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 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.

scholarly journals Stimulation of A2a adenosine receptor abolishes the inhibitory effect of arachidonic acid on the basolateral 50-pS K channel in the thick ascending limb

2011 ◽  
Vol 300 (4) ◽  
pp. F906-F913 ◽  
Author(s):  
Mingxiao Wang ◽  
Hongyu Sui ◽  
Wennan Li ◽  
Jing Wang ◽  
Yujie Liu ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Inhibitory Effect ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
A2a Adenosine Receptor ◽  
K Channels ◽  
Cgs 21680 ◽  
Stimulation Of

The basolateral 50-pS K channels are stimulated by a cAMP-dependent pathway and inhibited by cytochrome P-450-omega-hydroxylase-dependent metabolism of arachidonic acid (AA) in the rat thick ascending limb (TAL). We now used the patch-clamp technique to examine whether stimulation of adenosine A2a receptor modulates the inhibitory effect of AA on the basolateral 50-pS K channels in the medullary TAL. Stimulation of adenosine A2a receptor with CGS-21680 or inhibition of phospholipase A2 (PLA2) with AACOCF3 increased the 50-pS K channel activity in the TAL. Western blot demonstrated that application of CGS-21680 decreased the phosphorylation of PLA2 at serine residue 505, an indication of inhibiting PLA2 activity. In the presence of CGS-21680, inhibition of PLA2 had no further effect on the basolateral 50-pS K channels. The possibility that CGS-21680-induced stimulation of the basolateral 50-pS K channels was partially achieved by inhibition of PLA2 in the TAL was also supported by the observation that CGS-21680 had no additional effect in the presence of AACOCF3. Moreover, stimulation of adenosine A2a receptor with CGS-21680 also abolished the inhibitory effect of AA and 20-hydroxyeicosatetraenoic acid (20-HETE) on the 50-pS K channels. The effect of CGS-21680 on AA and 20-HETE-mediated inhibition of the 50-pS K channels was mediated by cAMP because application of membrane-permeable cAMP analog, dibutyryl-cAMP, not only increased the 50-pS K channel activity but also abolished the inhibitory effect of AA and 20-HETE. We conclude that stimulation of adenosine A2a receptor increased the 50-pS K channel activity in the TAL, an effect that is achieved by suppression of PLA2 activity and 20-HETE-induced inhibition.

Dietary K intake regulates the response of apical K channels to adenosine in the thick ascending limb

2004 ◽  
Vol 287 (5) ◽  
pp. F954-F959 ◽  
Author(s):  
Dimin Li ◽  
Yuan Wei ◽  
Wen-Hui Wang
Keyword(s):  
Adenosine Receptor ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Cgs 21680 ◽  
Adenosine Receptor Agonist ◽  
Dependent Pathway ◽  
Adenosine Analog

We used the patch-clamp technique to study the effect of adenosine on the apical 70-pS K channel in the thick ascending limb (TAL) of the rat kidney. Application of 1 μM cyclohexyladenosine (CHA), an adenosine analog, stimulated apical 70-pS K channel activity and increased the product of channel open probability and channel number ( NPo) from 0.34 to 0.7. Also, addition of CGS-21680, a specific A2a adenosine receptor agonist, mimicked the effect of CHA and increased NPo from 0.33 to 0.77. The stimulatory effect of CHA and CGS-21680 was completely blocked by H89, an inhibitor of protein kinase A (PKA), or by inhibition of adenylate cyclase with SQ-22536. This suggests that the stimulatory effect of adenosine analogs is mediated by a PKA-dependent pathway. The effect of adenosine analog was almost absent in the TAL from rats on a K-deficient (KD) diet for 7 days. Application of DDMS, an agent that inhibits cytochrome P-450 hydrolase, not only significantly increased the activity of the 70-pS K channel but also restored the stimulatory effect of CHA on the 70-pS K channel in the TAL from rats on a KD diet. Also, the effect of CHA was absent in the presence of 20-HETE. Inhibition of PKA blocked the stimulatory effect of CHA on the apical 70-pS K channel in the presence of DDMS in the TAL from rats on a KD diet. We conclude that stimulation of adenosine receptor increases the apical 70-pS K channel activity via a PKA-dependent pathway and that the effect of adenosine on the apical 70-pS K channel is suppressed by low-K intake. Moreover, the diminished response to adenosine is the result of increase in 20-HETE formation, which inhibits the cAMP-dependent pathway in the TAL from rats on a KD diet.

Role of Ca2+/CaMK II in Ca(2+)-induced K+ channel inhibition in rat CCD principal cell

1995 ◽  
Vol 268 (2) ◽  
pp. F211-F219 ◽  
Author(s):  
M. Kubokawa ◽  
W. Wang ◽  
C. M. McNicholas ◽  
G. Giebisch
Keyword(s):  
Protein Kinase ◽  
Collecting Duct ◽  
Inhibitory Effect ◽  
K Channel ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Pkc Inhibitor ◽  
Control Value ◽  
Gf 109203X

The apical low-conductance K+ channel of rat cortical collecting duct (CCD) is inhibited by increased intracellular Ca2+ concentrations. This effect has been shown to be mediated at least in part by activation of protein kinase C (PKC). In the present study, we used the patch-clamp technique to examine the role of Ca2+/calmodulin-dependent protein kinase II (CaMK II) in mediating the Ca(2+)-induced inhibitory effect. In cell-attached patches of principal cells of rat tubules, clamping of intracellular Ca2+ concentration at 400 nM by using 1 microM ionomycin reduced channel activity to 26.5% of the control value. A further reduction in channel activity, to 8.8% of the control value, was observed following the addition of phorbol 12-myristate 13-acetate (PMA), an agent known to activate PKC. Pretreatment of cells with KN-62 (CaMK II inhibitor) or GF-109203X (PKC inhibitor) attenuated the inhibitory effect of Ca2+ on K+ channel activity (83.2 and 50.7% of the control value, respectively). Even in the presence of KN-62, addition of 10 microM PMA significantly decreased channel activity to 57.2% of the control value. The Ca(2+)-induced inhibition was completely abolished by simultaneous incubation with both KN-62 and GF-109203X. In inside-out patches, addition of 20 micrograms/ml CaMK II in the presence of a PKC inhibitor reduced channel activity to 66.2% of control values. It is concluded that CaMK II is involved in mediating the Ca(2+)-induced inhibition of the activity of the apical K+ channel of rat CCD.

Arachidonic acid inhibits activity of cloned renal K+ channel, ROMK1

1996 ◽  
Vol 271 (3) ◽  
pp. F588-F594 ◽  
Author(s):  
C. M. Macica ◽  
Y. Yang ◽  
S. C. Hebert ◽  
W. H. Wang
Keyword(s):  
Arachidonic Acid ◽  
Membrane Fluidity ◽  
Collecting Duct ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Lipoxygenase Inhibitor ◽  
Open Probability ◽  
Apical Membranes

Arachidonic acid (AA) has been shown to inhibit the activity of the low-conductance ATP-sensitive K+ channel in the apical membrane of the cortical collecting duct [W. Wang, A. Cassola, and G. Giebisch. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F554-F559, 1992]. ROMK1, a K+ channel derived from the rat renal outer medulla, shares many biophysical properties of the native low-conductance K+ channel, which is localized to the apical membranes of the cortical collecting duct and thick ascending limb. This study was designed to determine whether the ROMK channel maintains the property of AA sensitivity of the native low-conductance K+ channel. Experiments were conducted in Xenopus oocytes injected with cRNA encoding the ROMK1 channel by use of patch-clamp techniques. We have confirmed previous reports that the cloned ROMK1 has similar channel kinetics, high open probability, and inward slope conductance as the native low-conductance K+ channel, respectively. Addition of 5 microM AA to an inside-out patch resulted in reversible inhibition of channel activity at a concentration similar to the inhibitor constant for AA on the native K+ channel. The effect of AA on channel activity was preserved in the presence of 10 microM indomethacin, a cyclooxygenase inhibitor, 4 microM cinnamyl-3,4-dihydroxycyanocinnamate, a lipoxygenase inhibitor, and 4 microM 17-octadecynoic acid, an inhibitor of cytochrome P-450 monooxygenases, thus indicating that the effect of AA was not mediated by metabolites of AA. The effect did not appear to be the result of changes in membrane fluidity, since 5 microM eicosatetraynoic acid, an AA analogue that is a potent modulator of membrane fluidity, had no effect. Furthermore, the addition of AA to the outside of the patch also had no effect on channel activity. These results indicate that, like the native low-conductance channel, AA is able to directly inhibit ROMK1 channel activity.

scholarly journals Arachidonic acid inhibits basolateral K channels in the cortical collecting duct via cytochrome P-450 epoxygenase-dependent metabolic pathways

2008 ◽  
Vol 294 (6) ◽  
pp. F1441-F1447 ◽  
Author(s):  
ZhiJian Wang ◽  
Yuan Wei ◽  
John R. Falck ◽  
Krishnam Raju Atcha ◽  
Wen-Hui Wang
Keyword(s):  
Arachidonic Acid ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Inhibitory Effect ◽  
K Channel ◽  
Dependent Manner ◽  
Cortical Collecting Duct ◽  
Patch Clamp Technique ◽  
K Channels ◽  
Cytochrome P 450

We used the patch-clamp technique to study the effect of arachidonic acid (AA) on basolateral 18-pS K channels in the principal cell of the cortical collecting duct (CCD) of the rat kidney. Application of AA inhibited the 18-pS K channels in a dose-dependent manner and 10 μM AA caused a maximal inhibition. The effect of AA on the 18-pS K channel was specific because application of 11,14,17-eicosatrienoic acid had no effect on channel activity. Also, the inhibitory effect of AA on the 18-pS K channels was abolished by blocking cytochrome P-450 (CYP) epoxygenase with N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH) but was not affected by inhibiting CYP ω-hydroxylase or cyclooxygenase. The notion that the inhibitory effect of AA was mediated by CYP epoxygenase-dependent metabolites was further supported by the observation that application of 100 nM 11,12-epoxyeicosatrienoic acid (EET) mimicked the effect of AA and inhibited the basolateral 18-pS K channels. In contrast, addition of either 5,6-, 8,9-, or 14,15-EET failed to inhibit the 18-pS K channels. Moreover, application of 11,12-EET was still able to inhibit the 18-pS K channels in the presence of MS-PPOH. This suggests that 11,12-EET is a mediator for the AA-induced inhibition of the 18-pS K channels. We conclude that AA inhibits basolateral 18-pS K channels by a CYP epoxygenase-dependent pathway and that 11,12-EET is a mediator for the effect of AA on basolateral K channels in the CCD.

scholarly journals Effect of arachidonic acid on activity of the apical K+ channel in the thick ascending limb of the rat kidney.

1995 ◽  
Vol 106 (4) ◽  
pp. 727-743 ◽  
Author(s):  
W Wang ◽  
M Lu
Keyword(s):  
Arachidonic Acid ◽  
Cytochrome P450 ◽  
Metabolic Pathway ◽  
Rat Kidney ◽  
Specific Inhibitor ◽  
Inhibitory Effect ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
Inside Out

We have used patch-clamp techniques to study the effects of arachidonic acid (AA) on the activity of the 70-pS K+ channel, the predominant type of the two apical K+ channels operating under physiological conditions in the thick ascending limb (TAL) of the rat kidney. Addition of 5-10 microM AA blocked the activity of the 70-pS K+ channel in both cell-attached and inside-out patches. The inhibitory effect of AA was specific, because application of 10 microM linoleic acid, oleic acid, or palmitic acid failed to mimic the effect of AA. The effect of AA could not be blocked by pretreatment of the TAL tubules with either 5 microM indomethacin (inhibitor of cyclooxygenase) or 4 microM cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC) (inhibitor of lipooxygenase). In contrast, addition of 5 microM 17-octadecynoic acid (17-ODYA), an inhibitor of P450 monooxygenases, abolised the effect of AA on the channel activity, indicating that the effect was mediated by cytochrome P450 metabolites of AA. Addition of 10 nM 20-hydroxyeicosatetraenoic acid (20-HETE), the main metabolite of the cytochrome P450 metabolic pathway in the medullary TAL, mimicked the inhibitory effect of 10 microM AA. However, addition of 100 nM 19-HETE or 17-HETE had no significant effects and 100 nM 20-carboxy AA (20-COOH) reduced the channel activity by only 20%, indicating that the inhibitory effect of 20-HETE was specific and responsible for the action of AA. Inhibition of the P450 metabolic pathway by either 5 microM 17-ODYA or 12, 12-dibromododec-11-enoic acid (DBDD) dramatically increased the channel activity by 280% in cell-attached patches. The stimulatory effect of 17-ODYA or DBDD was not observed in inside-out patches. The results strongly indicate that 20-HETE is a specific inhibitor for the 70-pS K+ channel and may play an important role in the regulation of the K+ channel activity in the TAL.

Arachidonic acid inhibits the secretory K+ channel of cortical collecting duct of rat kidney

1992 ◽  
Vol 262 (4) ◽  
pp. F554-F559 ◽  
Author(s):  
W. Wang ◽  
A. Cassola ◽  
G. Giebisch
Keyword(s):  
Arachidonic Acid ◽  
Oleic Acid ◽  
Unsaturated Fatty Acids ◽  
Enzyme Inhibitor ◽  
Collecting Duct ◽  
K Channel ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability

We used the patch-clamp technique to study the effects of arachidonic acid (AA) on the 35-pS secretory K+ channel in the apical membrane of rat cortical collecting duct (CCD). Application of 10 microM AA reversibly reduced channel activity to 1% of the control value [sum of open probability (NPo) decreased from 3.8 to 0.04]. AA inhibits the apical 35-pS K+ channel directly, because application of indomethacin (an inhibitor of cyclooxygenase), nordihydroguaiaretic acid (an enzyme inhibitor of lipoxygenase), and clotrimazole (an inhibitor of epoxygenase) failed to antagonize the AA-induced blocking effect on K+ channel activity. Oleic acid, a cis-unsaturated acid, also blocks K+ channel activity. However, the inhibitory constant (Ki) of oleic acid (5.1 microM) is significantly higher than that of AA (2.6 microM). These results indicate that AA and cis-unsaturated fatty acids are involved in downregulating the apical secretory K+ channel of rat CCD.

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