kn-62 and phorbol

The hypothesis that feedback inhibition of the apical Na+ channels in the cortical collecting tubule (CCT) is mediated by activation of a Ca(2+)-dependent protein kinase was tested using the patch-clamp technique. Na+ channel activity was monitored in cell-attached patches in principal cells of split-open rat tubules. Mean number of open channels (NPo) and single-channel current (i) were measured at 37 degrees C during continuous tubule superfusion. Phorbol 12-myristate 13-acetate (PMA; 50 nM), an activator of protein kinase C (PKC), decreased NPo to 33% of the control value. Staurosporine (200 nM), an inhibitor of PKC and of Ca(2+)-calmodulin kinase II, practically abolished the effect of PMA. Ouabain (1 mM), reduced NPo to 29% of control values and decreased i. Ouabain did not downregulate the channels in tubules exposed to staurosporine, although it still reduced i. Incubation of the tubules with 10 microM KN-62, a specific cell membrane-permeable inhibitor of Ca(2+)-calmodulin kinase II, did not interfere with the ouabain-dependent downregulation of the channels. The results support the view that the downregulation caused by ouabain involves the Ca(2+)-dependent phosphorylation of the channel itself or of proteins regulating the channel.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amiloride; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Down-Regulation; Enzyme Activation; Feedback; Female; Kidney Tubules, Collecting; Male; Ouabain; Patch-Clamp Techniques; Phorbols; Protein Kinase C; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Sodium Channels; Staurosporine; Tetradecanoylphorbol Acetate; Up-Regulation