Raising extracellular Ca2+ (Ca2+o) stimulating the Ca(2+)-sensing receptor (CaR) decreased the activity of the apical 70-pS K+ channel via a cytochrome P-450-dependent mechanism in the thick ascending limb (TAL) of the rat kidney [W. H. Wang, M. Lu, and S. C. Hebert. Am. J. Physiol. 270 (Cell Physiol. 39): C103-C111, 1996]. We have now used the patch-clamp technique and fluorescent dyes to investigate the signaling mechanism by which this effect is produced. Addition of 500 microM gadolinium (Gd3+), an agent which has been shown to activate the CaR (E. M. Brown, G. Gamba, D. Riccardi, M. Lombardi, R. Butters, O. Kifor, A. Sun, M. A. Hediger, J. Lytton, and S. C. Hebert. Nature 366: 575-580, 1993), mimics the inhibitory effect of raising Ca2+o from 1.1 to 5 mM on channel activity. Effects of the high Ca2+o and Gd3+ were abolished by blockade of phospholipase A2 (PLA2) but not by inhibition of phospholipase C (PLC). Raising Ca2+o also increased 20-hydroxyeicosatetraenoic acid production significantly. To investigate the effect of stimulation of the CaR on intracellular Ca2+ (Ca2+i), we used the acetoxymethyl ester of fura 2 to monitor the Ca2+i. Raising Ca2+o from 1.1 to 5 mM increased the Ca2+i significantly from 50 to 150 nM. However, addition of thapsigargin failed to abolish the effect of 5 mM Ca2+o on Ca2+i. Also, application of Gd3+ only slightly increased the Ca2+i, suggesting that elevation of the Ca2+i by high Ca2+o was the result of an influx of Ca2+ rather than enhanced Ca2+ release from Ca2+ stores. That the increase in Ca2+ influx is not mainly responsible for the effect of stimulating the CaR on channel activity is further supported by experiments in which 500 microM Gd3+ inhibited the K+ channel in cell-attached patches in a Ca(2+)-free bath. Furthermore, addition of 500 microM Gd3+ or 5 mM Ca2+o decreased intracellular Na+ measured with fluorescent sodium indicator, suggesting inhibition of Na+ transport. We conclude that PLA2 is involved in the stimulation of the CaR-induced inhibition of apical K+ channels in the TAL.
We have used the patch-clamp technique to study the regulation of the activity of the basolateral small-conductance K+ channel (SK) in the cortical collecting duct (CCD) of the rat kidney. Addition of 50-75 nM calphostin C, an agent which specifically inhibits protein kinase C (PKC), reduced channel activity by 90% in cell-attached patches. In contrast, addition of 1 microM phorbol 12-myristate 13-acetate, a stimulator of PKC, led to addition of "new" K+ channel currents in 9 of 20 patches in the basolateral membrane of the CCD, and the mean increase in NP0, a product of channel number (N) and open probability (Pzero), was 0.90 in these 9 patches. However, application of 1 nM exogenous PKC had no significant effect on channel activity in inside-out patches, suggesting that the PKC effect on the activity of the SK observed in cell-attached patches was not a result of a membrane-delimited action, such as a direct phosphorylation of the SK or closely associated proteins. The effect of calphostin C on the SK can be reversed by addition of either 10 microM S-nitroso-N-acetylpenicillamine, a donor of nitric oxide, or 100 microM 8-bromoguanosine 3',5'-cyclic monophosphate. In addition, the inhibitory effect of calphostin C on the SK was completely abolished by pretreatment of the cells with 1 microM okadaic acid, an inhibitor of protein phosphatase. However, 100 microM N omega-nitro-L-arginine methyl ester, an agent that inhibits nitric oxide synthases (NOS), blocked the SK in cell-attached patches in the presence of okadaic acid, suggesting that the effect of okadaic acid on calphostin C-induced inhibition of the SK was a step before formation of nitric oxide. We conclude that PKC is involved in the stimulation of the SK and that the effect of PKC on the SK may be mediated by regulation of NOS activity in the CCD of the rat kidney.
We used the patch-clamp technique in the split-open cortical collecting duct (CCD) to investigate the effect of nitric oxide (NO) on the low-conductance (6-pS) Na+ channel that can be blocked by 1 microM amiloride. We confirmed that the number of Na+ channels increased significantly in CCDs of rats on a low-Na+ diet (17). Application of 100 microM N(G)-nitro-L-arginine methyl ester (L-NAME), an agent that blocks endogenous NO synthase, reduced NPo [the product of channel number (N) and open probability (Po)] to 45% of the control value. The effect of L-NAME was specific, since addition of D-NAME, which does not inhibit NO synthase, did not change the activity of the Na+ channel. That the effect of L-NAME results from inhibition of NO synthase is further confirmed by experiments in which addition of an exogenous NO donor, either 10 microM S-nitroso-N-acetyl penicillamine or sodium nitroprusside (SNP), restored the Na+ channel activity when it had been blocked by L-NAME. The action of NO involves a guanosine 3',5'-cyclic monophosphate (cGMP)-dependent pathway, since 100 microM 8-bromo-cGMP (8-BrcGMP) mimicked the effect of SNAP on K+ channels. However, 100 microM 8-BrcGMP did not alter the activity of Na+ channels in inside-out patches, suggesting an indirect action. Because the Na+ channel is activated by hyperpolarization (19) and NO stimulates basolateral K+ channels (16), we tested whether hyperpolarization mediated the effect of NO. In perforated whole cell recordings, addition of L-NAME depolarized the cell membrane from -73 to 51 mV, and application of 10 microM SNP repolarized the membrane to -68 mV. Furthermore, the L-NAME-induced decrease in NPo was effectively restored by 25 mV hyperpolarization of the patch membranes, and addition of 2 mM Ba2+ also abolished the effect of L-NAME. We concluded that the stimulatory effect of NO on the Na+ channel is an indirect effect mediated by a NO-induced increase of basolateral K+ conductance.
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