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.

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.

Shear Stress Effect on the Production of Nitric Oxide in Cultured Rat Aorta Endothelial Cells

2002 ◽  
Author(s):  
Jianfeng Ye ◽  
Baoguo Chen ◽  
Lisa X. Xu
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Protein Tyrosine Kinase ◽  
Vascular Endothelial Cells ◽  
Free Calcium ◽  
Enos Gene ◽  
No Production ◽  
Stress Effect ◽  
Dependent Manner

Atherosclerotic lesions tend to develop in regions where there are separations from unidirectional laminar blood flow, typically near branches, bifurcations, regions of arterial narrowing, and curvatures in the arteries (1, 2). Obviously, homodynamic forces play a key role in atherosclerosis. Studies also indicate that vascular endothelium function disturbance, especially impairment of endothelium dependent vasodilation, is involved (3). Shear stress affects endothelial cells in many ways, such as cytoskeletal rearrangement, decrease of intracellular pH, release of PGI2 and some growth factors (PDGF, FGF, ECGF, TGF-b, etc), activation of IP3 and mitogen-activated protein kinases, and the significant increase in the production of nitric oxide (1,2,4,5). As an important function factor of vascular endothelial cells, nitric oxide (NO) is closely related to the endothelial dysfunction and atherosclerosis (6). Endothelial derived nitric oxide involves in many events in the vasculature, including vasodilation, inhibition of platelet aggregation, adhesion molecule expression, and vascular smooth muscle proliferation, which are directly or indirectly related to atherosclerosis. Endothelial cells release NO more potently in response to increased shear stress than to agonists that raise intracellular free calcium concentration [Ca2+]i. Studies have indicated that NO production increases with a calcium/CaM dependent manner in the first few minutes after exposed to shear stress, followed by a sustained NO production that occurs more than 30min which is Ca2+ independent (7). The activation of eNOS by shear stress, which modulated by Ca/CaM, G protein, tyrosine kinase phosphorylation and eNOS gene expression, is responsible for the increase of NO production (8). However, the contribution of extracellular calcium to the production of NO is somewhat contradictory.

Disruption of renal peritubular blood flow in lipopolysaccharide-induced renal failure: role of nitric oxide and caspases

2005 ◽  
Vol 289 (6) ◽  
pp. F1324-F1332 ◽  
Author(s):  
Manish M. Tiwari ◽  
Robert W. Brock ◽  
Judit K. Megyesi ◽  
Gur P. Kaushal ◽  
Philip R. Mayeux
Keyword(s):  
Nitric Oxide ◽  
Renal Failure ◽  
Blood Flow ◽  
Saline Group ◽  
No Production ◽  
Capillary Perfusion ◽  
Peritubular Capillary ◽  
Synthase Inhibitor ◽  
Peritubular Capillary Perfusion

Acute renal failure (ARF) is a frequent and serious complication of endotoxemia caused by lipopolysaccharide (LPS) and contributes significantly to mortality. The present studies were undertaken to examine the roles of nitric oxide (NO) and caspase activation on renal peritubular blood flow and apoptosis in a murine model of LPS-induced ARF. Male C57BL/6 mice treated with LPS ( Escherichia coli) at a dose of 10 mg/kg developed ARF at 18 h. Renal failure was associated with a significant decrease in peritubular capillary perfusion. Vessels with no flow increased from 7 ± 3% in the saline group to 30 ± 4% in the LPS group ( P < 0.01). Both the inducible NO synthase inhibitor l- N6-1-iminoethyl-lysine (l-NIL) and the nonselective caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD) prevented renal failure and reversed perfusion deficits. Renal failure was also associated with an increase in renal caspase-3 activity and an increase in renal apoptosis. Both l-NIL and Z-VAD prevented these changes. LPS caused an increase in NO production that was blocked by l-NIL but not by Z-VAD. Taken together, these data suggest NO-mediated activation of renal caspases and the resulting disruption in peritubular blood flow are an important mechanism of LPS-induced ARF.

Pregnancy enhances G protein activation and nitric oxide release from uterine arteries

2001 ◽  
Vol 280 (5) ◽  
pp. H2069-H2075 ◽  
Author(s):  
L. P. Thompson ◽  
C. P. Weiner
Keyword(s):  
Nitric Oxide ◽  
Cholera Toxin ◽  
G Protein ◽  
Pertussis Toxin ◽  
Nitric Oxide Synthase Inhibitor ◽  
Protein Activation ◽  
Nitric Oxide Release ◽  
G Protein Activation ◽  
Hormonal Modulation ◽  
Synthase Inhibitor

We hypothesized that pregnancy modulates receptor-mediated responses of the uterine artery (UA) by altering G protein activation or coupling. Relaxation and contraction to NaF (0.5–11.5 mM), acetylcholine (10−9–10−5 M), and bradykinin (10−12–3 × 10−5 M) were measured in isolated UA of pregnant and nonpregnant guinea pigs. Responses were measured in the presence and absence of either cholera toxin (2 μg/ml) or pertussis toxin (Gαs and Gαiinhibitors, respectively). NaF relaxation was endothelium dependent and nitro-l-arginine sensitive (a nitric oxide synthase inhibitor). Relaxation to NaF, acetylcholine, and bradykinin were potentiated by pregnancy. Cholera but not pertussis toxin increased relaxation to acetylcholine and bradykinin in UA from nonpregnant animals, had no effect in UA from pregnant animals, and abolished the pregnancy-induced differences in acetylcholine relaxation. Cholera toxin potentiated the bradykinin-induced contraction of UA of both pregnant and nonpregnant animals, whereas pertussis toxin inhibited contraction of UA from pregnant animals only. Therefore, pregnancy may enhance agonist-stimulated endothelium-dependent relaxation and bradykinin-induced contraction of UA by inhibiting GTPase activity or enhancing Gαs but not Gαi activation in pregnant animals. Thus the diverse effects of pregnancy on UA responsiveness may result from hormonal modulation of G proteins coupled to their specific receptors.

scholarly journals Mutations in the ‘DRY’ motif of the CB1 cannabinoid receptor result in biased receptor variants

2014 ◽  
Vol 54 (1) ◽  
pp. 75-89 ◽  
Author(s):  
Pál Gyombolai ◽  
András D Tóth ◽  
Dániel Tímár ◽  
Gábor Turu ◽  
László Hunyady
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Double Mutant ◽  
Cannabinoid Receptor ◽  
Camp Accumulation ◽  
Protein Activation ◽  
Cb1 Cannabinoid Receptor ◽  
G Protein Activation ◽  
Dry Motif

The role of the highly conserved ‘DRY’ motif in the signaling of the CB1cannabinoid receptor (CB1R) was investigated by inducing single-, double-, and triple-alanine mutations into this site of the receptor. We found that the CB1R-R3.50A mutant displays a partial decrease in its ability to activate heterotrimeric Goproteins (∼80% of WT CB1R (CB1R-WT)). Moreover, this mutant showed an enhanced basal β-arrestin2 (β-arr2) recruitment. More strikingly, the double-mutant CB1R-D3.49A/R3.50A was biased toward β-arrs, as it gained a robustly increased β-arr1 and β-arr2 recruitment ability compared with the WT receptor, while its G-protein activation was decreased. In contrast, the double-mutant CB1R-R3.50A/Y3.51A proved to be G-protein-biased, as it was practically unable to recruit β-arrs in response to agonist stimulus, while still activating G-proteins, although at a reduced level (∼70% of CB1R-WT). Agonist-induced ERK1/2 activation of the CB1R mutants showed a good correlation with their β-arr recruitment ability but not with their G-protein activation or inhibition of cAMP accumulation. Our results suggest that G-protein activation and β-arr binding of the CB1R are mediated by distinct receptor conformations, and the conserved ‘DRY’ motif plays different roles in the stabilization of these conformations, thus mediating both G-protein- and β-arr-mediated functions of CB1R.

Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity

2021 ◽  
Author(s):  
Sarah Basehore ◽  
Samantha Bohlman ◽  
Callie Weber ◽  
Swathi Swaminathan ◽  
Yuji Zhang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Laminar Flow ◽  
No Production ◽  
Disturbed Flow ◽  
Enos Activity ◽  
Enos Phosphorylation ◽  
Steady Laminar ◽  
In Cells

Rationale: In diabetic animals as well as high glucose cell culture conditions, endothelial nitric oxide synthase (eNOS) is heavily O-GlcNAcylated, which inhibits its phosphorylation and nitric oxide (NO) production. It is unknown, however, whether varied blood flow conditions, which affect eNOS phosphorylation, modulate eNOS activity via O-GlcNAcylation-dependent mechanisms. Objective: The goal of this study was to test if steady laminar flow, but not oscillating disturbed flow, decreases eNOS O-GlcNAcylation, thereby elevating eNOS phosphorylation and NO production. Methods and Results: Human umbilical vein endothelial cells (HUVEC) were exposed to either laminar flow (20 dynes/cm2 shear stress) or oscillating disturbed flow (4{plus minus}6 dynes/cm2 shear stress) for 24 hours in a cone-and-plate device. eNOS O-GlcNAcylation was almost completely abolished in cells exposed to steady laminar but not oscillating disturbed flow. Interestingly, there was no change in protein level or activity of key O-GlcNAcylation enzymes (OGT, OGA, or GFAT). Instead, metabolomics data suggest that steady laminar flow decreases glycolysis and hexosamine biosynthetic pathway (HBP) activity, thereby reducing UDP-GlcNAc pool size and consequent O-GlcNAcylation. Inhibition of glycolysis via 2-deoxy-2-glucose (2-DG) in cells exposed to disturbed flow efficiently decreased eNOS O-GlcNAcylation, thereby increasing eNOS phosphorylation and NO production. Finally, we detected significantly higher O-GlcNAcylated proteins in endothelium of the inner aortic arch in mice, suggesting that disturbed flow increases protein O-GlcNAcylation in vivo. Conclusions: Our data demonstrate that steady laminar but not oscillating disturbed flow decreases eNOS O-GlcNAcylation by limiting glycolysis and UDP-GlcNAc substrate availability, thus enhancing eNOS phosphorylation and NO production. This research shows for the first time that O-GlcNAcylation is regulated by mechanical stimuli, relates flow-induced glycolytic reductions to macrovascular disease, and highlights targeting HBP metabolic enzymes in endothelial cells as a novel therapeutic strategy to restore eNOS activity and prevent EC dysfunction in cardiovascular disease.

Adenosine enhances nitric oxide production by vascular endothelial cells

1995 ◽  
Vol 269 (2) ◽  
pp. C519-C523 ◽  
Author(s):  
J. M. Li ◽  
R. A. Fenton ◽  
B. S. Cutler ◽  
J. G. Dobson
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Vascular Tone ◽  
Vascular Endothelial Cells ◽  
Competitive Inhibitor ◽  
No Production ◽  
Dependent Manner ◽  
Bathing Medium ◽  
Adenosine Receptor Antagonist ◽  
Potent Vasodilator

Adenosine per se is a potent vasodilator of vascular smooth muscle. Endothelial cells modulate vascular tone via the release of nitric oxide (NO), which also elicits vasodilation. This study was undertaken to determine whether adenosine could directly stimulate endothelial cells to enhance NO production, which could subsequently reduce vascular tone. NO production was evaluated in porcine carotid artery endothelial cells (PCAEC) and human saphenous vein endothelial cells (HSVEC) seeded on multiwell plates, grown to confluence, and treated with adenosine for 1 h. The bathing medium was collected, and the NO production was determined as reflected by the formation of NO2- and NO3-. NO production by PCAEC was significantly increased by adenosine in a dose-dependent manner, whereas there was only an insignificant tendency for an increase by HSVEC. The addition of the NO synthase competitive inhibitor, NG-monomethyl-L-arginine (NMMA), or the adenosine receptor antagonist, theophylline, prevented the increase in NO production by adenosine. The results suggest that adenosine stimulates, by a receptor-mediated mechanism, the production of NO by arterial, but not by venous, endothelial cells.

Role of G proteins in stimulation of Na-H exchange by cell shrinkage

1992 ◽  
Vol 262 (2) ◽  
pp. C533-C536 ◽  
Author(s):  
B. A. Davis ◽  
E. M. Hogan ◽  
W. F. Boron
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Transport Processes ◽  
Cell Shrinkage ◽  
Protein Activation ◽  
Kinase C ◽  
G Protein Activation ◽  
Barnacle Muscle Fibers ◽  
Stimulation Of

Many cells respond to shrinkage by stimulating specific ion transport processes (e.g., Na-H exchange). However, it is not known how the cell senses this volume change, nor how this signal is transduced to an ion transporter. We have studied the activation of Na-H exchange in internally dialyzed barnacle muscle fibers, measuring intracellular pH (pHi) with glass microelectrodes. When cells are dialyzed to a pHi of approximately 7.2, Na-H exchange is active only in shrunken cells. We found that the shrinkage-induced stimulation of Na-H exchange, elicited by increasing medium osmolality from 975 to 1,600 mosmol/kgH2O, is inhibited approximately 72% by including in the dialysis fluid 1 mM guanosine 5'-O-(2-thiodiphosphate). The latter is an antagonist of G protein activation. Even in unshrunken cells, Na-H exchange is activated by dialyzing the cell with 1 mM guanosine 5'-O-(3-thiotriphosphate), which causes the prolonged activation of G proteins. Activation of Na-H exchange is also elicited in unshrunken cells by injecting cholera toxin, which activates certain G proteins. Neither exposing cells to 100 nM phorbol 12-myristate 13-acetate nor dialyzing them with a solution containing 20 microM adenosine 3',5'-cyclic monophosphate (cAMP) (or 50 microM dibutyryl cAMP) plus 0.5 mM 3-isobutyl-1-methylxanthine substantially stimulates the exchanger. Thus our data suggest that a G protein plays a key role in the transduction of the shrinkage signal to the Na-H exchanger via a pathway that involves neither protein kinase C nor cAMP.

Sources and implications of basal nitric oxide in spontaneous contractions of guinea pig taenia caeci

2003 ◽  
Vol 285 (4) ◽  
pp. G747-G753 ◽  
Author(s):  
Catalina Caballero-Alomar ◽  
Carmen Santos ◽  
Diego Lopez ◽  
M. Teresa Mitjavila ◽  
Pere Puig-Parellada
Keyword(s):  
Nitric Oxide ◽  
Guinea Pig ◽  
Inhibitory Effect ◽  
No Production ◽  
Paramagnetic Resonance ◽  
Synthase Inhibitor ◽  
Spontaneous Contractions ◽  
Electron Paramagnetic

We examined in vitro the source and role of basal nitric oxide (NO) in proximal segments of guinea pig taenia caeci in nonadrenergic, noncholinergic (NANC) conditions. Using electron paramagnetic resonance (EPR), we measured the effect of the NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME, 10–4 M), the neuronal blocker tetrodotoxin (TTX, 10–6 M), or both on spontaneous contractions and on the production of basal NO. Both l-NAME and TTX, when tested alone, increased the amplitude and frequency of contractions. NO production was abolished by l-NAME and was inhibited by 38% by TTX. When tested together, l-NAME in the presence of TTX or TTX in the presence of l-NAME had no further effect on the amplitude or frequency of spontaneous contractions, and the NO production was inhibited. These findings suggest that basal NO consists of TTX-sensitive and TTX-resistant components. The TTX-sensitive NO has an inhibitory effect on spontaneous contractions; the role of TTX-resistant NO is unknown.

Specific coupling of a cation-sensing receptor to G protein alpha-subunits in MDCK cells

1997 ◽  
Vol 273 (1) ◽  
pp. F129-F135 ◽  
Author(s):  
J. M. Arthur ◽  
G. P. Collinsworth ◽  
T. W. Gettys ◽  
L. D. Quarles ◽  
J. R. Raymond
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Plasma Membranes ◽  
Mdck Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cellular Activation ◽  
Protein Activation ◽  
A Cell ◽  
Cation Sensing

Extracellular cations such as Ca2+ stimulate a G protein-coupled, cation-sensing receptor (CaR). We used microphysiometry to determine whether an extracellular cation-sensing mechanism exists in Madin-Darby canine kidney (MDCK) cells. The CaR agonists Ca2+ and Gd3+ caused cellular activation in a concentration-dependent manner. mRNA for the CaR was identified by reverse transcription and polymerase chain reaction (PCR) using nested CaR-specific primers, identification of an appropriately located restriction site, and sequencing of the subcloned fragment obtained by PCR. G protein activation was evaluated using the GTP photoaffinity label [alpha-32P]GTP azidoanalide (AA-GTP). After stimulation with Gd3+ and cross-linking, plasma membranes were solubilized and immunoprecipitated with antisera specific for Gq/11 alpha and Gi alpha family members. Gd3+ increased incorporation of AA-GTP into Gq/11 alpha precipitates by 146 +/- 48% and into G alpha i-2 and G alpha i-3 to a lesser extent but not into G alpha i-1. Direct effects of Gd3+ on the G proteins were ruled out using partially purified mammalian G proteins expressed in Escherichia coli or Sf9 cells. We conclude that MDCK cells possess a cell-surface CaR that activates Gq/11 alpha, G alpha i-2, and G alpha i-3 but not G alpha i-1.

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