Biomedical subjects

Wen-Hui Wang

Publications and source records attributed to Wen-Hui Wang.

At least 19 recordsLinked to original sources

PubMed2006

Angiotensin II inhibits the ROMK-like small conductance K channel in renal cortical collecting duct during dietary potassium restriction.

Base-line urinary potassium secretion in the distal nephron is mediated by small conductance rat outer medullary K (ROMK)-like channels. We used the patch clamp technique applied to split-open cortical collecting ducts (CCDs) isolated from rats fed a normal potassium (NK) or low potassium (LK) diet to test the hypothesis that AngII directly inhibits ROMK channel activity. We found that AngII inhibited ROMK channel activity in LK but not NK rats in a dose-dependent manner. The AngII-induced reduction in channel activity was mediated by AT1 receptor (AT1R) binding, because pretreatment of CCDs with losartan but not PD123319 AT1 and AT2 receptor antagonists, respectively, blocked the response. Pretreatment of CCDs with U73122 and calphostin C, inhibitors of phospholipase C (PLC) and protein kinase C (PKC), respectively, abolished the AngII-induced decrease in ROMK channel activity, confirming a role of the PLC-PKC pathway in this response. Studies by others suggest that AngII stimulates an Src family protein-tyrosine kinase (PTK) via PKC-NADPH oxidase. PTK has been shown to regulate the ROMK channel. Inhibition of NADPH oxidase with diphenyliodonium abolished the inhibitory effect of AngII or the PKC activator phorbol 12-myristate 13-acetate on ROMK channels. Suppression of PTK by herbimycin A significantly attenuated the inhibitory effect of AngII on ROMK channel activity. We conclude that AngII inhibits ROMK channel activity through PKC-, NADPH oxidase-, and PTK-dependent pathways under conditions of dietary potassium restriction.

Angiotensin II↗

PubMed2006

Inhibition of MAPK stimulates the Ca2+ -dependent big-conductance K channels in cortical collecting duct.

The kidney plays a key role in maintaining potassium (K) homeostasis. K excretion is determined by the balance between K secretion and absorption in distal tubule segments such as the connecting tubule and cortical collecting duct. K secretion takes place by K entering principal cells (PC) from blood side through Na+, K+ -ATPase and being secreted into the lumen via both ROMK-like small-conductance K (SK) channels and Ca2+ -activated big-conductance K (BK) channels. K reabsorption occurs by stimulation of apical K/H-ATPase and inhibition of K recycling across the apical membrane in intercalated cells (IC). The role of ROMK channels in K secretion is well documented. However, the importance of BK channels in mediating K secretion is incompletely understood. It has been shown that their activity increases with high tubule flow rate and augmented K intake. However, BK channels have a low open probability and are mainly located in IC, which lack appropriate transporters for effective K secretion. Here we demonstrate that inhibition of ERK and P38 MAPKs stimulates BK channels in both PC and IC in the cortical collecting duct and that changes in K intake modulate their activity. Under control conditions, BK channel activity in PC was low but increased significantly by inhibition of both ERK and P38. Blocking MAPKs also increased channel open probability of BK in IC and thereby it may affect K backflux and net K absorption Thus, modulation of ERK and P38 MAPK activity is involved in controlling net K secretion in the distal nephron.

Animals↗

PubMed2006

Mitogen-activated protein kinases inhibit the ROMK (Kir 1.1)-like small conductance K channels in the cortical collecting duct.

It was demonstrated previously that low dietary potassium (K) intake stimulates Src family protein tyrosine kinase (PTK) expression via a superoxide-dependent signaling. This study explored the role of mitogen-activated protein kinase (MAPK) in mediating the effect of superoxide anions on PTK expression and ROMK (Kir 1.1) channel activity. Western blot analysis demonstrated that low K intake significantly increased the phosphorylation of P38 MAPK (P38) and extracellular signal-regulated kinase (ERK) but had no effect on phosphorylation of c-JUN N-terminus kinase in renal cortex and outer medulla. The stimulatory effect of low K intake on P38 and ERK was abolished by treatment of rats with tempol. The possibility that increases in superoxide and related products that are induced by low K intake were responsible for stimulating phosphorylation of P38 and ERK also was supported by the finding that application of H(2)O(2) increased the phosphorylation of ERK and P38 in the cultured mouse collecting duct cells. Simultaneous blocking of ERK and P38 completely abolished the effect of H(2)O(2) on c-Src expression in mouse collecting duct cells. For determination of the role of P38 and ERK in the regulation of ROMK-like small-conductance K (SK) channels, the patch-clamp technique was used to study the effect of inhibiting P38 and ERK on SK channels in the cortical collecting duct from rats that were on a control K diet (1.1%) and on a K-deficient diet for 1 d. Inhibition of ERK, c-JUN N-terminus kinase, or P38 alone had no effect on SK channels. In contrast, simultaneous inhibition of P38 and ERK significantly increased channel activity. The effect of inhibiting MAPK on SK channels was not affected in the presence of herbimycin A, a PTK inhibitor, and was larger in rats that were on a K-deficient diet than in rats that were on a normal-K diet. However, the stimulatory effect of inhibiting ERK and P38 on SK was absent in the cortical collecting duct that was treated with colchicine. It is concluded that low K intake-induced increases in superoxide levels are responsible for stimulation of P38 and ERK and that MAPK inhibit the SK channels by stimulating PTK expression and via a PTK-independent mechanism.

Adrenal Cortex↗

PubMed2006

Low Na intake suppresses expression of CYP2C23 and arachidonic acid-induced inhibition of ENaC.

We previously demonstrated that arachidonic acid (AA) inhibits epithelial Na channels (ENaC) through the cytochrome P-450 (CYP) epoxygenase-dependent pathway (34). In the present study, we tested the hypothesis that low Na intake suppresses the expression of CYP2C23, which is mainly responsible for converting AA to epoxyeicosatrienoic acid (EET) in the kidney (11) and attenuates the AA-induced inhibition of ENaC. Immunostaining showed that CYP2C23 is expressed in the Tamm-Horsfall protein (THP)-positive and aquaporin 2 (AQP2)-positive tubules. This suggests that CYP2C23 is expressed in the thick ascending limb (TAL) and collecting duct (CD). Na restriction significantly suppressed the expression of CYP2C23 in the TAL and CD. Western blot also demonstrated that the expression of CYP2C23 in renal cortex and outer medulla diminished in rats on Na-deficient diet (Na-D) but increased in those on high-Na diet (4%). Moreover, the content of 11,12-epoxyeicosatrienoic acid (EET) decreased in the isolated cortical CD from rats on Na-D compared with those on a normal-Na diet (0.5%). Patch-clamp study showed that application of 15 microM AA inhibited the activity of ENaC by 77% in the CCD of rats on a Na-D for 3 days. However, the inhibitory effect of AA on ENaC was significantly attenuated in rats on Na-D for 14 days. Furthermore, inhibition of CYP epoxygenase with MS-PPOH increased the ENaC activity in the CCD of rats on a control Na diet. We also used microperfusion technique to examine the effect of MS-PPOH on Na transport in the distal nephron. Application of MS-PPOH significantly increased Na absorption in the distal nephron of control rats but had no significant effect on Na absorption in rats on Na-D for 14 days. We conclude that low Na intake downregulates the activity and expression of CYP2C23 and attenuates the inhibitory effect of AA on Na transport.

8,11,14-Eicosatrienoic Acid↗

PubMed2006

Salt-sensitive hypertension is associated with dysfunctional Cyp4a10 gene and kidney epithelial sodium channel.

Functional and biochemical data have suggested a role for the cytochrome P450 arachidonate monooxygenases in the pathophysiology of hypertension, a leading cause of cardiovascular, cerebral, and renal morbidity and mortality. We show here that disruption of the murine cytochrome P450, family 4, subfamily a, polypeptide 10 (Cyp4a10) gene causes a type of hypertension that is, like most human hypertension, dietary salt sensitive. Cyp4a10-/- mice fed low-salt diets were normotensive but became hypertensive when fed normal or high-salt diets. Hypertensive Cyp4a10-/- mice had a dysfunctional kidney epithelial sodium channel and became normotensive when administered amiloride, a selective inhibitor of this sodium channel. These studies (a) establish a physiological role for the arachidonate monooxygenases in renal sodium reabsorption and blood pressure regulation, (b) demonstrate that a dysfunctional Cyp4a10 gene causes alterations in the gating activity of the kidney epithelial sodium channel, and (c) identify a conceptually novel approach for studies of the molecular basis of human hypertension. It is expected that these results could lead to new strategies for the early diagnosis and clinical management of this devastating disease.

Amiloride↗

PubMed2006

Ischemia impairs the association between connexin 43 and M3 subtype of acetylcholine muscarinic receptor (M3-mAChR) in ventricular myocytes.

We used Western blot analysis to examine the expression of connexin 43 and M2/M3 acetylcholine muscarinic receptors (mAChR) and their interaction in ventricular myocytes from control and the ischemic heart. We confirmed that the connexin 43 and M2/ M3-mAChR were expressed in ventricular myocytes. Moreover, we showed that M3-mAChR was expressed in non-glycosylated (72 kDa) and glycosylated forms (115 kDa). Immunostaining showed that connexin 43 is closely associated with M3-mAChR in parts of cell membranes of myocytes. Immunoprecipitation of lysate of cardiac myocytes with M2/M3-mAChR antibody pulled down a 44 kDa protein recognized by connexin 43 antibody. Ischemia increased the expression of M3-mAChR in myocytes. The ischemiainduced increase in the M3-mAChR expression was specific because ischemia did not affect the expression of M1, M2, M4 and M5- mAChR in the heart. On the other hand, ischemia decreased the expression of connexin 43 in myocardium. We also examined the effect of ischemia on the interaction between M2/M3-mAChR and connexin 43. Ischemia suppressed the association of M3-mAChR with connexin 43 but did not affect the association of connexin 43 with M2-mAChR. Administration of choline before ischemia not only partially restored the expression of connexin 43 but also attenuated the ischemia-induced suppression of the association between connexin 43 and M3-mAChR. We conclude that connexin 43 interacts with M2/M3-mAChR and that ischemia specifically impairs the association between M3-mAChR and connexin 43.

Animals↗

PubMed2006

Clinical and pathological study of lichen-planus-like keratosis in China.

Lichen-planus-like keratosis is usually diagnosed pathologically; rarely, a definitive diagnosis can be made grossly in the clinic. Only a few cases of lichen-planus-like keratosis have been reported in China. The purpose of this study was to investigate the clinical and pathological features of lichen-planus-like keratosis in China. Fifty cases of lichen-planus-like keratosis patients diagnosed pathologically during a 5-year period in our clinic were analyzed. Clinical features were recorded. Sectioned specimens were subjected to hematoxylin and eosin staining to observe pathological changes. Results showed that there were 34 males and 16 females (ratio 2:1) with an average age of 61.2 years. Most of the lesions were single papules or plaques with rough surfaces. They were distributed on the face, larger than 1 cm, and dark red to brown in color. Only one case (2%) was considered to be lichen-planus-like keratosis clinically. By hematoxylin and eosin staining, solar lentigo and solar elastosis could be found in 68% and 32% of lichen-planus-like keratosis lesions, respectively. Eosinophil (42%) and plasma cell (36%) infiltration was also found frequently. Exoerythrocytes could be detected in 50% of the cases. Lichen-planus-like keratosis is not uncommon in clinical practice in China, the diagnosis of lichen-planus-like keratosis should be made by a combination of clinical manifestations and pathological changes. It is better to classify lichen-planus-like keratosis as a benign skin tumor. More attention should be paid to lichen-planus-like keratosis in China.

Aged↗

PubMed2006

Regulation of ROMK (Kir1.1) channels: new mechanisms and aspects.

This brief review attempts to provide an overview regarding recent developments in the regulation of ROMK channels. Studies performed in ROMK null mice suggest that ROMK cannot only form hometetramers such as the small-conductance (30-pS) K channels but also construct heterotetramers such as the 70-pS K channel in the thick ascending limb (TAL). The expression of ROMK channels in the plasma membrane is regulated by protein tyrosine kinase (PTK), serum and glucorticoid-induced kinase (SGK), and with-no-lysine-kinase 4. PTK is involved in mediating the effect of low K intake on ROMK channel activity. Increases in superoxide anions induced by low dietary K intake are responsible for the stimulation of PTK expression and tyrosine phosphorylation of ROMK channels. Finally, a recent study indicated that ROMK channels can be monoubiquitinated and monoubiquitination regulates the surface expression of ROMK channels.

Animals↗

PubMed2005

Adenosine inhibits ENaC via cytochrome P-450 epoxygenase-dependent metabolites of arachidonic acid.

We used the patch-clamp technique to examine the effect of adenosine on epithelial sodium channel (ENaC) activity in rat cortical collecting duct (CCD). Application of adenosine inhibits ENaC activity, and the effect of adenosine was mimicked by cyclohexyladenosine (CHA), an A(1) adenosine-receptor agonist that reduced channel activity from 1.32 to 0.64. The inhibitory effect of CHA on ENaC was mimicked by cyclopentyladenosine (CPA), which reduced channel activity from 1.1 to 0.55. In contrast, application of CGS-21680, an A(2a) adenosine-receptor agonist, had no effect on ENaC and increased channel activity from 0.96 to 1.22. This suggests that the inhibitory effect of adenosine analogs resulted from stimulation of the A(1) adenosine receptor. Inhibition of PLC with U-73122 failed to abolish the effect of CHA on ENaC. In contrast, the inhibitory effect of CHA on ENaC was absent in the presence of the PLA(2) inhibitor arachidonyl trifluoromethyl ketone (AACOCF(3)). This suggests a role of arachidonic acid (AA) in mediating the effect of adenosine on ENaC. To determine the metabolic pathway of AA responsible for the effect of adenosine, we examined the effect of CHA in the presence of indomethacin or N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH). Inhibition of cytochrome P-450 (CYP) epoxygenase with MS-PPOH blocked the effect of CHA on ENaC. In contrast, CHA reduced ENaC activity in the presence of indomethacin. This suggests that CYP epoxygenase-dependent metabolites of AA mediate the effect of adenosine. Because 11,12-epoxyeicosatrienoic acid (11,12-EET) inhibits ENaC activity in the CCD (Wei Y, Lin DH, Kemp R, Yaddanapudi GSS, Nasjletti A, Falck JR, and Wang WH. J Gen Physiol 124: 719-727, 2004), we examined the role of 11,12-EET in mediating the effect of adenosine on ENaC. Addition of 11,12-EET inhibited ENaC channels in the CCD in which adenosine-induced inhibition was blocked by AACOCF3. We conclude that adenosine inhibits ENaC activity by stimulation of the A(1) adenosine receptor in the CCD and that the effect of adenosine is mediated by 11,12-EET.

Adenosine↗

PubMed2005

Inhibition of phosphatidylinositol 3-kinase stimulates activity of the small-conductance K channel in the CCD.

We used Western blotting to examine the expression of phosphatidylinositol 3-kinase (PI3K) in the renal cortex and outer medulla and employed the patch-clamp technique to study the effect of PI3K on the ROMK-like small-conductance K (SK) channels in the cortical collecting duct (CCD). Low K intake increased the expression of the 110-kDa alpha-subunit (p110alpha) of PI3K compared with rats on a normal-K diet. Because low K intake increases superoxide levels (2), the possibility that increases in superoxide anions may be responsible for the effect of low K intake on the expression of PI3K is supported by finding that addition of H(2)O(2) stimulates the expression of p110alpha in M1 cells. Inhibition of PI3K with either wortmannin or LY-294002 significantly increased channel activity in the CCD from rats on a K-deficient (KD) diet or on a normal-K diet. The stimulatory effect of wortmannin on ROMK channel activity cannot be mimicked by inhibition of phospholipase C with U-73122. This suggests that the effect of inhibiting PI3K was not the result of increasing the phosphatidylinositol 4,5-bisphosphate level. Moreover, application of the exogenous phosphatidylinositol 3,4,5-trisphosphate analog had no effect on channel activity in excised patches. Because low K intake has been shown to increase the activity of protein tyrosine kinase (PTK), we explored the role of the interaction between PTK and PI3K in the regulation of the SK channel activity. Inhibition of PTK increased SK channel activity in the CCD from rats on a KD diet. However, addition of wortmannin did not further increase ROMK channel activity. Also, the effect of wortmannin was abolished by treatment of CCD with phalloidin. We conclude that PI3K is involved in mediating the effect of low K intake on ROMK channel activity in the CCD and that the effect of PI3K on SK channels requires the involvement of PTK and the cytoskeleton.

Animals↗

PubMed2005

ROMK1 channel activity is regulated by monoubiquitination.

The ubiquitination of proteins can signal their degradation, modify their activity or target them to specific membranes or cellular organelles. Here, we show that monoubiquitination regulates the plasma membrane abundance and function of the potassium channel, ROMK. Immunoprecipitation of proteins obtained from renal cortex and outer medulla with ROMK antibody revealed that this channel was monoubiquitinated. To determine the ubiquitin binding site on ROMK1, all intracellular lysine (Lys) residues of ROMK1 were individually mutated to arginine (Arg), and a two-electrode voltage clamp was used to measure the ROMK1 channel activity in Xenopus oocytes. ROMK1 channel activity increased from 8.1 to 27.2 microA only when Lys-22 was mutated to Arg. Furthermore, Western blotting failed to detect the ubiquitinated ROMK1 in oocytes injected with R1K22R. Patch-clamp experiments showed that biophysical properties of R1K22R were identical to those of wild-type ROMK1. Although total protein expression levels of GFP-ROMK1 and GFP-R1K22R in oocytes were similar, confocal microscopy showed that the surface fluorescence intensity in oocytes injected with GFP-R1K22R was higher than that of GFP-ROMK1. In addition, biotin labeling of ROMK1 and R1K22R proteins expressed in HEK293 cells showed increased surface expression of the Lys-22 mutant channel. Finally, expression of R1K22R in COS7 cells significantly stimulated the surface expression of ROMK1. We conclude that ROMK1 can be monoubiquitinated and that Lys-22 is an ubiquitin-binding site. Thus, monoubiquitination of ROMK1 regulates channel activity by reducing the surface expression of channel protein. This finding implicates the linking of a single ubiquitin molecule to channels as an important posttranslational regulatory signal.

Animals↗

PubMed2005

Superoxide anions are involved in mediating the effect of low K intake on c-Src expression and renal K secretion in the cortical collecting duct.

We previously demonstrated that low K intake stimulated the expression of c-Src and that stimulation of protein tyrosine kinase inhibited ROMK channel activity (Wei, Y., Bloom, P., Lin, D. H., Gu, R. M., and Wang, W. H. (2001) Am. J. Physiol. 281, F206-F212). Decreases in dietary K content significantly increased O(2)(-) levels and the phosphorylation of c-Jun, a transcription factor, in renal cortex and outer medulla. The role of O(2)(-) and related products such as H(2)O(2) in stimulating the expression of protein tyrosine kinase is suggested by the observation that addition of 50-200 microm H(2)O(2) increased the phosphorylation of c-Jun and the expression of c-Src in M1 cells, a mouse collecting duct principal cell line. The effect of H(2)O(2) on c-Src expression was completely abolished with cyclohexamide or actinomycin D. The treatment of animals on a K-deficient (KD) diet with tempol for 7 days significantly decreased the production of O(2)(-), c-Jun phosphorylation, and c-Src expression. Moreover, low K intake decreased the activity of ROMK-like small conductance channels from 1.37 (control K diet) to 0.5 in the cortical collecting duct and increased the tyrosine phosphorylation of ROMK in the renal cortex and outer medulla. In contrast, the tempol treatment not only increased channel activity to 1.1 in the cortical collecting duct but also decreased the tyrosine phosphorylation of ROMK from rats on a KD diet. Finally, suppressing O(2)(-) production with tempol significantly increased renal K excretion measured with metabolic cage and lowered the plasma K concentration in comparison with those on a KD diet alone without tempol. We conclude that O(2)(-) and related products play a role in mediating the effect of low K intake on c-Src expression and in suppressing ROMK channel activity and renal K secretion.

Animals↗

PubMed2005

Cosmetic dermatitis in Chinese eczema patients patch tested with a modified European standard series of allergens.

The prevalence of cosmetic allergic contact dermatitis (CACD) in Chinese eczema patients (CEP) has not been reported. The purpose of our study was to analyse CACD in CEP and examine the frequency of patch test (PT) reactions to common cosmetic-related allergens (CRA). 378 consecutive CEP patch tested with a modified European standard series of allergens during a 2-year period in our clinic were analysed. 73 patients (19.3%) were considered as suspected CACD and 37 patients (9.8%) were confirmed. The frequencies of the positive PT reactions in suspected CACD and confirmed CACD to at least 1 CRA were 64.4% and 89.2%, to para-phenylenediamine (PPD) were 31.5% and 59.5%, to fragrance mix (FM) were 27.4% and 32.4% and to imidazolidinylurea were 5.5% and 8.1%, respectively. These results were much higher than those of CEP without cosmetic reactions (26.3% for at least 1 CRA, 5.8% for PPD, 8.8% for FM, and 0 for imidazolidinylurea) (P < 0.01 for all, Chi-square test and Fisher's exact test). These results suggested that CACD is very common in CEP patch tested. PPD and FM are the leading allergens identified. CACD should be strongly considered in CEP with positive PT reactions to PPD, FM and imidazolidinylurea.

Adult↗

PubMed2005

Mineralocorticoids decrease the activity of the apical small-conductance K channel in the cortical collecting duct.

We used the patch-clamp technique to examine the effect of DOCA treatment (2 mg/kg) on the apical small-conductance K (SK) channels, epithelial Na channels (ENaC), and the basolateral 18-pS K channels in the cortical collecting duct (CCD). Treatment of rats with DOCA for 6 days significantly decreased the plasma K from 3.8 to 3.1 meq and reduced the activity of the SK channel, defined as NP(o), from 1.3 in the CCD of control rats to 0.6. In contrast, DOCA treatment significantly increased ENaC activity from 0.01 to 0.53 and the basolateral 18-pS K channel activity from 0.67 to 1.63. Moreover, Western blot analysis revealed that DOCA treatment significantly increased the expression of the nonreceptor type of protein tyrosine kinase (PTK), cSrc, and the tyrosine phosphorylation of ROMK in the renal cortex and outer medulla. The possibility that decreases in apical SK channel activity induced by DOCA treatment were the result of stimulation of PTK activity was further supported by experiments in which inhibition of PTK with herbimycin A significantly increased NP(o) from 0.6 to 2.1 in the CCD from rats receiving DOCA. Also, when rats were fed a high-K (10%) diet, DOCA treatment did not increase the expression of c-Src and decrease the activity of the SK channel in the CCD. We conclude that DOCA treatment decreased the apical SK channel activity in rats on a normal-K diet and that an increase in PTK expression may be responsible for decreased channel activity in the CCD from DOCA-treated rats.

Animal Feed↗

PubMed2005

The protein tyrosine kinase-dependent pathway mediates the effect of K intake on renal K secretion.

Dietary K intake plays an important role in the regulation of K secretion: a decrease stimulates and an increase suppresses kidney expression of protein tyrosine kinase (PTK), which plays a role in regulating Kir1.1 (ROMK), which is responsible for K secretion in the cortical collecting duct (CCD) and K recycling in the thick ascending limb. Tyrosine phosphorylation of ROMK channels increases with low dietary K and decreases with high dietary K. Moreover, stimulation of tyrosine phosphorylation of ROMK1 enhances ROMK1 internalization and reduces the K channel number in the cell surface in the CCD.

Animals↗

PubMed2004

Arachidonic acid inhibits epithelial Na channel via cytochrome P450 (CYP) epoxygenase-dependent metabolic pathways.

We used the patch-clamp technique to study the effect of arachidonic acid (AA) on epithelial Na channels (ENaC) in the rat cortical collecting duct (CCD). Application of 10 microM AA decreased the ENaC activity defined by NPo from 1.0 to 0.1. The dose-response curve of the AA effect on ENaC shows that 2 microM AA inhibited the ENaC activity by 50%. The effect of AA on ENaC is specific because neither 5,8,11,14-eicosatetraynoic acid (ETYA), a nonmetabolized analogue of AA, nor 11,14,17-eicosatrienoic acid mimicked the inhibitory effect of AA on ENaC. Moreover, inhibition of either cyclooxygenase (COX) with indomethacin or cytochrome P450 (CYP) omega-hydroxylation with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) failed to abolish the effect of AA on ENaC. In contrast, the inhibitory effect of AA on ENaC was absent in the presence of N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH), an agent that inhibits CYP-epoxygenase activity. The notion that the inhibitory effect of AA is mediated by CYP-epoxygenase-dependent metabolites is also supported by the observation that application of 200 nM 11,12-epoxyeicosatrienoic acid (EET) inhibited ENaC in the CCD. In contrast, addition of 5,6-, 8,9-, or 14,15-EET failed to decrease ENaC activity. Also, application of 11,12-EET can still reduce ENaC activity in the presence of MS-PPOH, suggesting that 11,12-EET is a mediator for the AA-induced inhibition of ENaC. Furthermore, gas chromatography mass spectrometry analysis detected the presence of 11,12-EET in the CCD and CYP2C23 is expressed in the principal cells of the CCD. We conclude that AA inhibits ENaC activity in the CCD and that the effect of AA is mediated by a CYP-epoxygenase-dependent metabolite, 11,12-EET.

Animals↗

PubMed2004

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

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 microM cyclohexyladenosine (CHA), an adenosine analog, stimulated apical 70-pS K channel activity and increased the product of channel open probability and channel number (NP(o)) from 0.34 to 0.7. Also, addition of CGS-21680, a specific A(2a) adenosine receptor agonist, mimicked the effect of CHA and increased NP(o) 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.

Adenosine↗

PubMed2004

Dual effect of insulin-like growth factor on the apical 70-pS K channel in the thick ascending limb of rat kidney.

We used the patch-clamp technique to study the effect of insulin-like growth factor I (IGF-I) on the apical 70-pS K channel in the isolated thick ascending limb (TAL) of the rat kidney. The isolated TAL was cut open to gain access to the apical membrane. Addition of 25 nM IGF-I stimulates the apical 70-pS K channel and increases channel activity, defined by the product of channel open probability and channel number, from 0.31 to 1.21. The stimulatory effect of IGF-I is not mediated by nitric oxide- or protein tyrosine phosphatase-dependent mechanisms, because inhibition of nitric oxide synthase or blocking protein tyrosine phosphatase did not abolish the stimulatory effect of IGF-I on the 70-pS K channel. In contrast, inhibition of mitogen-activated protein (MAP) kinase with PD-98059 or U0126 abolished the stimulatory effect of IGF-I. This suggests that MAP kinase is responsible for mediating the effect of IGF-I on the apical K channels. Moreover, the effect of IGF-I on the apical 70-pS K channel is biphasic because high concentrations (>200 nM) inhibit apical 70-pS K channels. Application of 400 nM IGF-I decreased channel activity from 1.45 to 0.2. The inhibitory effect of IGF-I is not blocked by calphostin C (an inhibitor of PKC), but inhibition of protein tyrosine kinase with herbimycin A abolished the IGF-induced inhibition. We conclude that IGF-I has a dual effect on the apical 70-pS K channel in the TAL: low concentrations of IGF-I stimulate, whereas high concentrations inhibit the channel activity. The stimulatory effect of IGF-I is mediated by a MAP kinase-dependent pathway, whereas the inhibitory effect is the result of stimulation of protein tyrosine kinase.

Animals↗