal and mineralocorticoid-treated rats respectively. These values and those measured with the nystatin-perforated slow-whole-cell configuration (-79 +/- 1 mV, n = 23) are comparable to those measured in principal cells of isolated CCD segments. The cells hyperpolarized after the addition of amiloride and depolarized with the addition of adiuretin to the bath. The amiloride effect was enhanced when cells were isolated from deoxycorticosterone-acetate-treated rats. The cells were strongly depolarized upon elevation of the extracellular K(+)-concentration and did not demonstrate a measurable Cl- conductance. A large-conductance K+ channel (174 pS, n = 5, cell-attached, 145 mmol/l K+ in the pipette; 140 pS, n = 12, cell-free, 3.6 mmol/l K+ in the bath) was seen. It had a very low activity on the cell, but a high open probability when excised into a solution with 1 mmol/l Ca2+ on the cytosolic side.(ABSTRACT TRUNCATED AT 250 WORDS)