Duel effects of nitric oxide on K⁺ channels in the basolateral membrane of rat CCD

Nitric oxide (NO) stimulates the basolateral small-conductance K⁺ channel (SK) via a cGMP-dependent pathway (AJP, 270:01336-42,96). NO can also exert its effect through forming peroxynitrite via interaction with free radicals such as O₂^- when concentration of NO increases. We extended our study to examine the effect of high concentration of NO on the SK in the rat CCD using the patch-clamp technique. Addition of 0.1 mM S-nitroso-N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP) reduced channel activity that is defined by NP_o, a product of channel number (N) and open probability (P_o), to 5% of the control value. The inhibitory effect of NO was completely abolished in the presence of 10 mM 4,5-Dihydroxy-1,3-benzene-disulfonic acid, which is a scavenger for free radicals and has no effect on channel activity. In addition, the stimulatory effect of 10 μM SNAP or SNP on SK was reversed into an inhibitory effect in the presence of pyrogallol (an O₂^- donor) or diethyldithiocarbamic acid (an inhibitor of superoxide dismutase). Thus, data suggest that the inhibitory effect of NO results from interaction between NO and free radicals. Since basolateral K⁺ channels participate in generating cell membrane potential, inhibitory effects of NO can affect Na⁺ transport in the CCD. We examined the effect of 10 μM SNAP on intracellular Na⁺ in the presence of pyrogallol using Na⁺-sensitive fluorescent dye (SBFI). In the presence of pyrogallol, 10 μM SNAP decreased intracellular Na⁺ from 15 mM to 11 mM. In contrast, SNAP increased intracellular Na⁺ in the absence of pyrogallol. We conclude that the stimulatory effect of NO is mediated by cGMP-dependent pathway whereas the inhibitory effect results from interaction between NO and O₂^-.