FEEDBACK-REGULATION OF NA CHANNELS IN RAT CCT .3. RESPONSE TO CAMP

The effects of exogenous adenosine 3',5'-cyclic monophosphate (cAMP) on apical Na channels in the rat cortical collecting tubule were studied using the patch-clamp technique and fura 2 fluorescence measurements of intracellular Ca2+ (Ca-i(2+)). When the permeant analogue, 8-(4-chlorophenylthio)-cAMP (CPT-cAMP, 200 mu M), was added to the superfusate during recording from cell-attached patches, both the mean number of open channels (NP,) and the single-channel current (i) decreased within 3 min. When the superfusate also contained amiloride (10 mu M), there was no effect of CPT-cAMP on either NP0 or i. When CPT-cAMP was added to the bath before formation of the patch, the density of conducting channels was increased from 10 +/- 2 to 37 +/- 6 per patch, as estimated by analysis of channel-induced noise. This suggests that cAMP increases open-channel density in the regions of the apical membrane outside the patch but not within the patch. Channels already active in the patch before stimulation with the nucleotide are subject to feedback inhibition secondary to increased Na entry into the cell. CPT-cAMP increased Ca-i(2+) from 104 to 198 nM. This increase in Ca-i(2+) was abolished by benzamil (0.5 mu M) or by low extracellular Ca2+. The cAMP-dependent reduction in NP0 was still observed in Ca2+-free medium, indicating that a rise in Ca-i(2+) was not essential for the feedback response. The decrease in NP, was attenuated, however, when cAMP was added in the absence of Ca2+ and in the presence of ouabain (1 mM) in the superfusate. This response may be mediated in part by changes in cellular metabolism induced by increased Na transport.