scholarly journals PKC expression is regulated by dietary K intake and mediates internalization of SK channels in the CCD

2004 ◽  
Vol 286 (6) ◽  
pp. F1072-F1078 ◽  
Author(s):  
Hyacinth Sterling ◽  
Dao-Hong Lin ◽  
Yu-Jung Chen ◽  
Yuan Wei ◽  
Zhi-Jian Wang ◽  
...  
Keyword(s):  
Western Blot ◽  
Renal Cortex ◽  
Collecting Duct ◽  
Control Diet ◽  
Sk Channels ◽  
Patch Clamp Technique ◽  
Outer Medulla ◽  
Protein Tyrosine ◽  
Pkc Isoforms ◽  
Low K

We have used Western blot analysis and immunocytochemistry to determine the effect of dietary K intake on the expression of protein kinase C (PKC) isoforms in the kidney. Western blot has demonstrated that conventional PKC isoforms (α and β), novel PKC isoforms (δ, ε, and η), and atypical PKC isoforms (ζ) are expressed in the renal cortex and outer medulla. Moreover, a low K intake significantly increases the expression of PKC-ε in the renal cortex and outer medulla but does not change the expression of PKC-α, PKC-β, PKC-δ, PKC-η, and PKC-ζ. Also, immunocytochemistry shows that PKC-ε isoform is expressed in the cortical collecting duct (CCD) and outer medullary collecting duct (OMCD) and that the intensity of PKC-ε staining is higher in the kidney from rats on a K-deficient diet than those on a control diet. Also, we used the patch-clamp technique to study the role of PKC in mediating internalization of ROMK (Kir 1.1)-like small-conductance K (SK) channels induced by phenylarsine oxide (PAO), an agent that inhibits protein tyrosine phosphatase and has been shown to stimulate the internalization of the SK channel in the CCD (Sterling H, Lin DH, Qu RM, Dong K, Herbert SC, and Wang WH. J Biol Chem 277: 4317–4323, 2002). Inhibition of PKC with calphostin C and GF-109203x had no significant effect on channel activity but abolished the inhibitory effect of PAO on SK channels. In conclusion, a low K intake increases the expression of PKC-ε isoform in the renal cortex and outer medulla, and PKC is involved in mediating the internalization of SK channels in the CCD induced by stimulation of protein tyrosine kinase activity.

Effect of dietary K intake on apical small-conductance K channel in CCD: role of protein tyrosine kinase

2001 ◽  
Vol 281 (2) ◽  
pp. F206-F212 ◽  
Author(s):  
Yuan Wei ◽  
Peter Bloom ◽  
Daohong Lin ◽  
Ruimin Gu ◽  
Wen Hui Wang
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Renal Cortex ◽  
Collecting Duct ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Protein Tyrosine ◽  
Herbimycin A ◽  
Small Conductance

We have used Western blot to examine the expression of cSrc protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP)-1D in the renal cortex, and the patch-clamp technique to determine the role of PTK in mediating the effect of dietary K intake on the small-conductance K (SK) channel in the cortical collecting duct (CCD). When rats were on a K-deficient (KD) diet for 1, 3, 5, and 7 days, the expression of cSrc increased by 40, 90, 140, and 135%, respectively. In contrast, the expression of cSrc in the renal cortex from rats on a high-K (HK) diet for 1, 2, and 3 days decreased by 40, 60, and 75%, respectively. However, the protein level of PTP-1D was not significantly changed by dietary K intake. The addition of 1 μM herbimycin A increased NP o, a product of channel number ( N) and open probability ( P o) in the CCD from rats on a normal diet or on a KD diet. The increase in NP o was 0.30 (normal), 0.45 (1-day KD), 0.65 (3-day KD), 1.55 (5-day KD), and 1.85 (7-day KD), respectively. Treatment of the CCD with herbimycin A from rats on a KD diet increased NP o per patch from the control value (0.7) to 1.4 (1-day KD), 1.6 (3-day KD), 2.6 (5-day KD), and 3.5 (7-day KD), respectively. In contrast, HK intake for as short as 1 day abolished the effect of herbimycin A. Furthermore, the expression of ROMK channels in the renal cortex was the same between rats on a KD diet or on a HK diet. Moreover, treatment with herbimycin A did not further increase NP o in the CCDs from rats on a HK diet. We conclude that dietary K intake plays a key role in regulating the activity of the SK channels and that PTK is involved in mediating the effect of the K intake on channel activity in the CCD.

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

2006 ◽  
Vol 290 (4) ◽  
pp. F806-F812 ◽  
Author(s):  
Dimin Li ◽  
Yuan Wei ◽  
Elisa Babilonia ◽  
Zhijian Wang ◽  
Wen-Hui Wang
Keyword(s):  
Collecting Duct ◽  
Sk Channels ◽  
Channel Activity ◽  
Phosphatidylinositol 3 Kinase ◽  
Patch Clamp Technique ◽  
Α Subunit ◽  
Sk Channel ◽  
Low K ◽  
Small Conductance ◽  
Phosphatidylinositol 3

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 α-subunit (p110α) 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 H2O2 stimulates the expression of p110α 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.

Micropuncture study of renal potassium excretion in the rat

1964 ◽  
Vol 206 (4) ◽  
pp. 674-686 ◽  
Author(s):  
Gerhard Malnic ◽  
Ruth M. Klose ◽  
Gerhard Giebisch
Keyword(s):  
Collecting Duct ◽  
Control Diet ◽  
Electrical Potential ◽  
Potential Gradient ◽  
Distal Tubule ◽  
Relative Increase ◽  
Dual Channel ◽  
High K ◽  
Tubular Lumen ◽  
Low K

Samples of proximal and distal tubular fluid were collected from rats maintained on a control, a low-K, or a high-K, low-Na diet. All animals received inulin-C14. Plasma (P) and tubular fluid (TF) were analyzed for Na and K by dual-channel microflame photometry and assayed for radioactivity. Transtubular electrical potential differences were measured by means of glass microelectrodes. Mean TF/P ratios for potassium in the proximal tubule were slightly below unity in all groups of animals. A comparison of the relative increase in K and inulin-C14 along the distal tubule indicates: 1) net movement of potassium into the tubular lumen in most control animals; 2) net movement of K into the tubular lumen of high-K, low-Na, sulfate-loaded animals, and in dichlorphenamide-treated animals on a control diet; and 3) the possibility of continued net reabsorption of potassium along the distal tubule and, particularly, the collecting duct in animals kept on a low-K diet. Distal tubular entry of potassium occurs down an electrochemical potential gradient.

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

2005 ◽  
Vol 289 (5) ◽  
pp. F1065-F1071 ◽  
Author(s):  
Yuan Wei ◽  
Elisa Babilonia ◽  
Hyacinth Sterling ◽  
Yan Jin ◽  
Wen-Hui Wang
Keyword(s):  
Protein Tyrosine Kinase ◽  
Collecting Duct ◽  
K Channel ◽  
Sk Channels ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Herbimycin A ◽  
Sk Channel ◽  
Small Conductance

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 NPo, 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 NPo 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.

scholarly journals SPAK-mediated NCC regulation in response to low-K+ diet

2015 ◽  
Vol 308 (8) ◽  
pp. F923-F931 ◽  
Author(s):  
James B. Wade ◽  
Jie Liu ◽  
Richard Coleman ◽  
P. Richard Grimm ◽  
Eric Delpire ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Western Blot ◽  
Control Diet ◽  
Distal Convoluted Tubule ◽  
Wild Type ◽  
Unknown Mechanism ◽  
Specific Antibodies ◽  
Important Mediator ◽  
Low Potassium ◽  
Low K

The NaCl cotransporter (NCC) of the renal distal convoluted tubule is stimulated by low-K+ diet by an unknown mechanism. Since recent work has shown that the STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) can function to stimulate NCC by phosphorylation of specific N-terminal sites, we investigated whether the NCC response to low-K+ diet is mediated by SPAK. Using phospho-specific antibodies in Western blot and immunolocalization studies of wild-type and SPAK knockout (SPAK−/−) mice fed a low-K+ or control diet for 4 days, we found that low-K+ diet strongly increased total NCC expression and phosphorylation of NCC. This was associated with an increase in total SPAK expression in cortical homogenates and an increase in phosphorylation of SPAK at the S383 activation site. The increased pNCC in response to low-K+ diet was blunted but not completely inhibited in SPAK−/− mice. These findings reveal that SPAK is an important mediator of the increased NCC activation by phosphorylation that occurs in the distal convoluted tubule in response to a low-K+ diet, but other low-potassium-activated kinases are likely to be involved.

PGE2 inhibits apical K channels in the CCD through activation of the MAPK pathway

2007 ◽  
Vol 293 (4) ◽  
pp. F1299-F1307 ◽  
Author(s):  
Yan Jin ◽  
Zhijian Wang ◽  
Yan Zhang ◽  
Baofeng Yang ◽  
Wen-Hui Wang
Keyword(s):  
Mapk Pathway ◽  
Collecting Duct ◽  
Bk Channels ◽  
Mitogen Activated Protein Kinase ◽  
Sk Channels ◽  
Patch Clamp Technique ◽  
Deficient Diet ◽  
Mapk Phosphorylation ◽  
K Channels ◽  
Ep1 Receptor

We used the patch-clamp technique and Western blot analysis to explore the effect of PGE2 on ROMK-like small-conductance K (SK) channels and Ca2+-activated big-conductance K channels (BK) in the cortical collecting duct (CCD). Application of 10 μM PGE2 inhibited SK and BK channels in the CCD. Moreover, either inhibition of PKC or blocking mitogen-activated protein kinase (MAPK), P38 and ERK, abolished the effect of PGE2 on SK channels in the CCD. The effect of PGE2 on SK channels was completely blocked in the presence of SC-51089, a specific EP1 receptor antagonist, and mimicked by application of sulprostone, an agonist for EP1 and EP3 receptors. To determine whether PGE2 stimulates the phosphorylation of P38 and ERK, we treated mouse CCD cells (M-1) with PGE2. Application of PGE2 significantly stimulated the phosphorylation of P38 and ERK within 5 min. The dose-response curve of PGE2 effect shows that 1, 5, and 10 μM PGE2 increased the phosphorylation of P38 and ERK by 20–21, 50–80, and 80–100%, respectively. The stimulatory effect of PGE2 on MAPK phosphorylation was not affected by indomethacin but abolished by inhibition of PKC. This suggests that the effect of PGE2 on MAPK phosphorylation is PKC dependent. Also, the expression of cyclooxygenase II and PGE2 concentration in renal cortex and outer medulla was significantly higher in rats fed a K-deficient diet than those on a normal-K diet. We conclude that PGE2 inhibits SK and BK channels and that there is an effect of PGE2 on SK channels in the CCD through activation of EP1 receptor and MAPK pathways. Also, high concentrations of PGE2 induced by K restriction may be partially responsible for increasing MAPK activity during K restriction.

Role of the cytoskeleton in mediating effect of vasopressin and herbimycin A on secretory K channels in CCD

2002 ◽  
Vol 282 (4) ◽  
pp. F680-F686 ◽  
Author(s):  
Yuan Wei ◽  
Wen-Hui Wang
Keyword(s):  
Collecting Duct ◽  
Cytochalasin D ◽  
Mediating Effect ◽  
Microtubule Assembly ◽  
Sk Channels ◽  
Cortical Collecting Duct ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Herbimycin A

We have previously demonstrated that inhibiting protein tyrosine kinase (PTK) and stimulating protein kinase A (PKA) increase the activity of the small-conductance K (SK) channel in the cortical collecting duct (CCD) of rat kidneys (Cassola AC, Giebisch G, and Wang WH. Am J Physiol Renal Fluid Electrolyte Physiol 264: F502–F509, 1993; Wang WH, Lerea KM, Chan M, and Giebisch G. Am J Physiol Renal Physiol 278: F165–F171, 2000). In the present study, we used the patch-clamp technique to study the role of the cytoskeleton in mediating the effect of herbimycin A, an inhibitor of PTK, and vasopressin on the SK channels in the CCD. The addition of colchicine, an inhibitor of microtubule assembly, or taxol, an agent that blocks microtubule reconstruction, had no significant effect on channel activity. However, colchicine and taxol treatment completely abolished the stimulatory effect of herbimycin A on the SK channels in the CCD. Removal of the microtubule inhibitors restored the stimulatory effect of herbimycin A. In contrast, treatment of the tubules with either taxol or colchicine did not block the stimulatory effect of vasopressin on the SK channels. Moreover, the effect of herbimycin A on the SK channels was also absent in the CCDs treated with either cytochalasin D or phalloidin. In contrast, the stimulatory effect of vasopressin was still observed in the tubules treated with phalloidin. However, cytochalasin D treatment abolished the effect of vasopressin on the SK channels. Finally, the effects of vasopressin and herbimycin A are additive because inhibiting PTK can still increase the channel activity in CCD that has been challenged by vasopressin. We conclude that an intact cytoskeleton is required for the effect on the SK channels of inhibiting PTK and that the SK channels that are activated by inhibiting PTK were differently regulated from those stimulated by vasopressin.

Immunolocalization of the Na+/H+exchanger isoform NHE2 in rat kidney

1998 ◽  
Vol 275 (3) ◽  
pp. F379-F386 ◽  
Author(s):  
Régine Chambrey ◽  
David G. Warnock ◽  
René-Alexandre Podevin ◽  
Patrick Bruneval ◽  
Chantal Mandet ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Western Blot ◽  
Blot Analysis ◽  
Renal Cortex ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Strong Staining ◽  
Convoluted Tubules

Four Na+/H+exchangers (NHE1 to NHE4) have been detected in the kidney. Renal NHE2 expression sites have not been fully established. We have raised rabbit antisera against an oligopeptide related to the amino acids 652 to 661 of rat NHE2. Western blot analysis of plasma membrane fractions isolated from rat renal cortex showed that affinity-purified anti-NHE2 antibody detected an 85-kDa protein in apical but not in basolateral membranes. The labeling of this 85-kDa protein was specifically blocked by preincubation of the antibody with its monomeric peptide, indicating specific recognition. Indirect immunolabeling was performed on sections of paraformaldehyde-fixed rat kidney embedded in paraffin. Strong staining was seen in the apical membrane of cortical thick ascending limbs, distal convoluted tubules, and connecting tubules. Much weaker apical staining was found in medullary thick ascending limbs of Henle. In the inner medulla, some thin limbs were intensively labeled by the anti-NHE2 antibody. No staining could be detected in any segments of the proximal tubule and collecting duct.

scholarly journals Effect of hydrogen peroxide on ROMK channels in the cortical collecting duct

2007 ◽  
Vol 292 (4) ◽  
pp. F1151-F1156 ◽  
Author(s):  
Yuan Wei ◽  
ZhiJian Wang ◽  
Elisa Babilonia ◽  
Hyacinth Sterling ◽  
Peng Sun ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Inhibitory Effect ◽  
Mitogen Activated Protein Kinase ◽  
Hek293 Cells ◽  
Sk Channels ◽  
Cortical Collecting Duct ◽  
Direct Oxidation ◽  
Patch Clamp Technique ◽  
Sk Channel

We used the patch-clamp technique to study the effect of H2O2 on the apical ROMK-like small-conductance K (SK) channel in the cortical collecting duct (CCD). The addition of H2O2 decreased the activity of the SK channels and the inhibitory effect of H2O2 was larger in the CCD from rats on a K-deficient diet than that from rats on a normal-K or a high-K diet. However, application of H2O2 did not inhibit the SK channels in inside-out patches. This suggests that the H2O2-mediated inhibition of SK channels was not due to direct oxidation of the SK channel protein. Because a previous study showed that H2O2 stimulated the expression of Src family protein tyrosine kinase (PTK) which inhibited SK channels ( 3 ), we explored the role of PTK in mediating the effect of H2O2 on SK channels. The application of H2O2 stimulated the activity of endogenous PTK in M-1 cells and increased tyrosine phosphorylation of ROMK in HEK293 cells transfected with GFP-ROMK1 and c-Src. However, blockade of PTK only attenuated but did not completely abolish the inhibitory effect of H2O2 on SK channels. Since H2O2 has also been demonstrated to activate mitogen-activated protein kinase, P38, and ERK ( 3 ), we examined the role of P38 and ERK in mediating the effect of H2O2 on SK channels. Similar to blockade of PTK, suppression of P38 and ERK did not completely abolish the H2O2-induced inhibition of SK channels. However, combined use of ERK, P38, and PTK inhibitors completely abolished the effect of H2O2 on SK channels. Also, treatment of the CCDs with concanavalin A, an agent which has been shown to inhibit endocytosis ( 19 ), abolished the inhibitory effect of H2O2. We conclude that addition of H2O2 inhibited SK channels by stimulating PTK activity, P38, and ERK in the CCD and that H2O2 enhances the internalization of the SK channels.

Apical potassium channels in the rat connecting tubule

2004 ◽  
Vol 287 (5) ◽  
pp. F1030-F1037 ◽  
Author(s):  
Gustavo Frindt ◽  
Lawrence G. Palmer
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Membrane Voltage ◽  
Sk Channels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
K Channels ◽  
Connecting Tubule ◽  
Channel Type

Apical membrane K channels in the rat connecting tubule (CNT) were studied using the patch-clamp technique. Tubules were isolated from the cortical labyrinth of the kidney and split open to provide access to the apical membrane. Cell-attached patches were formed on presumed principal and/or connecting tubule cells. The major channel type observed had a single-channel conductance of 52 pS, high open probability and kinetics that were only weakly dependent on voltage. These correspond closely to the “SK”-type channels in the cortical collecting duct, identified with the ROMK (Kir1.1) gene product. A second channel type, which was less frequently observed, mediated larger currents and was strongly activated by depolarization of the apical membrane voltage. These were identified as BK or maxi-K channels. The density of active SK channels revealed a high degree of clustering. Although heterogeneity of tubules or of cell types within a tubule could not be excluded, the major factor underlying the distribution appeared to be the presence of channel clusters on the membrane of individual cells. The overall density of channels was higher than that previously found in the cortical collecting tubule (CCT). In contrast to results in the CCT, we did not detect an increase in the overall density of SK channels in the apical membrane after feeding the animals a high-K diet. However, the activity of amiloride-sensitive Na channels was undetectable under control conditions but was increased after both 1 day (90 ± 24 pA/cell) or 7 days (385 ± 82 pA/cell) of K loading. Thus one important factor leading to an increased K secretion in the CNT in response to increased dietary K is an increased apical Na conductance, leading to depolarization of the apical membrane voltage and an increased driving force for K movement out into the tubular lumen.

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