Regulation of an inwardly rectifying K+ channel by nitric oxide in cultured human proximal tubule cells

2004 ◽  
Vol 287 (3) ◽  
pp. F411-F417 ◽  
Author(s):  
Kazuyoshi Nakamura ◽  
Junko Hirano ◽  
Manabu Kubokawa
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Low Dose ◽  
Specific Inhibitor ◽  
High Dose ◽  
Channel Activity ◽  
No Donor ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Inwardly Rectifying

We investigated the effects of nitric oxide (NO) on activity of the inwardly rectifying K+ channel in cultured human proximal tubule cells, using the cell-attached mode of the patch-clamp technique. An inhibitor of NO synthases, Nω-nitro-l-arginine methyl ester (l-NAME; 100 μM), reduced channel activity, which was restored by an NO donor, sodium nitroprusside (SNP; 10 μM) or 8-bromo-cGMP (8-BrcGMP; 100 μM). However, SNP failed to activate the channel in the presence of an inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 μM). Similarly, the SNP effect was abolished by a protein kinase G (PKG)-specific inhibitor, KT-5823 (1 μM), but not by a protein kinase A-specific inhibitor, KT-5720 (500 nM). Another NO donor, S-nitroso- N-acetyl-d,l-penicillamine (10 μM), mimicked the SNP-induced channel activation. In contrast to the stimulatory effect of SNP at a low dose (10 μM), a higher dose of SNP (1 mM) reduced channel activity, which was not restored by 8-BrcGMP. Recordings of membrane potential with the slow whole cell configuration demonstrated that l-NAME (100 μM) and the high dose of SNP (1 mM) depolarized the cell by 10.1 ± 2.6 and 9.2 ± 1.0 mV, respectively, whereas the low dose of SNP (10 μM) hyperpolarized it by 7.1 ± 0.7 mV. These results suggested that the endogenous NO would contribute to the maintenance of basal activity of this K+ channel and hence the potential formation via a cGMP/PKG-dependent mechanism, whereas a high dose of NO impaired channel activity independent of cGMP/PKG-mediated processes.

Protein kinase G activates inwardly rectifying K+ channel in cultured human proximal tubule cells

2002 ◽  
Vol 283 (4) ◽  
pp. F784-F791 ◽  
Author(s):  
Kazuyoshi Nakamura ◽  
Junko Hirano ◽  
Shun-Ichi Itazawa ◽  
Manabu Kubokawa
Keyword(s):  
Proximal Tubule ◽  
Specific Inhibitor ◽  
K Channel ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Proximal Tubule Cells ◽  
Bath Application ◽  
Tubule Cells ◽  
Inwardly Rectifying ◽  
Human Proximal Tubule

An ATP-regulated inwardly rectifying K+ channel, whose activity is enhanced by PKA, is present in the plasma membrane of cultured human proximal tubule cells. In this study, we investigated the effects of PKG on this K+ channel, using the patch-clamp technique. In cell-attached patches, bath application of a membrane-permeant cGMP analog, 8-bromoguanosine 3′,5′-monophosphate (8-BrcGMP; 100 μM), stimulated channel activity, whereas application of a PKG-specific inhibitor, KT-5823 (1 μM), reduced the activity. Channel activation induced by 8-BrcGMP was observed even in the presence of a PKA-specific inhibitor, KT-5720 (500 nM), which was abolished by KT-5823. Direct effects of cGMP and PKG were examined with inside-out patches in the presence of 1 mM MgATP. Although cytoplasmic cGMP (100 μM) alone had little effect on channel activity, subsequent addition of PKG (500 U/ml) enhanced it. Furthermore, bath application of atrial natriuretic peptide (ANP; 20 nM) in cell-attached patches stimulated channel activity, which was blocked by KT-5823. In conclusion, cGMP/PKG-dependent processes participate in activating the ATP-regulated K+ channel and producing the stimulatory effect of ANP on channel activity.

scholarly journals Testosterone-induced relaxation of coronary arteries: activation of BKCa channels via the cGMP-dependent protein kinase

2012 ◽  
Vol 302 (1) ◽  
pp. H115-H123 ◽  
Author(s):  
Viju Deenadayalu ◽  
Yashoda Puttabyatappa ◽  
Alexander T. Liu ◽  
John N. Stallone ◽  
Richard E. White
Keyword(s):  
Nitric Oxide ◽  
Coronary Artery ◽  
Protein Kinase ◽  
Coronary Arteries ◽  
Specific Inhibitor ◽  
No Production ◽  
Channel Activity ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein

Androgens are reported to have both beneficial and detrimental effects on human cardiovascular health. The aim of this study was to characterize nongenomic signaling mechanisms in coronary artery smooth muscle (CASM) and define the ionic basis of testosterone (TES) action. TES-induced relaxation of endothelium-denuded porcine coronary arteries was nearly abolished by 20 nM iberiotoxin, a highly specific inhibitor of large-conductance, calcium-activated potassium (BKCa) channels. Molecular patch-clamp studies confirmed that nanomolar concentrations of TES stimulated BKCa channel activity by ∼100-fold and that inhibition of nitric oxide synthase (NOS) activity by NG-monomethyl-l-arginine nearly abolished this effect. Inhibition of nitric oxide (NO) synthesis or guanylyl cyclase activity also attenuated TES-induced coronary artery relaxation but did not alter relaxation due to 8-bromo-cGMP. Furthermore, we detected TES-stimulated NO production in porcine coronary arteries and in human CASM cells via stimulation of the type 1 neuronal NOS isoform. Inhibition of the cGMP-dependent protein kinase (PKG) attenuated TES-stimulated BKCa channel activity, and direct assay determined that TES increased activity of PKG in a concentration-dependent fashion. Last, the stimulatory effect of TES on BKCa channel activity was mimicked by addition of purified PKG to the cytoplasmic surface of a cell-free membrane patch from CASM myocytes (∼100-fold increase). These findings indicate that TES-induced relaxation of endothelium-denuded coronary arteries is mediated, at least in part, by enhanced NO production, leading to cGMP synthesis and PKG activation, which, in turn, opens BKCa channels. These findings provide a molecular mechanism that could help explain why androgens have been reported to relax coronary arteries and relieve angina pectoris.

scholarly journals Involvement of Endogenous Nitric Oxide in the Regulation of K+ Channel Activity in Cultured Human Proximal Tubule Cells

2006 ◽  
Vol 56 (6) ◽  
pp. 407-413 ◽  
Author(s):  
Kazuyoshi Nakamura ◽  
Wataru Habano ◽  
Toshiyuki Kojo ◽  
You Komagiri ◽  
Takahiro Kubota ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Proximal Tubule ◽  
K Channel ◽  
Channel Activity ◽  
Proximal Tubule Cells ◽  
Endogenous Nitric Oxide ◽  
Tubule Cells ◽  
Human Proximal Tubule

Evidence suggesting that nitric oxide mediates iron-induced toxicity in cultured proximal tubule cells

1998 ◽  
Vol 274 (1) ◽  
pp. F18-F25 ◽  
Author(s):  
Liguang Chen ◽  
Bao-Hong Zhang ◽  
David C. H. Harris
Keyword(s):  
Nitric Oxide ◽  
Lipid Peroxidation ◽  
Proximal Tubule ◽  
Cell Damage ◽  
No Production ◽  
Dependent Manner ◽  
No Donor ◽  
Variable Effect ◽  
Proximal Tubule Cells ◽  
Tubule Cells

The potential role of nitric oxide (NO) in iron-induced toxicity was studied in proximal tubule cells in primary culture. NO production ([Formula: see text]/[Formula: see text]) was significantly increased in iron-treated compared with control cells (3.43 ± 0.08 vs. 1.56 ± 0.08 nmol/dish, P < 0.01). NO synthase (NOS) activity was also induced by iron treatment (16.2 ± 2.0 vs. 0.4 ± 0.2 nmol of [Formula: see text]citrulline/mg protein, P < 0.01).l-Arginine, a substrate for NOS, augmented iron-induced NO production and cell damage [lactate dehydrogenase (LDH) leakage], whereas aminoguanidine, an inhibitor of NOS, reduced iron-induced NO production and LDH leakage. Sodium nitroprusside, an exogenous NO donor, induced LDH leakage in a dose-dependent manner, but no effect on lipid peroxidation {malondialdehyde bis[dimethyl acetal] (MDA) production} was observed. Superoxide dismutase and catalase decreased iron-induced MDA production but did not affect LDH leakage or NO production. Dimethylpyrroline N-oxide and desferal prevented MDA production, LDH leakage, and NO production. We concluded that NO is one of the mediators of iron-induced toxicity in proximal tubule cells. NO-induced toxicity is not dependent on lipid peroxidation. This may explain the variable effect of different antioxidants on cell damage and lipid peroxidation in iron-induced cytotoxicity.

Delayed and acute effects of interferon-γ on activity of an inwardly rectifying K+ channel in cultured human proximal tubule cells

2009 ◽  
Vol 296 (1) ◽  
pp. F46-F53 ◽  
Author(s):  
Kazuyoshi Nakamura ◽  
You Komagiri ◽  
Toshiyuki Kojo ◽  
Manabu Kubokawa
Keyword(s):  
Proximal Tubule ◽  
Acute Effect ◽  
No Production ◽  
Channel Activity ◽  
Acute Effects ◽  
Prolonged Incubation ◽  
Proximal Tubule Cells ◽  
Ifn Γ ◽  
Tubule Cells ◽  
Inwardly Rectifying

The activity of an inwardly rectifying K+ channel in cultured human renal proximal tubule cells (RPTECs) is stimulated and inhibited by nitric oxide (NO) at low and high concentrations, respectively. In this study, we investigated the effects of IFN-γ, one of the cytokines which affect the expression of inducible NO synthase (iNOS), on intracellular NO and channel activity of RPTECs, using RT-PCR, NO imaging, and the cell-attached mode of the patch-clamp technique. Prolonged incubation (24 h) of cells with IFN-γ (20 ng/ml) enhanced iNOS mRNA expression and NO production. In these cells, a NOS inhibitor, Nω-nitro-l-arginine methyl ester (l-NAME; 100 μM), elevated channel activity, suggesting that NO production was so high as to suppress the channel. This indicated that IFN-γ would chronically suppress channel activity by enhancing NO production. Acute effects of IFN-γ was also examined in control cells. Simple addition of IFN-γ (20 ng/ml) to the bath acutely stimulated channel activity, which was abolished by inhibitors of IFN-γ receptor-associated Janus-activated kinase [P6 (1 μM) and AG490 (10 μM)]. However, l-NAME did not block the acute effect of IFN-γ. Indeed, IFN-γ did not acutely affect NO production. Moreover, the acute effect was not blocked by inhibition of PKA, PKG, and phosphatidylinositol 3-kinase (PI3K). We conclude that IFN-γ exerted a delayed suppressive effect on K+ channel activity by enhancing iNOS expression and an acute stimulatory effect, which was independent of either NO pathways or phosphorylation processes mediated by PKA, PKG, and PI3K in RPTECs.

scholarly journals Hydrogen Sulfide Increases Nitric Oxide Production and Subsequent S-Nitrosylation in Endothelial Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Ping-Ho Chen ◽  
Yaw-Syan Fu ◽  
Yun-Ming Wang ◽  
Kun-Han Yang ◽  
Danny Ling Wang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Hydrogen Sulfide ◽  
Protein Kinase ◽  
Fluorescent Probe ◽  
Acid Methyl Ester ◽  
High Specificity ◽  
Protein S ◽  
No Production ◽  
No Donor

Hydrogen sulfide (H2S) and nitric oxide (NO), two endogenous gaseous molecules in endothelial cells, got increased attention with respect to their protective roles in the cardiovascular system. However, the details of the signaling pathways between H2S and NO in endothelia cells remain unclear. In this study, a treatment with NaHS profoundly increased the expression and the activity of endothelial nitric oxide synthase. Elevated gaseous NO levels were observed by a novel and specific fluorescent probe, 5-amino-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid methyl ester (FA-OMe), and quantified by flow cytometry. Further study indicated an increase of upstream regulator for eNOS activation, AMP-activated protein kinase (AMPK), and protein kinase B (Akt). By using a biotin switch, the level of NO-mediated protein S-nitrosylation was also enhanced. However, with the addition of the NO donor, NOC-18, the expressions of cystathionine-γ-lyase, cystathionine-β-synthase, and 3-mercaptopyruvate sulfurtransferase were not changed. The level of H2S was also monitored by a new designed fluorescent probe, 4-nitro-7-thiocyanatobenz-2-oxa-1,3-diazole (NBD-SCN) with high specificity. Therefore, NO did not reciprocally increase the expression of H2S-generating enzymes and the H2S level. The present study provides an integrated insight of cellular responses to H2S and NO from protein expression to gaseous molecule generation, which indicates the upstream role of H2S in modulating NO production and protein S-nitrosylation.

Nitric oxide stimulates the stress-activated protein kinase p38 in rat renal mesangial cells

1999 ◽  
Vol 202 (6) ◽  
pp. 655-660
Author(s):  
A. Huwiler ◽  
J. Pfeilschifter
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
P38 Mapk ◽  
Mesangial Cells ◽  
Mapk Pathway ◽  
Mapk Cascade ◽  
No Donor ◽  
No Donors ◽  
Mitogen Activated Protein ◽  
Rapid Activation

Nitric oxide (NO) has gained increased attention as a diffusible universal messenger that plays a crucial role in the pathogenesis of inflammatory and autoimmune diseases. Recently, we reported that exogenous NO is able to activate the stress-activated protein kinase (SAPK) cascade in mesangial cells. Here, we demonstrate that exposure of glomerular mesangial cells to compounds releasing NO, including spermine-NO and (Z)-1-?N-methyl-N-[6-(N-methylammoniohexyl)amino]diazen?-1-ium+ ++-1,2-diolate (MAHMA-NO), results in an activation of the stress-activated p38-mitogen-activated protein kinase (p38-MAPK) cascade as measured by the phosphorylation of the activator of transcription factor-2 (ATF2) in an immunocomplex kinase assay. Activation of the p38-MAPK cascade by a short stimulation (10 min) with the NO donor MAHMA-NO causes a large increase in ATF2 phosphorylation that is several times greater than that observed after stimulation with interleukin-1beta, a well-known activator of the p38-MAPK pathway. Time course studies reveal that MAHMA-NO causes rapid and maximal activation of p38-MAPK after 10 min of stimulation and that activation declines to basal levels within 60 min. The longer-lived NO donor spermine-NO causes a comparable rapid activation of the p38-MAPK pathway; however, the increased activation state of p38-MAPK was maintained for several hours before control values were reattained after 24 h of stimulation. Furthermore, the NO donors also activated the classical extracellular signal-regulated kinase (ERK) p44-MAPK cascade as shown by phosphorylation of the specific substrate cytosolic phospholipase A2 in an immunocomplex kinase reaction. Both MAHMA-NO and spermine-NO cause a rapid activation of p44-MAPK after 10 min of stimulation. Interestingly, there is a second delayed peak of p44-MAPK activation after 4–24 h of stimulation with NO donors. These results suggest that there is a differential activation pattern for stress-activated and mitogen-activated protein kinases by NO and that the integration of these signals may lead to specific cell responses.

Abstract P169: Antenatal Betamethasone Attenuates the Angiotensin-(1-7)/Nitric Oxide Axis in Adult Male but not Female Renal Proximal Tubule Cells

Hypertension ◽  
2015 ◽  
Vol 66 (suppl_1) ◽  
Author(s):  
Yixin Su ◽  
Victor M Pulgar ◽  
Jianli Bi ◽  
Mark Chappell ◽  
James C Rose
Keyword(s):  
Nitric Oxide ◽  
Proximal Tubule ◽  
Adult Male ◽  
Fetal Programming ◽  
Cell Model ◽  
Specific Effect ◽  
Proximal Tubules ◽  
Proximal Tubule Cells ◽  
Tubule Cells ◽  
Na Uptake

Our studies have revealed a sex-specific effect of fetal programming on sodium (Na+) excretion in adult sheep whereby the males exhibit reduced Na+ excretion and an attenuated natriuretic response to Ang-(1-7) as compared to the females. We hypothesize that the renal proximal tubules are a key target for the early programming effects of glucocorticoids exposure to regulate Na+ handling in the adult males. Therefore, we isolated and cultured cortical proximal tubule cells (RPTC) from adult male and female sheep antenatally exposed to betamethasone (Beta) or vehicle. Na+ uptake and nitric oxide (NO) were assessed with Sodium Green and DAF fluorescence prior to and following a low dose of Ang-(1-7) (1x10-11 M) in isolated RPTC from sheep at ~1.5 years of age. Data are expressed as % of basal uptake or area under the curve (AUC) for Na+ or % of control for NO. Male Beta RPTC exhibit greater Na+ uptake than male vehicle cells (427±32%, n=13, vs. 315±28%, n=14, p<0.05; however, Beta had no effect on Na+ uptake in the female cells (242±18%, n=9, vs. 250±15%, n=10, p>0.05). Ang-(1-7) inhibited Na+ uptake in RPTC from vehicle male (255±40%) and from both vehicle (191±14%) and Beta (209±11%) females (Figure 1B), but failed to attenuate Na+ uptake in Beta male cells (Figure 1A). Beta exposure also abolished NO stimulation by Ang-(1-7) in male but not female RPTC (Figure 1C). We conclude that an Ang-(1-7)-NO-dependent pathway contributes to the sex-dependent consequences of programming on Na+ regulation in the proximal tubules of the kidney. Moreover, the RPTC retain both the sex and Beta-induced phenotype of the adult and may reflect an appropriate cell model of fetal programming.

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