Amiloride-sensitive sodium channels in rabbit cortical collecting tubule primary cultures

Patch-clamp methodology was applied to principal cell apical membranes of rabbit cortical collecting tubule (CCT) primary cultures grown on collagen supports in the presence of aldosterone (1.5 microM). The most frequently observed channel had a unit conductance of 3-5 pS, nonlinear current-voltage (I-V) relationship, Na permeability (PNa)-to-K permeability (PK) ratio greater than 19:1, and inward current at all applied potentials (Vapp) less than +80 mV (n = 41). Less frequently, an 8- to 10-pS channel with a linear I-V curve, PNa/PK less than 5:1, and inward current at Vapp less than +40 mV was also observed (n = 7). Luminal amiloride (0.75 microM) decreased the open probability (Po) for both of these channels. Mean open time for the high-selectivity Na+ channel was 2.1 +/- 0.5 s and for the low-selectivity Na+ channel was 50 +/- 12 ms. In primary cultures grown without aldosterone the high-selectivity Na+ channel was rarely observed (1 of 32 patches). Lastly, a 26- to 35-pS channel, nonselective for Na+ over K+, was not activated by cytoplasmic Ca2+ or voltage nor inhibited by amiloride (n = 17). We conclude that under specific growth conditions, namely permeable transporting supports and chronic mineralocorticoid hormone exposure, principal cell apical membranes of rabbit CCT primary cultures contain 1) both high-selectivity and low-selectivity, amiloride-inhibitable Na+ channels and 2) amiloride-insensitive, nonselective cation channels.

Volume regulatory Cl- loss after Na+ pump inhibition in CCT principal cells

Ouabain caused rabbit cortical collecting tubule (CCT) principal cells to swell 53% and then undergo regulatory volume decrease (RVD) at a rate of 4%/min to a new steady-state volume 10% below control. Reduction of peritubular Cl- concentration transiently depolarized transepithelial potential (Vte) by 36 mV and stimulated the rate of RVD 30-fold. Peritubular application of 0.5 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited RVD 74%. In contrast, luminal Cl- reduction or application of DIDS had no effect on RVD. A 10-fold elevation of perfusate K+ caused volume-regulated cells to swell 23% at a rate of 60%/min. Removal of luminal Cl- had no effect on either the rate or magnitude of K+ swelling. Peritubular or bilateral Cl- removal, however, inhibited the rate of K+ swelling by 96 and 99%, respectively. Substitution of bath Cl- for Br-, SCN-, or I- inhibited the rate of K+ swelling by 40, 38, and 98%, respectively. Surprisingly, NO3- inhibited the rate of K+ swelling by 82%. All Cl- substitutes tested transiently depolarized Vte by 3–49 mV. These results suggest strongly that RVD is mediated by a basolateral Cl- channel with a high selectivity for Cl- over other anions.

Cell volume regulation of rabbit cortical collecting tubule in anisotonic media

Volume regulation of nonperfused rabbit cortical collecting tubules in anisotonic bathing media was examined in vitro. When media osmolality is abruptly increased by 150 mosmol/kgH2O with the addition of NaCl, tubules shrink by 20% but do not volume regulate. However, volume regulatory increase (VRI) is observed when 1 mM butyrate is present in the bathing media or when tubules are pretreated with hypotonic media. When media osmolality is increased, butyrate-treated tubules shrink to 74% of their isotonic control volume. As evidence of volume regulation, butyrate-treated tubules swell while still bathed in hypertonic media, recovering in 30 min 78% of the volume lost due to osmotic shrinkage. The butyrate effect requires external Na+ and is inhibited by amiloride. When media osmolality is lowered to 150 mosmol/kgH2O, nonbutyrate tubules swell before showing typical volume regulatory decrease. When these tubules are returned to isotonic media, they immediately shrink to 78% of control volume before showing evidence of VRI. These results suggest that, under the appropriate conditions, cortical collecting tubules are capable of VRI.