Mechanisms of decreased tubular flow-induced nitric oxide in Dahl salt-sensitive rat thick ascending limbs

2021 ◽  
Author(s):  
Nancy J. Hong ◽  
Agustin Gonzalez-Vicente ◽  
Fara Saez ◽  
Jeffrey L. Garvin
Keyword(s):  
Nitric Oxide ◽  
No Synthase ◽  
Thick Ascending Limb ◽  
Western Blots ◽  
Luminal Flow ◽  
Measured Flow ◽  
Nos Inhibitor ◽  
The Difference ◽  
Tubular Flow ◽  
Superoxide Scavenger

Dahl salt-sensitive rat (SS) kidneys produce less nitric oxide (NO) than those of salt-resistant rats (SR). Thick ascending limb (THAL) NO synthase 3 (NOS3) is a major source of renal NO, and luminal flow enhances its activity. We hypothesized that flow-induced NO is reduced in SS THALs primarily due to NOS uncoupling and diminished NOS3 expression rather than scavenging. Rats were fed normal (NS) or high-salt (HS) diets. We measured flow-induced NO and superoxide in perfused THALs, and performed Western blots of renal outer medullas. For rats on NS, flow-induced NO was 35±6 arbitrary units (AU)/min in SR but only 11±2 AU/min in SS THALs (p<0.008). The superoxide scavenger tempol decreased the difference in flow-induced NO between strains by about 36% (p<0.020). The NOS inhibitor L-NAME decreased flow-induced superoxide 36 ± 8% in SS (p<0.02) but had no effect in SR THALs. NOS3 expression was not different between strains on NS. For rats on HS, the difference in flow-induced NO between strains was enhanced (SR: 44±10 vs SS: 9±2 AU/min; p<0.005). Tempol decreased the difference in flow-induced NO between strains by about 37% (p<0.012). L-NAME did not significantly reduce flow-induced superoxide in either strain. HS increased NOS3 expression in SR but not in SS THALs (p<0.003). We conclude: 1) on NS, flow-induced NO is diminished in SS THALs mainly due to NOS3 uncoupling such that it produces superoxide; and 2) on HS, the difference is enhanced due to failure of SS THALs to increase NOS3 expression.

scholarly journals Luminal flow induces eNOS activation and translocation in the rat thick ascending limb

2004 ◽  
Vol 287 (2) ◽  
pp. F274-F280 ◽  
Author(s):  
Pablo A. Ortiz ◽  
Nancy J. Hong ◽  
Jeffrey L. Garvin
Keyword(s):  
Nitric Oxide ◽  
Apical Membrane ◽  
Renal Medulla ◽  
Cytochalasin D ◽  
No Synthase ◽  
No Production ◽  
Thick Ascending Limb ◽  
Loop Of Henle ◽  
Luminal Flow ◽  
Nos Inhibitor

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) acts as an autacoid to inhibit NaCl absorption in the thick ascending limb of the loop of Henle (THAL). In the vasculature, shear stress activates eNOS. We hypothesized that increasing luminal flow activates eNOS and enhances NO production in the THAL. We measured NO production by isolated, perfused THALs using a NO-sensitive microelectrode. Increasing luminal flow from 0 to 20 nl/min increased NO production by 43.1 ± 4.1 pA/mm of tubule ( n = 10, P < 0.05), and this response was blunted (92%) by the NOS inhibitor l-ωnitro-methylarginine ( P < 0.05). We studied the effect of flow on eNOS subcellular localization. In the absence of flow, eNOS was diffusely localized throughout the cell (basolateral = 33 ± 4%; middle = 27 ± 3%; apical = 40 ± 4% of total eNOS). Increasing luminal flow induced eNOS translocation to the apical membrane, as evidenced by a 60% increase in eNOS immunoreactivity in the apical membrane (from 40 ± 4 to 65 ± 2%; n = 6; P < 0.05). Disrupting the actin cytoskeleton with cytochalasin D (10 μM) reduced flow-induced NO production by 62% (from 37.1 ± 3.4 to 14.0 ± 2.4 pA/mm tubule, n = 7, P < 0.04) and blocked flow-induced eNOS translocation. Flow also increased the amount of phosphorylated eNOS (Ser1179) at the apical membrane (from 25 ± 2 to 56 ± 2%; P < 0.05). We conclude that increasing luminal flow induces eNOS activation and translocation to the apical membrane in THALs. These are the first data showing that flow regulates eNOS in epithelial cells. This may be an important mechanism for regulation of NO levels in the renal medulla.

scholarly journals Shear stress increases nitric oxide production in thick ascending limbs

2010 ◽  
Vol 299 (5) ◽  
pp. F1185-F1192 ◽  
Author(s):  
Pablo D. Cabral ◽  
Nancy J. Hong ◽  
Jeffrey L. Garvin
Keyword(s):  
Nitric Oxide ◽  
Shear Stress ◽  
Primary Cultures ◽  
Physiological Saline ◽  
Shear Stresses ◽  
No Production ◽  
Thick Ascending Limb ◽  
Luminal Flow ◽  
Nos Inhibitor ◽  
Stimulation Of

We showed that luminal flow stimulates nitric oxide (NO) production in thick ascending limbs. Ion delivery, stretch, pressure, and shear stress all increase when flow is enhanced. We hypothesized that shear stress stimulates NO in thick ascending limbs, whereas stretch, pressure, and ion delivery do not. We measured NO in isolated, perfused rat thick ascending limbs using the NO-sensitive dye DAF FM-DA. NO production rose from 21 ± 7 to 58 ± 12 AU/min ( P < 0.02; n = 7) when we increased luminal flow from 0 to 20 nl/min, but dropped to 16 ± 8 AU/min ( P < 0.02; n = 7) 10 min after flow was stopped. Flow did not increase NO in tubules from mice lacking NO synthase 3 (NOS 3). Flow stimulated NO production by the same extent in tubules perfused with ion-free solution and physiological saline (20 ± 7 vs. 24 ± 6 AU/min; n = 7). Increasing stretch while reducing shear stress and pressure lowered NO generation from 42 ± 9 to 17 ± 6 AU/min ( P < 0.03; n = 6). In the absence of shear stress, increasing pressure and stretch had no effect on NO production (2 ± 8 vs. 8 ± 8 AU/min; n = 6). Similar results were obtained in the presence of tempol (100 μmol/l), a O2− scavenger. Primary cultures of thick ascending limb cells subjected to shear stresses of 0.02 and 0.55 dyne/cm2 produced NO at rates of 55 ± 10 and 315 ± 93 AU/s, respectively ( P < 0.002; n = 7). Pretreatment with the NOS inhibitor l-NAME (5 mmol/l) blocked the shear stress-induced increase in NO production. We concluded that shear stress rather than pressure, stretch, or ion delivery mediates flow-induced stimulation of NO by NOS 3 in thick ascending limbs.

VEGF increases endothelial permeability by separate signaling pathways involving ERK-1/2 and nitric oxide

2003 ◽  
Vol 284 (1) ◽  
pp. H92-H100 ◽  
Author(s):  
Jerome W. Breslin ◽  
Peter J. Pappas ◽  
Joaquim J. Cerveira ◽  
Robert W. Hobson ◽  
Walter N. Durán
Keyword(s):  
Nitric Oxide ◽  
Signaling Pathways ◽  
Mapk Pathway ◽  
Umbilical Vein ◽  
Endothelial Permeability ◽  
Western Blots ◽  
Cell Monolayers ◽  
Dna Oligonucleotides ◽  
Dextran 70 ◽  
Nos Inhibitor

We tested the hypothesis that VEGF regulates endothelial hyperpermeability to macromolecules by activating the ERK-1/2 MAPK pathway. We also tested whether PKC and nitric oxide (NO) mediate VEGF-induced increases in permeability via the ERK-1/2 pathway. FITC-Dextran 70 flux across human umbilical vein endothelial cell monolayers served as an index of permeability, whereas Western blots assessed the phosphorylation of ERK-1/2. VEGF-induced hyperpermeability was inhibited by antisense DNA oligonucleotides directed against ERK-1/2 and by blockade of MEK and Raf-1 activities (20 μM PD-98059 and 5 μM GW-5074). These blocking agents also reduced ERK-1/2 phosphorylation. The PKC inhibitor bisindolylmaleimide I (10 μM) blocked both VEGF-induced ERK-1/2 activation and hyperpermeability. The NO synthase (NOS) inhibitor N G-nitro-l-arginine methyl ester (200 μM) and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidiazoline-1-oxyl-3-oxide (100 μM) abolished VEGF-induced hyperpermeability but did not block ERK-1/2 phosphorylation. These observations demonstrate VEGF-induced hyperpermeability involves activation of PKC and NOS as well as Raf-1, MEK, and ERK-1/2. Furthermore, our data suggest that ERK-1/2 and NOS are elements of different signaling pathways in VEGF-induced hyperpermeability.

scholarly journals Nitric oxide mediates anomalous tubuloglomerular feedback in rats fed high-NaCl diet after subtotal nephrectomy

2019 ◽  
Vol 316 (2) ◽  
pp. F223-F230 ◽  
Author(s):  
Scott C. Thomson
Keyword(s):  
Nitric Oxide ◽  
Tubuloglomerular Feedback ◽  
Cross Term ◽  
Nitric Oxide Signaling ◽  
Subtotal Nephrectomy ◽  
Single Nephron ◽  
Nos Inhibitor ◽  
Tubular Flow ◽  
Fluid Collections ◽  
Distal Delivery

Tubuloglomerular feedback (TGF) responses become anomalous in rats fed high-NaCl diet after subtotal nephrectomy (STN), such that stimulating TGF causes single nephron GFR (SNGFR) to increase rather than decrease. Micropuncture experiments were performed to determine whether this anomaly results from heightened nitric oxide response to distal delivery, which is a known mechanism for resetting TGF, or from connecting tubule TGF (cTGF), which is a novel amiloride-inhibitable system for offsetting TGF responses. Micropuncture was done in Wistar Froemter rats fed high-NaCl diet (HS) for 8–10 days after STN or sham nephrectomy. TGF was manipulated by orthograde microperfusion of Henle’s loop with artificial tubular fluid with or without NOS inhibitor, LNMMA, or the cell-impermeant amiloride analog, benzamil. SNGFR was measured by inulin clearance in tubular fluid collections from the late proximal tubule. TGF responses were quantified as the increase in SNGFR that occurred when the perfusion rate was reduced from 50 to 8 nl/min in STN or 40 to 8 nl/min in sham animals. The baseline TGF response was anomalous in STN HS (−4 ± 3 vs 14 ± 3 nl/min, P < 0.001). TGF response was normalized by perfusing STN nephron with LNMMA (14 ± 3 nl/min, P < 0.005 for ANOVA cross term) but not with benzamil (−3 ± 4 nl/min, P = 0.4 for ANOVA cross term). Anomalous TGF occurs in STN HS due to heightened effect of tubular flow on nitric oxide signaling, which increases to the point of overriding the normal TGF response. There is no role for cTGF in this phenomenon.

Effects of NG-substituted arginines on coronary vascular function after endotoxin

1993 ◽  
Vol 75 (1) ◽  
pp. 424-431 ◽  
Author(s):  
M. J. Winn ◽  
B. Vallet ◽  
N. K. Asante ◽  
S. E. Curtis ◽  
S. M. Cain
Keyword(s):  
Nitric Oxide ◽  
Vascular Function ◽  
Endotoxic Shock ◽  
No Synthase ◽  
Relaxing Factor ◽  
Nos Inhibitors ◽  
Nos Inhibitor ◽  
Endothelium Derived Relaxing Factor ◽  
Constrictor Response

We investigated the responses of canine coronary rings to endothelium-derived relaxing factor-nitric oxide- (EDRF-NO) dependent agonists and NO synthase (NOS) inhibitors 3 h after endotoxic shock was induced in dogs by lipopolysaccharide infusion (LPS; 2 mg/kg). EDRF-NO-dependent relaxation to thrombin [control maximum response produced after administration of thrombin (Emax) was -85.2 +/- 7.0% of the constrictor response produced by the thromboxane analogue U-46619], acetylcholine (control Emax -88.4 +/- 3.4%), or bradykinin (control Emax -80.5 +/- 2.2%) was not inhibited by LPS (Emax thrombin -75.9 +/- 9.5%; Emax acetylcholine -90.2 +/- 2.4%; Emax bradykinin -91.6 +/- 3.4%). The NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) (10(-6)-3 x 10(-4) M) caused constriction of rings with endothelium (Emax 36.3 +/- 5.6%), an effect that was greater after LPS (Emax 59.2 +/- 4.1%; P < 0.05). D-NMMA had no effect in control, but it increased tension after LPS (Emax 20.8 +/- 9.7%). Contrary to expectations, L- and D-NMMA relaxed endothelium-denuded rings (-30.4 +/- 8.7% L-NMMA; -45.1 +/- 11.7% D-NMMA; P < 0.05). However, neither agent caused relaxation after in vivo LPS (10.2 +/- 3.4% L-NMMA; 8.9 +/- 5.2% D-NMMA). N omega-nitro-L-arginine-methylester (L-NAME) and nitro-L-arginine (10(-6)-3 x 10(-4) M) increased tension (Emax 82.3 +/- 23.9 and 73.1 +/- 8.8%, respectively) but only when endothelium was present, and the increases were no greater in LPS-treated groups than in controls (with LPS: Emax L-NAME 87.3 +/- 16.5%; Emax nitro-L-arginine 65.7 +/- 3.3%).(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Nitric oxide synthase, ADMA, SDMA, and nitric oxide activity in the paraventricular nucleus throughout the etiology of renal wrap hypertension

2012 ◽  
Vol 302 (11) ◽  
pp. H2276-H2284 ◽  
Author(s):  
Carrie A. Northcott ◽  
Scott Billecke ◽  
Teresa Craig ◽  
Carmen Hinojosa-Laborde ◽  
Kaushik P. Patel ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Blood Pressure ◽  
Paraventricular Nucleus ◽  
No Synthase ◽  
Elevated Blood ◽  
Inhibitory Effects ◽  
Symmetric Dimethylarginine ◽  
Inhibitory Neurotransmitters ◽  
Nos Inhibitor ◽  
Oxide Synthase

Within the paraventricular nucleus (PVN), there is a balance between the excitatory and inhibitory neurotransmitters that regulate blood pressure; in hypertension, the balance shifts to enhanced excitation. Nitric oxide (NO) is an atypical neurotransmitter that elicits inhibitory effects on cardiovascular function. We hypothesized that reduced PVN NO led to elevations in blood pressure during both the onset and sustained phases of hypertension due to decreased NO synthase (NOS) and increased asymmetrical dimethylarginine (ADMA; an endogenous NOS inhibitor) and symmetric dimethylarginine (SDMA). Elevated blood pressure, in response to PVN bilateral microinjections of a NO inhibitor, nitro-l-arginine methyl ester, was blunted in renal wrapped rats during the onset of hypertension ( day 7) and sustained renal wrap hypertension ( day 28) compared with sham-operated rats. Adenoviruses (Ad) encoding endothelial NOS (eNOS) or LacZ microinjected into the PVN [1 × 109 plaque-forming units, bilateral (200 nl/site)] reduced mean arterial pressure compared with control ( Day 7, Ad LacZ wrap: 144 ± 7 mmHg and Ad eNOS wrap: 117 ± 5 mmHg, P ≤ 0.05) throughout the study ( Day 28, Ad LacZ wrap: 123 ± 1 mmHg and Ad eNOS wrap: 108 ± 4 mmHg, P ≤ 0.05). Western blot analyses of PVN NOS revealed significantly lower PVN neuronal NOS during the onset of hypertension but not in sustained hypertension. Reduced SDMA was found in the PVN during the onset of hypertension; however, no change in ADMA was observed. In conclusion, functional indexes of NO activity indicated an overall downregulation of NO in renal wrap hypertension, but the mechanism by which this occurs likely differs throughout the development of hypertension.

scholarly journals Nitric oxide inhibits basolateral 10-pS Cl− channels through the cGMP/PKG signaling pathway in the thick ascending limb of C57BL/6 mice

2016 ◽  
Vol 310 (8) ◽  
pp. F755-F762 ◽  
Author(s):  
Peng Wu ◽  
Zhongxiuzi Gao ◽  
Shiwei Ye ◽  
Zhi Qi
Keyword(s):  
Nitric Oxide ◽  
Soluble Guanylyl Cyclase ◽  
Inhibitory Effect ◽  
No Synthase ◽  
Thick Ascending Limb ◽  
Channel Activity ◽  
No Donor ◽  
Cgmp Analog ◽  
Synthase Inhibitor

We used patch-clamp techniques to examine whether nitric oxide (NO) decreases NaCl reabsorption by suppressing basolateral 10-pS Cl− channels in the thick ascending limb (TAL). Both the NO synthase substrate l-arginine (l-Arg) and the NO donor S-nitroso- N-acetylpenicillamine significantly inhibited 10-pS Cl− channel activity in the TAL. The inhibitory effect of l-Arg on Cl− channels was completely abolished in the presence of the NO synthase inhibitor or NO scavenger. Moreover, inhibition of soluble guanylyl cyclase abrogated the effect of l-Arg on Cl− channels, whereas the cGMP analog 8-bromo-cGMP (8-BrcGMP) mimicked the effect of l-Arg and significantly decreased 10-pS Cl− channel activity, indicating that NO inhibits basolateral Cl− channels by increasing cGMP production. Furthermore, treatment of the TAL with a PKG inhibitor blocked the effect of l-Arg and 8-BrcGMP on Cl− channels, respectively. In contrast, a phosphodiesterase 2 inhibitor had no significant effect on l-Arg or 8-BrcGMP-induced inhibition of Cl− channels. Therefore, we conclude that NO decreases basolateral 10-pS Cl− channel activity through a cGMP-dependent PKG pathway, which may contribute to the natriuretic and diuretic effects of NO in vivo.

Bioenergetics in cardiac hypertrophy: mitochondrial respiration as a pathological target of NO·

2001 ◽  
Vol 281 (6) ◽  
pp. H2261-H2269 ◽  
Author(s):  
Lijun Dai ◽  
Paul S. Brookes ◽  
Victor M. Darley-Usmar ◽  
Peter G. Anderson
Keyword(s):  
Nitric Oxide ◽  
Cardiac Hypertrophy ◽  
Mitochondrial Respiration ◽  
Specific Inhibitor ◽  
No Synthase ◽  
Pathological Features ◽  
Aortic Banding ◽  
Hypertrophied Heart ◽  
Nos Inhibitor ◽  
Perfused Hearts

A rat aortic banding model of cardiac hypertrophy was used to test the hypothesis that reversible inhibition of mitochondrial respiration by nitric oxide (NO·) elicits a bioenergetic defect in the hypertrophied heart. In support of this hypothesis, the respiration of myocytes isolated from hypertrophied hearts was more sensitive to exogenous NO· (IC50 200 ± 10 nM vs. 290 ± 30 nM in controls, P = 0.0064). Hypertrophied myocytes also exhibited significantly elevated inducible NO· synthase (iNOS). Consistent with this endogenous source for NO·, the respiration of hypertrophied myocytes was significantly inhibited at physiological O2 tensions versus controls. Both the nonspecific NOS inhibitor nitro-l-arginine and the iNOS-specific inhibitor N-[3-(aminomethyl)- benzyl]acetamidine · 2HCl reversed this inhibition, with no effect on respiration of control myocytes. Consistent with an NO·-mediated mitochondrial dysfunction, the ability of intact perfused hearts to respond to a pacing workload was impaired in hypertrophy, and this effect was reversed by NOS inhibition. We conclude that endogenously generated NO· can modulate mitochondrial function in the hypertrophied heart and suggest that this bioenergetic defect may underlie certain pathological features of hypertrophy.

scholarly journals Gastrointestinal nitric oxide generation in germ-free and conventional rats

2004 ◽  
Vol 287 (5) ◽  
pp. G993-G997 ◽  
Author(s):  
Tanja Sobko ◽  
Claudia Reinders ◽  
Elisabeth Norin ◽  
Tore Midtvedt ◽  
Lars E. Gustafsson ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Gastrointestinal Tract ◽  
Intestinal Microflora ◽  
No Synthase ◽  
No Production ◽  
Germ Free ◽  
Qualitative And Quantitative ◽  
Nitrate Load ◽  
Nos Inhibitor

Nitric oxide (NO) is a central mediator of various physiological events in the gastrointestinal tract. The influence of the intestinal microflora for NO production in the gut is unknown. Bacteria could contribute to this production either by stimulating the mucosa to produce NO, or they could generate NO themselves. Using germ-free and conventional rats, we measured gaseous NO directly in the gastrointestinal tract and from the luminal contents using a chemiluminescence technique. Mucosal NO production was studied by using an NO synthase (NOS) inhibitor, and to evaluate microbial contribution to the NO generation, nitrate was given to the animals. In conventional rats, luminal NO differed profoundly along the gastrointestinal tract with the greatest concentrations in the stomach [>4,000 parts per billion (ppb)] and cecum (≈200 ppb) and lower concentrations in the small intestine and colon (≤20 ppb). Cecal NO correlated with the levels in incubated luminal contents. NOS inhibition lowered NO levels in the colon, without affecting NO in the stomach and in the cecum. Gastric NO increased greatly after a nitrate load, proving it to be a substrate for NO generation. In germ-free rats, NO was low (≤30 ppb) throughout the gastrointestinal tract and absent in the incubated luminal contents. NO also remained low after a nitrate load. Our results demonstrate a pivotal role of the intestinal microflora in gastrointestinal NO generation. Distinctly compartmentalized qualitative and quantitative NO levels in conventional and germ-free rats reflect complex host microbial cross talks, possibly making NO a regulator of the intestinal eco system.

Peroxynitrite triggers a delayed resistance of coronary endothelial cells against ischemia-reperfusion injury

2002 ◽  
Vol 283 (4) ◽  
pp. H1418-H1423 ◽  
Author(s):  
Karine Laude ◽  
Christian Thuillez ◽  
Vincent Richard
Keyword(s):  
Nitric Oxide ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
No Synthase ◽  
No Donor ◽  
Rat Hearts ◽  
Nos Inhibitor ◽  
Coronary Endothelial Cells ◽  
Peroxynitrite Scavengers ◽  
Inducible Nos

Experiments were designed to test whether nitric oxide (NO) and peroxynitrite trigger delayed coronary endothelial protection induced by preconditioning (PC) in rats. Prolonged ischemia reperfusion markedly reduced the response of isolated coronary arteries to acetylcholine, and this was prevented by PC performed 24 h earlier. The NO synthase (NOS) inhibitor N G-nitro-l-arginine methyl ester (l-NAME) administered during PC abolished its delayed endothelial protective effect, whereas the inducible NOS inhibitor N-(3(aminomethyl)benzyl)acetaminide had no effect. Delayed endothelial PC was also abolished by the peroxynitrite scavengers selenomethionine or uric acid given during PC. In parallel, the NO/peroxynitrite donor S-morpholinosydnonimine and authentic peroxynitrite, administered 24 h before prolonged ischemia-reperfusion mimicked endothelial PC, whereas the NO donor S-nitroso- N-acetylpencillamine had no effect. This suggests that peroxynitrite is an essential trigger of the delayed coronary endothelial protection induced by PC in rat hearts.

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