Role of calcium and calmodulin in flow-induced nitric oxide production in endothelial cells

1994 ◽  
Vol 266 (3) ◽  
pp. C628-C636 ◽  
Author(s):  
M. J. Kuchan ◽  
J. A. Frangos
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
No Synthase ◽  
Rate Of Change ◽  
No Production ◽  
Rapid Production ◽  
Nos Inhibitor ◽  
Elevated Rate ◽  
Stimulation Of

These experiments demonstrate that exposure of cultured endothelial cells (EC) to well-defined laminar fluid flow results in an elevated rate of NO production. NO production was monitored by release of NOx (NO2- + NO3(2-) and by cellular guanosine 3',5'-cyclic monophosphate (cGMP) concentration. NO synthase (NOS) inhibitor blocked the flow-mediated stimulation of both NOx and cGMP, indicating that both measurements reflect NO production. Exposure to laminar flow increased NO release in a biphasic manner, with an initial rapid production consequent to the onset of flow followed by a less rapid, sustained production. A similar rapid increase in NO production resulted from an increase in flow above a preexisting level. The rapid initial production of NO was not dependent on shear stress within a physiological range (6-25 dyn/cm2) but may be dependent on the rate of change in shear stress. The sustained release of NO was dependent on physiological levels of shear stress. The calcium (Ca2+) or calmodulin (CaM) dependence of the initial and sustained production of NO was compared with bradykinin (BK)-mediated NO production. Both BK and the initial production were inhibited by Ca2+ and CaM antagonists. In contrast, the sustained shear stress-mediated NO production was not affected, despite the continued functional presence of the antagonists. Dexamethasone had no effect on either the initial or the sustained shear stress-mediated NO production. An inducible NOS does not, therefore, explain the apparent Ca2+/CaM independence of the sustained shear stress-mediated NO production.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of G proteins in shear stress-mediated nitric oxide production by endothelial cells

1994 ◽  
Vol 267 (3) ◽  
pp. C753-C758 ◽  
Author(s):  
M. J. Kuchan ◽  
H. Jo ◽  
J. A. Frangos
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
G Protein ◽  
G Proteins ◽  
No Production ◽  
Dependent Manner ◽  
Protein Activation ◽  
Synthase Inhibitor

Exposure of cultured endothelial cells to shear stress resulting from well-defined fluid flow stimulates the production of nitric oxide (NO). We have established that an initial burst in production is followed by sustained steady-state NO production. The signal transduction events leading to this stimulation are not well understood. In the present study, we examined the role of regulatory guanine nucleotide binding proteins (G proteins) in shear stress-mediated NO production. In endothelial cells not exposed to shear stress, AIF4-, a general activator of G proteins, markedly elevated the production of guanosine 3',5'-cyclic monophosphate (cGMP). Pretreatment with NO synthase inhibitor N omega-nitro-L-arginine completely blocked this stimulation. Incubation with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), a general G protein inhibitor, blocked the flow-mediated burst in cGMP production in a dose-dependent manner. Likewise, GDP beta S inhibited NOx (NO2 + NO3) production for the 1st h. However, inhibition was not detectable between 1 and 3 h. Pertussis toxin (PTx) had no effect on the shear response at any time point. The burst in NO production caused by a change in shear stress appears to be dependent on a PTx-refractory G protein. Sustained shear-mediated production is independent of G protein activation.

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.

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.

scholarly journals In Endothelial Cells, the Activation or Stimulation of Soluble Guanylyl Cyclase Induces the Nitric Oxide Production by a Mechanism Dependent of Nitric Oxide Synthase Activation

2018 ◽  
Vol 21 ◽  
pp. 38-45 ◽  
Author(s):  
Ariane Migliato Martinelli ◽  
Carla Nascimento dos Santos Rodrigues ◽  
Thiago Francisco de Moraes ◽  
Gerson Jhonatan Rodrigues
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Soluble Guanylate Cyclase ◽  
Soluble Guanylyl Cyclase ◽  
No Production ◽  
Selective Electrode ◽  
Nos Inhibitor ◽  
Oxide Synthase ◽  
Stimulation Of

Purpose. In endothelial cells, investigate if the soluble guanylate cyclase (sGC) activation or stimulation is able to potentiate the relaxation in vessels. Methods. Aortic and coronary rings with and without endothelium were placed in a myograph and cumulative concentration-effect curves for DETA-NO or ataciguat were performed. Nitric oxide (NO) were measured by fluorescence or by selective electrode in human umbilical endothelial cells (HUVECs) in response to some treatments, including ataciguat, 8-Br-cGMP and A23187. Results. The presence of the endothelium potentiated the relaxation induced by DETA-NO in aortic and coronary rings. In addition, in aortic rings the endothelium potentiated the relaxation induced by ataciguat. In the presence of nitric oxide synthase (NOS) inhibitor, the endothelium effect was abolished to DETA-NO or ataciguat, in both vessels. Ataciguat, 8-Br-cGMP and A23187 were able to induce NO production in HUVECs cells. In the presence of NOS inhibitor, the NO production induced by ataciguat and 8-Br-cGMP was abolished. Conclusions. Our results suggest that in aortic and coronary rings the endothelium potentiates the relaxation induced by activation or stimulation of sGC through a mechanism dependent of NOS activation. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

Role of cytoskeleton in shear stress-induced endothelial nitric oxide production

1997 ◽  
Vol 273 (1) ◽  
pp. H347-H355 ◽  
Author(s):  
H. L. Knudsen ◽  
J. A. Frangos
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Plasma Membrane ◽  
Cytochalasin D ◽  
Induced Response ◽  
No Production ◽  
Dependent Manner ◽  
Mechanochemical Transduction

To study the role of the cytoskeleton in mechanochemical transduction, human umbilical vein endothelial cells were exposed to cytoskeleton-disrupting or -stabilizing agents, and the flow-induced production of nitric oxide (NO) as monitored by intracellular levels of guanosine 3',5'-cyclic monophosphate (cGMP) was examined. A shear stress of 20 dyn/cm2 elevated cGMP levels approximately twofold relative to basal (stationary) levels (1.9 +/- 0.1 pmol cGMP in stationary controls; P < 0.01). Treatment with 1 microM phalloidin and 0.5 microM cytochalasin D did not significantly affect the flow-induced response (1.77 +/- 0.23 and 2.89 +/- 0.18 pmol cGMP in stationary controls, respectively), whereas disruption of microtubules with 0.5 microM colchicine significantly elevated the response (3.64 +/- 0.18 pmol cGMP in stationary controls; P < 0.01). The NO synthase inhibitor NG-amino-L-arginine abrogated all flow-induced elevations of cGMP, indicating that increased cGMP levels were mediated by NO. Cytoskeletal disruption with 0.2 microM cytochalasin D or 0.5 microM colchicine did not alter cGMP levels in response to 10 nM bradykinin. The role of the plasma membrane in mechanochemical transduction was examined by treatment with cholesteryl hemisuccinate, which attenuated the flow-induced response in a dose-dependent manner. In conclusion, the pathways of flow- and bradykinin-mediated NO production in endothelial cells did not require actin filament turnover or intact actin or microtubule cytoskeletons, and cholesterol, possibly by stiffening the plasma membrane, attenuated the flow response.

Hypoxic contraction of small pulmonary arteries from normal and endotoxemic rats: fundamental role of NO

1999 ◽  
Vol 276 (4) ◽  
pp. H1207-H1214 ◽  
Author(s):  
Sylvain Terraz ◽  
François Baechtold ◽  
Delphine Renard ◽  
Attila Barsi ◽  
Anne Rosselet ◽  
...  
Keyword(s):  
Vascular Tone ◽  
Pulmonary Arteries ◽  
Isometric Tension ◽  
No Synthase ◽  
Severe Hypoxia ◽  
No Production ◽  
Outer Diameter ◽  
Nos Inhibitor ◽  
Severe Degree

The present study was aimed at examining the role of nitric oxide (NO) in the hypoxic contraction of isolated small pulmonary arteries (SPA) in the rat. Animals were treated with either saline (sham experiments) or Escherichia coli lipolysaccharide [LPS, to obtain expression of the inducible NO synthase (iNOS) in the lung] and killed 4 h later. SPA (300- to 600-μm outer diameter) were mounted as rings in organ chambers for the recording of isometric tension, precontracted with PGF2α, and exposed to either severe (bath [Formula: see text] 8 ± 3 mmHg) or milder (21 ± 3 mmHg) hypoxia. In SPA from sham-treated rats, contractions elicited by severe hypoxia were completely suppressed by either endothelium removal or preincubation with an NOS inhibitor [ N G-nitro-l-arginine methyl ester (l-NAME), 10−3 M]. In SPA from LPS-treated rats, contractions elicited by severe hypoxia occurred irrespective of the presence or absence of endothelium and were largely suppressed by l-NAME. The milder hypoxia elicited no increase in vascular tone. These results indicate an essential role of NO in the hypoxic contractions of precontracted rat SPA. The endothelium independence of HPV in arteries from LPS-treated animals appears related to the extraendothelial expression of iNOS. The severe degree of hypoxia required to elicit any contraction is consistent with a mechanism of reduced NO production caused by a limited availability of O2 as a substrate for NOS.

Ca2+-independent PLA2 controls endothelial store-operated Ca2+ entry and vascular tone in intact aorta

2008 ◽  
Vol 295 (6) ◽  
pp. H2466-H2474 ◽  
Author(s):  
François-Xavier Boittin ◽  
Françoise Gribi ◽  
Karima Serir ◽  
Jean-Louis Bény
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Muscle Relaxation ◽  
Physiological Mechanism ◽  
Smooth Muscle Relaxation ◽  
No Production ◽  
Arterial Tone ◽  
Endothelium Dependent Relaxation ◽  
Stimulation Of

During an agonist stimulation of endothelial cells, the sustained Ca2+ entry occurring through store-operated channels has been shown to significantly contribute to smooth muscle relaxation through the release of relaxing factors such as nitric oxide (NO). However, the mechanisms linking Ca2+ stores depletion to the opening of such channels are still elusive. We have used Ca2+ and tension measurements in intact aortic strips to investigate the role of the Ca2+-independent isoform of phospholipase A2 (iPLA2) in endothelial store-operated Ca2+ entry and endothelium-dependent relaxation of smooth muscle. We provide evidence that iPLA2 is involved in the activation of endothelial store-operated Ca2+ entry when Ca2+ stores are artificially depleted. We also show that the sustained store-operated Ca2+ entry occurring during physiological stimulation of endothelial cells with the circulating hormone ATP is due to iPLA2 activation and significantly contributes to the amplitude and duration of ATP-induced endothelium-dependent relaxation. Consistently, both iPLA2 metabolites arachidonic acid and lysophosphatidylcholine were found to stimulate Ca2+ entry in native endothelial cells. However, only the latter triggered endothelium-dependent relaxation through NO release, suggesting that lysophosphatidylcholine produced by iPLA2 upon Ca2+ stores depletion may act as an intracellular messenger that stimulates store-operated Ca2+ entry and subsequent NO production in endothelial cells. Finally, we found that ACh-induced endothelium relaxation also depends on iPLA2 activation, suggesting that the iPLA2-dependent control of endothelial store-operated Ca2+ entry is a key physiological mechanism regulating arterial tone.

scholarly journals Protein kinase B/Akt activates c-Jun NH2-terminal kinase by increasing NO production in response to shear stress

2001 ◽  
Vol 91 (4) ◽  
pp. 1574-1581 ◽  
Author(s):  
Young-Mi Go ◽  
Yong Chool Boo ◽  
Heonyong Park ◽  
Matthew C. Maland ◽  
Rakesh Patel ◽  
...  
Keyword(s):  
Shear Stress ◽  
Protein Kinase ◽  
Transient Expression ◽  
Protein Kinase B ◽  
No Synthase ◽  
No Production ◽  
Endothelial No Synthase ◽  
Jnk Activation ◽  
Adenoviral Construct ◽  
Stimulation Of

Laminar shear stress activates c-Jun NH2-terminal kinase (JNK) by the mechanisms involving both nitric oxide (NO) and phosphatidylinositide 3-kinase (PI3K). Because protein kinase B (Akt), a downstream effector of PI3K, has been shown to phosphorylate and activate endothelial NO synthase, we hypothesized that Akt regulates shear-dependent activation of JNK by stimulating NO production. Here, we examined the role of Akt in shear-dependent NO production and JNK activation by expressing a dominant negative Akt mutant (AktAA) and a constitutively active mutant (AktMyr) in bovine aortic endothelial cells (BAEC). As expected, pretreatment of BAEC with the PI3K inhibitor (wortmannin) prevented shear-dependent stimulation of Akt and NO production. Transient expression of AktAA in BAEC by using a recombinant adenoviral construct inhibited the shear-dependent stimulation of NO production and JNK activation. However, transient expression of AktMyr by using a recombinant adenoviral construct did not induce JNK activation. This is consistent with our previous finding that NO is required, but not sufficient on its own, to activate JNK in response to shear stress. These results and our previous findings strongly suggest that shear stress triggers activation of PI3K, Akt, and endothelial NO synthase, leading to production of NO, which (along with O[Formula: see text], which is also produced by shear) activates Ras-JNK pathway. The regulation of Akt, NO, and JNK by shear stress is likely to play a critical role in its antiatherogenic effects.

scholarly journals Protective Role of Nitric Oxide in a Model of Thrombotic Microangiopathy in Rats

2001 ◽  
Vol 12 (10) ◽  
pp. 2088-2097
Author(s):  
JING SHAO ◽  
TOSHIO MIYATA ◽  
KOEI YAMADA ◽  
NORIO HANAFUSA ◽  
TAKEHIKO WADA ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Thrombotic Microangiopathy ◽  
Periodic Acid ◽  
Minor Role ◽  
Protective Mechanism ◽  
No Production ◽  
Nos Inhibitor ◽  
Inducible Nos

Abstract. A new model of thrombotic microangiopathy (TMA) was previously developed, and it was demonstrated that endothelial nitric oxide (NO) synthase (NOS) is upregulated in glomeruli in this model. It was hypothesized that the synthesis of NO, a potent vasodilator and platelet inhibitory factor, is induced as a defense mechanism. The goal of this study was to clarify the role of NO in this model.Ex vivoexperiments using Western blotting and functional assays demonstrated upregulation of endothelial NOS in isolated glomeruli from TMA rats. Inin vivoexperiments, five groups of rats were studied, including rats with TMA treated with vehicle,NG-nitro-L-arginine methyl ester (L-NAME) (a NOS inhibitor), or L-N6-(1-iminoethyl)lysine (L-NIL) (a specific inducible NOS inhibitor) and normal control rats treated with vehicle or L-NAME. Blood urea nitrogen levels, BP, urinary nitrate/nitrite excretion, and proteinuria were measured. Histologic assessments using periodic acid-Schiff staining and immunohistologic studies with markers for endothelium, platelets, fibrin, cell proliferation, and apoptosis were also performed. L-NAME inhibition of NO synthesis in rats with TMA resulted in more severe glomerular and tubulointerstitial injury, which was accompanied by thrombus formation and a marked loss of endothelial cells, with more apoptotic cells. These changes were associated with severe renal function deterioration. In contrast, these features were less pronounced in the vehicle- or L-NIL-treated rats with TMA and were absent in the control animals. In conclusion, inhibition of NO production in this model of TMA markedly exacerbated renal injury. The absence of effects with L-NIL treatment suggests a minor role for inducible NOS in this model. These results suggest that production of NO, most likely by endothelial cells, is an important protective mechanism in TMA.

Arginase inhibition increases nitric oxide production in bovine pulmonary arterial endothelial cells

2004 ◽  
Vol 287 (1) ◽  
pp. L60-L68 ◽  
Author(s):  
Louis G. Chicoine ◽  
Michael L. Paffett ◽  
Tamara L. Young ◽  
Leif D. Nelin
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
No Synthase ◽  
No Production ◽  
Urea Production ◽  
Pulmonary Arterial ◽  
Factor Α ◽  
Arterial Endothelial Cells ◽  
Regular Medium ◽  
Necrosis Factor

Nitric oxide (NO) is produced by NO synthase (NOS) from l-arginine (l-Arg). Alternatively, l-Arg can be metabolized by arginase to produce l-ornithine and urea. Arginase (AR) exists in two isoforms, ARI and ARII. We hypothesized that inhibiting AR with l-valine (l-Val) would increase NO production in bovine pulmonary arterial endothelial cells (bPAEC). bPAEC were grown to confluence in either regular medium (EGM; control) or EGM with lipopolysaccharide and tumor necrosis factor-α (L/T) added. Treatment of bPAEC with L/T resulted in greater ARI protein expression and ARII mRNA expression than in control bPAEC. Addition of l-Val to the medium led to a concentration-dependent decrease in urea production and a concentration-dependent increase in NO production in both control and L/T-treated bPAEC. In a second set of experiments, control and L/T bPAEC were grown in EGM, EGM with 30 mM l-Val, EGM with 10 mM l-Arg, or EGM with both 10 mM l-Arg and 30 mM l-Val. In both control and L/T bPAEC, treatment with l-Val decreased urea production and increased NO production. Treatment with l-Arg increased both urea and NO production. The addition of the combination l-Arg and l-Val decreased urea production compared with the addition of l-Arg alone and increased NO production compared with l-Val alone. These data suggest that competition for intracellular l-Arg by AR may be involved in the regulation of NOS activity in control bPAEC and in response to L/T treatment.

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