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

1996 ◽  
Vol 108 (6) ◽  
pp. 537-547 ◽  
Author(s):  
M Lu ◽  
Y Zhu ◽  
M Balazy ◽  
K M Reddy ◽  
J R Falck ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Angiotensin Ii ◽  
Rat Kidney ◽  
Inhibitory Effect ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Control Value

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 NPo, a product of channel open probability (Po) 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 NPo was partially reversed by addition of 5 microM 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 microM 17-ODYA and 5 microM calphostin C, an inhibitor of protein kinase C. However, addition of 100 microM NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, abolished completely the AII (50-100 nM)-induced increase 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 dual effects on the activity of the apical 70 pS K+ channel. The inhibitory effect of AII is mediated by P450-dependent metabolites whereas the stimulatory effect may be mediated via NO.

Two types of K+ channel in thick ascending limb of rat kidney

1994 ◽  
Vol 267 (4) ◽  
pp. F599-F605 ◽  
Author(s):  
W. H. Wang
Keyword(s):  
Apical Membrane ◽  
Rat Kidney ◽  
Inhibitory Effect ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
K Channels ◽  
Inside Out

We have used the patch-clamp technique to study the apical K+ channels in the thick ascending limb (TAL) of the rat kidney. Two types of K+ channels, a low-conductance and an intermediate-conductance K+ channel, were identified in both cell-attached and inside-out patches. We confirmed the previously reported intermediate-conductance K+ channel (72 pS), which is inhibited by millimolar cell ATP, acidic pH, Ba2+, and quinidine (4). We now report a second K+ channel in apical membrane of the TAL. The slope conductance of this low-conductance K+ channel is 30 pS, and its open probability is 0.80 in cell-attached patches. This channel is not voltage dependent, and application of 2 mM ATP in the bath inhibits channel activity in inside-out patches. In addition, 250 microM glyburide, an ATP-sensitive K+ channel inhibitor, blocks channel activity, whereas the same concentration of glyburide has no inhibitory effect on the 72-pS K+ channel. Channel activity of the 30-pS K+ channel decreases rapidly upon excision of patches (channel run down). Application of 0.1 mM ATP and the catalytic subunit of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) restores channel activity. Furthermore, addition of 0.1 mM 8-(4-chlorophenylthio)-cAMP or 50-100 pM vasopressin in the cell-attached patches increases channel activity. In conclusion, two types of K+ channels are present in the apical membrane of TAL of rat kidney, and PKA plays an important role in modulation of the low-conductance K+ channel activity.

Cytochrome P-450 metabolites mediate extracellular Ca(2+)-induced inhibition of apical K+ channels in the TAL

1996 ◽  
Vol 271 (1) ◽  
pp. C103-C111 ◽  
Author(s):  
W. H. Wang ◽  
M. Lu ◽  
S. C. Hebert
Keyword(s):  
Arachidonic Acid ◽  
Inhibitory Effect ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Control Value ◽  
Calphostin C ◽  
Cytochrome P 450

We used the patch-clamp technique to study the effect of extracellular Ca2+ (Ca2+o) on the activity of the apical 70-pS K+ channel in the isolated split-open thick ascending limb (TAL) of the rat kidney. Raising Ca2+o from 1.1 to 5 mM reversibly reduced the activity of the 70-pS K+ channel in cell-attached patches to 16 +/- 2% of the control value within 300 s. In addition, 50 microM neomycin mimicked the effect of an increase in Ca2+o on channel activity in cell-attached patches and completely inhibited channel activity. The effect of neomycin on the channel activity in cell-attached patches is an indirect effect, since addition of 50 microM neomycin on the 70-pS K+ channel in inside-out patches reduced only the apparent amplitude of the channel current without changing channel open probability. We examined further the role of protein kinase C (PKC) and the cytochrome P-450-dependent metabolites of arachidonic acid in mediating the Ca2+o -induced inhibition of channel activity. Addition of phorbol 12-myristate 13-acetate (2 microM) reversibly blocked channel activity in cell-attached patches to 4 +/- 1% of the control value, whereas 75 nM calphostin C increased the channel activity by 115 +/- 10%. Moreover, addition of 1 nM exogenous PKC reversibly and completely inhibited the 70-pS K+ channel. However, inhibition of PKC with calphostin C (75 nM) only slightly prolonged the time course of the effect of Ca2+o on channel activity (370 +/- 40 s) and failed to abolish the inhibitory effect of 5 mM Ca2+o on channel activity in cell-attached patches, indicating that PKC was not mainly responsible for the effect of Ca2+o on channel activity. In contrast, the effect of 5 mM Ca2+o on the apical 70-pS K+ channel was completely abolished when TAL tubules were first incubated in the 17-octadecynoic acid (5 microM)-containing solution, an agent that specifically blocks cytochrome P-450 monooxygenase. In conclusion, these data indicate that Ca2+o is an important regulator of the apical 70-pS K+ channel and that a cytochrome P-450-dependent metabolite of arachidonic acid is involved in mediating this inhibitory effect.

Arachidonic acid inhibits K channels in basolateral membrane of the thick ascending limb

2002 ◽  
Vol 283 (3) ◽  
pp. F407-F414 ◽  
Author(s):  
Rui-Min Gu ◽  
Wen-Hui Wang
Keyword(s):  
Arachidonic Acid ◽  
Basolateral Membrane ◽  
Inhibitory Effect ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
K Channels ◽  
Cytochrome P 450

We have used the patch-clamp technique to study the effect of arachidonic acid (AA) on the basolateral K channels in the medullary thick ascending limb (mTAL) of rat kidney. An inwardly rectifying 50-pS K channel was identified in cell-attached and inside-out patches in the basolateral membrane of the mTAL. The channel open probability ( P o) was 0.51 at the spontaneous cell membrane potential and decreased to 0.25 by 30 mV hyperpolarization. The addition of 5 μM AA decreased channel activity, identified as NP o, from 0.58 to 0.08 in cell-attached patches. The effect of AA on the 50-pS K channel was specific because 10 μM cis-11,14,17-eicosatrienoic acid had no significant effect on channel activity. To determine whether the effect of AA was mediated by AA per se or by its metabolites, we examined the effect of AA on channel activity in the presence of indomethacin, an inhibitor of cyclooxygenase, or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), an inhibitor of cytochrome P-450 monooxygenase. Inhibition of cyclooxygenase increased channel activity from 0.54 to 0.9. However, indomethacin did not abolish the inhibitory effect of AA on the 50-pS K channel. In contrast, inhibition of cytochrome P-450 metabolism not only increased channel activity from 0.49 to 0.83 but also completely abolished the effect of AA. Moreover, addition of DDMS can reverse the inhibitory effect of AA on channel activity. The notion that the effect of AA was mediated by cytochrome P-450-dependent metabolites of AA is also supported by the observation that addition of 100 nM of 20-hydroxyeicosatetraenoic acid, a main metabolite of AA in the mTAL, can mimic the effect of AA. We conclude that AA inhibits the 50-pS K channel in the basolateral membrane of the mTAL and that the effect of AA is mainly mediated by cytochrome P-450-dependent metabolites of AA.

scholarly journals Angiotensin II stimulates basolateral 50-pS K channels in the thick ascending limb

2014 ◽  
Vol 306 (5) ◽  
pp. F509-F516 ◽  
Author(s):  
Mingxiao Wang ◽  
Haiyan Luan ◽  
Peng Wu ◽  
Lili Fan ◽  
Lijun Wang ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Protein Tyrosine Kinase ◽  
Rat Kidney ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Ang Ii ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
K Channels ◽  
Family Protein

We used the patch-clamp technique to examine the effect of angiotensin II (ANG II) on the basolateral K channels in the thick ascending limb (TAL) of the rat kidney. Application of ANG II increased the channel activity and the current amplitude of the basolateral 50-pS K channel. The stimulatory effect of ANG II on the K channels was completely abolished by losartan, an inhibitor of type 1 angiotensin receptor (AT1R), but not by PD123319, an AT2R antagonist. Moreover, inhibition of phospholipase C (PLC) and protein kinase C (PKC) also abrogated the stimulatory effect of ANG II on the basolateral K channels in the TAL. This suggests that the stimulatory effect of ANG II on the K channels was induced by activating PLC and PKC pathways. Western blotting demonstrated that ANG II increased the phosphorylation of c-Src at tyrosine residue 416, an indication of c-Src activation. This effect was mimicked by PKC stimulator but abolished by calphostin C. Moreover, inhibition of NADPH oxidase (NOX) also blocked the effect of ANG II on c-Src tyrosine phosphorylation. The role of Src-family protein tyrosine kinase (SFK) in mediating the effect of ANG II on the basolateral K channel was further suggested by the experiments in which inhibition of SFK abrogated the stimulatory effect of ANG II on the basolateral 50-pS K channel. We conclude that ANG II increases basolateral 50-pS K channel activity via AT1R and that activation of AT1R stimulates SFK by a PLC-PKC-NOX-dependent mechanism.

Reaction of nitric oxide with superoxide inhibits basolateral K+ channels in the rat CCD

1998 ◽  
Vol 275 (1) ◽  
pp. C309-C316 ◽  
Author(s):  
Ming Lu ◽  
Wen-Hui Wang
Keyword(s):  
Nitric Oxide ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Inhibitory Effect ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
High Concentration ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
No Donors

We previously demonstrated that nitric oxide (NO) stimulates the basolateral small-conductance K+channel (SK) via a cGMP-dependent pathway [M. Lu and W. H. Wang. Am. J. Physiol. 270 ( Cell Physiol. 39): C1336–C1342, 1996]. Because NO at high concentration has been shown to react with superoxide ([Formula: see text]) to form peroxynitrite (OONO−) [W. A. Pryor and G. L. Squadrito. Am. J. Physiol. 268 ( Lung Cell. Mol. Physiol. 12): L699–L722, 1995 and M. S. Wolin. Microcirculation 3: 1–17, 1996], we extended our study to examine, using patch-clamp technique, the effect of high concentrations of NO on SK in cortical collecting duct (CCD) of rat kidney. Addition of NO donors [100–200 μM S-nitroso- N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP)] reduced channel activity, defined as the product of channel number and open probability, to 15 and 25% of the control value, respectively. The inhibitory effect of NO was completely abolished in the presence of 10 mM Tiron, an intracellular scavenger of [Formula: see text]. NO donors, 10 μM SNAP or SNP, which stimulate channel activity under control conditions, can also inhibit SK in the presence of an[Formula: see text] donor, pyrogallol, or in the presence of an inhibitor of superoxide dismutase, diethyldithiocarbamic acid. The inhibitory effect of NO is still observed in the presence of exogenous cGMP, suggesting that the NO-induced inhibition is not the result of decreased cGMP production. We conclude that the inhibitory effect of NO on channel activity results from an interaction between NO and [Formula: see text].

Nitric oxide regulates the low-conductance K+ channel in basolateral membrane of cortical collecting duct

1996 ◽  
Vol 270 (5) ◽  
pp. C1336-C1342 ◽  
Author(s):  
M. Lu ◽  
W. H. Wang
Keyword(s):  
Nitric Oxide ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Inhibitory Effect ◽  
K Channel ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Dependent Pathway

Two types of K+ channels, low conductance (28 pS) and intermediate conductance (85 pS), have been previously identified in the basolateral membrane of the cortical collecting duct (CCD) of the rat kidney (31, 32). In the present study, we used the patch-clamp technique to explore further the mechanism by which the low-conductance K+ channel is regulated. The conductance of the low-conductance K+ channel is inward rectifying, with an inward slope conductance of 30 pS between 0 and -20 mV and an outward slope conductance of 16 pS between 0 and 50 mV in symmetrical 140 mM KCl in the bath and in the pipette. This K+ channel was not sensitive to ATP (10 mM), tetraethylammonium chloride (5 mM), and quinidine (1 mM). Addition of 100 microM N omega-nitro-L-arginine methyl ester (L-NAME) or N omega-(imonoethyl)-L-ornithine (L-NIO), an inhibitor of nitric oxide synthase (NOS), completely blocked channel activity in cell-attached patches. In contrast, addition of 200 microM-D-NAME, which does not block NOS, had no effect on channel activity. The inhibitory effect of L-NAME or L-NIO was fully reversible and completely overcome by addition of exogenous nitric oxide (NO) donors, such as 10 microM S-nitroso-N-acetyl-penicillamine or sodium nitroprusside. Furthermore, addition of 100 microM 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) restored the activity of the channel when it had been inhibited by either L-NAME or L-NIO, indicating that the effect of NO on the channel activity was mediated by a cGMP-dependent pathway. In conclusion, NO plays a key role in the regulation of the basolateral 30-pS K+ channel and the effect of NO on channel activity is mediated by a cGMP-dependent pathway.

Nitric oxide increases the activity of the apical 70-pS K+ channel in TAL of rat kidney

1998 ◽  
Vol 274 (5) ◽  
pp. F946-F950 ◽  
Author(s):  
Ming Lu ◽  
Xiaohong Wang ◽  
Wenhui Wang
Keyword(s):  
Nitric Oxide ◽  
Rat Kidney ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
No Donor ◽  
Oxide Synthase ◽  
Dependent Pathway

We have previously shown that nitric oxide (NO) mediates the stimulatory effect of angiotensin II on the apical 70-pS K+ channel in the thick ascending limb (TAL) of Henle’s loop of the rat kidney (12). In the present study, we used the patch-clamp technique to examine the effects of NO on the 70-pS K+ channel. Addition of 10 μM S-nitroso- N-acetylpenicillamine (SNAP), a NO donor, increased the channel activity in cell-attached patches. In contrast, application of 100 μM N ω-nitro-l-arginine methyl ester (l-NAME), an inhibitor of nitric oxide synthase (NOS), reduced the channel activity by 75 ± 7%. The effect of l-NAME was the result of inhibiting NOS, since d-NAME, which does not block NOS activity, had no effect on the channel activity. In addition, the effect ofl-NAME was abolished in the presence of 1 mM l-arginine or by addition of 10 μM SNAP, further supporting the role of NO. Finally, the l-NAME-induced inhibition was also reversed by adding 8-bromoguanosine 3′,5′-cyclic monophosphate (8-BrcGMP). That the effect of NO is mediated by the cGMP-dependent pathway is also suggested by experiments in which inhibition of guanylate cyclase abolished the effect of SNAP. Finally, 10 μM SNAP significantly increased cGMP concentration of the medullary TAL from 12.4 fM/μg protein to 38.9 fM/μg protein, as measured with ELISA. We conclude that NO is involved in regulating the activity of the apical 70-pS K+ channel in the TAL of the rat kidney.

scholarly journals Vasopressin and PGE2 regulate activity of apical 70 pS K+ channel in thick ascending limb of rat kidney

2000 ◽  
Vol 278 (5) ◽  
pp. C905-C913 ◽  
Author(s):  
Hua Jun Liu ◽  
Yuan Wei ◽  
Nicholas R. Fererri ◽  
Alberto Nasjletti ◽  
Wen Hui Wang
Keyword(s):  
Rat Kidney ◽  
Inhibitory Effect ◽  
K Channel ◽  
Prostaglandin E ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Camp Production ◽  
Patch Clamp Technique ◽  
High Concentrations ◽  
Dependent Pathway

Vasopressin and prostaglandin E2 (PGE2) are involved in regulating NaCl reabsorption in the thick ascending limb (TAL) of the rat kidney. In the present study, we used the patch-clamp technique to study the effects of vasopressin and PGE2 on the apical 70 pS K+ channel in the rat TAL. Addition of vasopressin increased the channel activity, defined as NP o, from 1.11 to 1.52 (200 pM) and 1.80 (500 pM), respectively. The effect of vasopressin can be mimicked by either forskolin (1–5 μM) or 8-bromo-cAMP/dibutyryl-cAMP (8-Br-cAMP/DBcAMP) (200–500 μM). Moreover, the effects of cAMP and vasopressin were not additive and application of 10 μM H-89 abolished the effect of vasopressin. This suggests that the effect of vasopressin is mediated by a cAMP-dependent pathway. Applying 10 nM PGE2 alone had no significant effect on the channel activity. However, PGE2 (10 nM) abolished the stimulatory effect of vasopressin. The PGE2-induced inhibition of the vasopressin effect was the result of decreasing cAMP production because addition of 200 μM 8-Br-cAMP/DBcAMP reversed the PGE2-induced inhibition. In addition to antagonizing the vasopressin effect, high concentrations of PGE2 reduced channel activity in the absence of vasopressin by 33% (500 nM) and 51% (1 μM), respectively. The inhibitory effect of high concentrations of PGE2 was not the result of decreasing cAMP production because adding the membrane-permeant cAMP analog failed to restore the channel activity. In contrast, inhibiting protein kinase C (PKC) with calphostin C (100 nM) abolished the effect of 1 μM PGE2. We conclude that PGE2 inhibits apical K+ channels by two mechanisms: 1) low concentrations of PGE2 attenuate the vasopressin-induced stimulation mainly by reducing cAMP generation, and 2) high concentrations of PGE2 inhibit the channel activity by a PKC-dependent pathway.

scholarly journals K Depletion Enhances the Extracellular Ca2+-Induced Inhibition of the Apical K Channels in the Mtal of Rat Kidney

2001 ◽  
Vol 119 (1) ◽  
pp. 33-44 ◽  
Author(s):  
Rui-Min Gu ◽  
Yuan Wei ◽  
Ho-Lin Jiang ◽  
Dao-Hong Lin ◽  
Hyacinth Sterling ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Inhibitory Effect ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
No Donor ◽  
P450 Monooxygenase ◽  
High K ◽  
K Depletion

We have shown previously that raising extracellular Ca2+ 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 Ca2+ on the 70-pS K channel in the mTAL from rats on a different K diet. Increasing the extracellular Ca2+ from 10 μM 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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+. 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 Ca2+ 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 Ca2+ 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-nitroso-N-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 Ca2+, and that a decrease in NOS activity is involved in enhancing the inhibitory effect of the extracellular Ca2+ on channel activity in the mTAL during K depletion.

scholarly journals Nitric Oxide Links the Apical Na+ Transport to the Basolateral K+ Conductance in the Rat Cortical Collecting Duct

1997 ◽  
Vol 110 (6) ◽  
pp. 717-726 ◽  
Author(s):  
Ming Lu ◽  
Gerhard Giebisch ◽  
WenHui Wang
Keyword(s):  
Nitric Oxide ◽  
Collecting Duct ◽  
K Channel ◽  
Na Channel ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Na Transport ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Control Value

We have used the patch clamp technique to study the effects of inhibiting the apical Na+ transport on the basolateral small-conductance K+ channel (SK) in cell-attached patches in cortical collecting duct (CCD) of the rat kidney. Application of 50 μM amiloride decreased the activity of SK, defined as nPo (a product of channel open probability and channel number), to 61% of the control value. Application of 1 μM benzamil, a specific Na+ channel blocker, mimicked the effects of amiloride and decreased the activity of the SK to 62% of the control value. In addition, benzamil reduced intracellular Na+ concentration from 15 to 11 mM. The effect of amiloride was not the result of a decrease in intracellular pH, since addition 50 μM 5-(n-ethyl-n-isopropyl) amiloride (EIPA), an agent that specifically blocks the Na/H exchanger, did not alter the channel activity. The inhibitory effect of amiloride depends on extracellular Ca2+ because removal of Ca2+ from the bath abolished the effect. Using Fura-2 AM to measure the intracellular Ca2+, we observed that amiloride and benzamil significantly decreased intracellular Ca2+ in the Ca2+-containing solution but had no effect in a Ca2+-free bath. Furthermore, raising intracellular Ca2+ from 10 to 50 and 100 nM with ionomycin increased the activity of the SK in cell-attached patches but not in excised patches, suggesting that changes in intracellular Ca2+ are responsible for the effects on SK activity of inhibition of the Na+ transport. Since the neuronal form of nitric oxide synthase (nNOS) is expressed in the CCD and the function of the nNOS is Ca2+ dependent, we examined whether the effects of amiloride or benzamil were mediated by the NO-cGMP–dependent pathways. Addition of 10 μM S-nitroso-n-acetyl-penicillamine (SNAP) or 100 μM 8-bromoguanosine 3′:5′-cyclic monophosphate (8Br-cGMP) completely restored channel activity when it had been decreased by either amiloride or benzamil. Finally, addition of SNAP caused a significant increase in channel activity in the Ca2+-free bath solution. We conclude that Ca2+-dependent NO generation mediates the effect of inhibiting the apical Na+ transport on the basolateral SK in the rat CCD.

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