-induced Inhibition of the Apical K Channels in the mTAL of Rat Kidney

We have shown previously that raising extracellular Ca 2 inhibited the apical 70-pS K channel in the thick ascending limb (TAL; Wang, W.H., M. Lu, and S.C. Hebert. 1996. Am. J. Physiol. 270:C103–C111). We now used the patch-clamp technique to study the effect of increasing the extracellular Ca 2 on the 70-pS K channel in the mTAL from rats on a different K diet. Increasing the extracellular Ca 2 from 10 M to 0.5, 1, and to 1.5 mM in the mTAL from rats on a K-deficient (KD) diet inhibited the channel activity by 30, 65, and 90%, respectively. In contrast, raising the extracellular Ca 2 to 1.5 mM had no significant effect on channel activity in the mTAL from animals on a high K (HK) diet and further increasing the extracellular Ca 2 to 2.5, 3.5, and 5.5 mM decreased the channel activity by 29, 55, and 90%, respectively. Inhibition of the cytochrome P450 monooxygenase completely abolished the effect of the extracellular Ca 2 on channel activity in the mTAL from rats on a different K diet. In contrast, blocking cyclooxygenase did not significantly alter the responsiveness of the 70-pS K channel to the extracellular Ca 2 . Moreover, addition of sodium nitropruside, a nitric oxide (NO) donor, not only increased the channel activity, but also blunted the inhibitory effect of the extracellular Ca 2 on the 70-pS K channel and decreased 20-hydroxyeicosatetraenoic acid (20-HETE) concentration in the mTAL from rats on a KD diet. In contrast, inhibiting NOS with L-NAME enhanced the inhibitory effect of the extracellular Ca 2 on the channel activity and increased 20-HETE concentration in the mTAL from rats on a high K diet. Western blot has further shown that the expression of inducible NO synthase (iNOS) is significantly higher in the renal medulla from rats on an HK diet than that on a KD diet. Also, addition of S-nitrosoN -acetylpenicillamine abolished the inhibitory effect of arachidonic acid on channel activity in the mTAL, whereas it did not block the inhibitory effect of 20-HETE. We conclude that a low dietary K intake increases the sensitivity of the 70-pS K channel to the extracellular Ca 2 , and that a decrease in NOS activity is involved in enhancing the inhibitory effect of the extracellular Ca 2 on channel activity in the mTAL during K depletion. key words: 20-hydroxyeicosatetraenoic acid • iNOS • cytochrome P450 • calcium-sensing receptor • K recycling I N T R O D U C T I O N We have previously reported that raising the extracellular Ca 2 inhibited the apical 70-pS K channel in the thick ascending limb (TAL;* Wang et al., 1996). The effect of increasing the extracellular Ca 2 is most likely mediated by stimulating the Ca 2 -sensing receptor (CaR) in the mTAL. Moreover, the effect of the extracellular Ca 2 on channel activity was induced by stimulating the cytochrome P450 metabolic pathway of arachidonic acid (AA) because inhibiting -hydroxylation of cytochrome P450 abolished the effect of the extracellular Ca 2 . Furthermore, we have demonstrated that increasing the extracellular Ca 2 enhanced the formation of 20-hydroxyeicosatetraenoic acid (20-HETE), a major metabolite of cytochrome P450 -hydroxylation of AA in the mTAL (Carroll et al., 1991; Wang et al., 1997a). This suggests that 20-HETE may be responsible for mediating the effect of the extracellular Ca 2 . We recently reported that a dietary K intake affects 20-HETE production: a low K intake stimulates, whereas a high K intake suppresses the 20-HETE generation (Gu et al., 2001). Since 20-HETE is the mediator for the effect of increasing the extracellular Ca 2 , it is possible that the response of the apical 70-pS K channel to the extracellular Ca 2 differs in mTAL harvested from animals on a high K (HK) or a K-deficient (KD) diet. In addition, NOS has been shown to be expressed in the renal mTAL (Ahn et al., 1994; Mohaupt et al., 1994; Shin et al., 1999). Our previous experiments illustrated that NO stimulates the 70-pS K channel in the mTAL by a cGMP-dependent protein kinase (Lu et al., 1998). Moreover, as NO has been demonstrated to inhibit the cytochrome P450 -hydroxylation (Sun et al., 1998; Ito and Roman, 1999; Oyekan et al., 1999), it is conceivable that NO may be involved in modifying the effect of increasing the extracellular Ca 2 by either increasing cGMP or suppressing cytochrome P450 metabAddress correspondence to Dr. Wen-Hui Wang, Department of Pharmacology, New York Medical College, Valhalla, NY 10595. Fax: (914) 347-4956; E-mail: [email protected] * Abbreviations used in this paper: AA, arachidonic acid; CaR, Ca 2 sensing receptor; HK, high K; iNOS, inducible NO synthase; KD, K-deficient; NO, nitric oxide; P o , open probability; SNP, sodium nitropruside; TAL, thick ascending limb. on A ril 8, 2017 D ow nladed fom Published December 17, 2001