Effect of angiotensin II on the apical K⁺ channel in the thick ascending limb of the rat kidney

We have used the patch-clamp technique to study the effect of angiotensin II (AII) on the activity of the apical 70 pS K⁺ channel and used Na⁺ -sensitive fluorescent dye (SBFI) to investigate the effect of AII on intracellular Na⁺ concentration (Na⁺(i)) in the thick ascending limb (TAL) of the rat kidney. Addition of 50 pM AII reversibly reduced NP(o), a product of channel open probability (P(o)) and channel number (N), to 40% of the control value and reduced the Na⁺(i) by 26%. The AII (50 pM)-induced decrease in channel activity defined by NP(o) was partially reversed by addition of 5 μM 17-octadecynoic acid (17-ODYA), an agent which blocks the cytochrome P450 monooxygenase. The notion that P450 metabolites of arachidonic acid (AA) may mediate the inhibitory effect of AII was further suggested by experiments in which addition of 10 nM of 20- hydroxyeicosatetraenoic acid (20-HETE) blocked the channel activity in cell- attached patches in the presence of 17-ODYA. We have used gas chromatography mass spectrometry (GC/MS) to measure the production of 20-HETE, a major AA metabolite of the P450-dependent pathway in the TAL of the rat. Addition of 50 pM AII increased the production of 20-HETE to 260% of the control value, indicating that 20-HETE may be involved in mediating the effect of AII (50 pM). In contrast to the inhibitory effect of 50 pM AII, addition of 50-100 nM AII increased the channel activity to 270% of the control value and elevated the Na⁺(i) by 45%. The effect of AII on the activity of the 70 pS K⁺ channel was also observed in the presence of 5 μM 17-ODYA and a 5 μM calphostin C, an inhibitor of protein kinase C. However, addition of 100 μM N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, abolished completely the AII (50-100 nM)-induced increased in channel activity and addition of an exogenous nitric oxide (NO) donor, S- nitroso-N-acetyl-penicillamine (SNAP), increased channel activity in the presence of L-NAME. These data suggest that the stimulatory effect of AII is mediated by NO. We conclude that AII has duel effects on the activity of the apical 70 pS K⁺ channel. The inhibitory effect of AII is mediated by 450- dependent metabolites whereas the stimulatory effect may be mediated via NO.