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

The effects of exogenous adenosine 3',5'-cyclic monophosphate (cAMP) o n apical Na channels in the rat cortical collecting tubule were studie d using the patch-clamp technique and fura 2 fluorescence measurements of intracellular Ca2+ (Ca-i(2+)). When the permeant analogue, 8-(4-ch lorophenylthio)-cAMP (CPT-cAMP, 200 mu M), was added to the superfusat e during recording from cell-attached patches, both the mean number of open channels (NP,) and the single-channel current (i) decreased with in 3 min. When the superfusate also contained amiloride (10 mu M), the re was no effect of CPT-cAMP on either NP0 or i. When CPT-cAMP was add ed to the bath before formation of the patch, the density of conductin g channels was increased from 10 +/- 2 to 37 +/- 6 per patch, as estim ated by analysis of channel-induced noise. This suggests that cAMP inc reases open-channel density in the regions of the apical membrane outs ide 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 inc reased Ca-i(2+) from 104 to 198 nM. This increase in Ca-i(2+) was abol ished by benzamil (0.5 mu M) or by low extracellular Ca2+. The cAMP-de pendent reduction in NP0 was still observed in Ca2+-free medium, indic ating that a rise in Ca-i(2+) was not essential for the feedback respo nse. 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 su perfusate. This response may be mediated in part by changes in cellula r metabolism induced by increased Na transport.