Platelet-activating factor increases endothelial [Ca2+]i and NO production in individually perfused intact microvessels

2005 ◽  
Vol 288 (6) ◽  
pp. H2869-H2877 ◽  
Author(s):  
Longkun Zhu ◽  
Pingnian He
Keyword(s):  
Endothelial Cells ◽  
Platelet Activating Factor ◽  
No Production ◽  
Caveolin 1 ◽  
Microvessel Permeability ◽  
Nos Inhibitor ◽  
The Mean ◽  
Induced Changes ◽  
Dependent Signaling Pathway ◽  
Peak Value

We have demonstrated that inhibition of NO synthase (NOS) in endothelial cells by either the NOS inhibitor Nω-monomethyl-l-arginine (l-NMMA) or the internalization of caveolin-1 scaffolding domain attenuated platelet-activating factor (PAF)-induced increases in microvessel permeability ( Am J Physiol Heart Circ Physiol 286: H195–H201, 2004) indicating the involvement of an NO-dependent signaling pathway. To investigate whether an increase in endothelial cytoplasmic Ca2+ concentration ([Ca2+]i) is the initiating event and Ca2+-dependent NO production is crucial for permeability increases, PAF (10 nM)-induced changes in endothelial [Ca2+]i and NO production were measured in individually perfused rat mesenteric venular microvessels via fluorescence microscopy. When venular microvessels were exposed to PAF, endothelial [Ca2+]i increased from 69 ± 8 nM to a peak value of 374 ± 26 nM within 3 min and then declined to a sustained level at 190 ± 12 nM after 15 min. Inhibition of NOS did not modify PAF-induced increases in endothelial [Ca2+]i. PAF-induced NO production was visualized and quantified at cellular levels in individually perfused microvessels using 4,5-diaminofluorescein diacetate and fluorescence imaging. Increased fluorescence intensity (FI), which is an indication of increased NO production, occurred in 75 ± 7% of endothelial cells in each vessel. The mean maximum FI increase was 140 ± 7% of baseline value. This increased FI was abolished by pretreatment of the vessel with l-NMMA and attenuated in the absence of extracellular Ca2+. These results provide direct evidence from intact microvessels that increased endothelial [Ca2+]i is the initial signal that activates endothelial NOS, and the subsequent increased NO production contributes to PAF-induced increases in microvessel permeability.

scholarly journals Temporal and spatial correlation of platelet-activating factor-induced increases in endothelial [Ca2+]i, nitric oxide, and gap formation in intact venules

2011 ◽  
Vol 301 (5) ◽  
pp. H1788-H1797 ◽  
Author(s):  
Xueping Zhou ◽  
Pingnian He
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Platelet Activating Factor ◽  
Cellular Level ◽  
Confocal Imaging ◽  
No Production ◽  
Molecular Signaling ◽  
Gap Formation ◽  
Microvessel Permeability ◽  
Endothelial Gaps

We have previously demonstrated that platelet-activating factor (PAF)-induced increases in microvessel permeability were associated with endothelial gap formation and that the magnitude of peak endothelial intracellular Ca2+ concentration ([Ca2+]i) and nitric oxide (NO) production at the single vessel level determines the degree of the permeability increase. This study aimed to examine whether the magnitudes of PAF-induced peak endothelial [Ca2+]i, NO production, and gap formation are correlated at the individual endothelial cell level in intact rat mesenteric venules. Endothelial gaps were quantified by the accumulation of fluorescent microspheres at endothelial clefts using confocal imaging. Endothelial [Ca2+]i was measured on fura-2- or fluo-4-loaded vessels, and 4,5-diaminofluorescein (DAF-2) was used for NO measurements. The results showed that increases in endothelial [Ca2+]i, NO production, and gap formation occurred in all endothelial cells when vessels were exposed to PAF but manifested a spatial heterogeneity in magnitudes among cells in each vessel. PAF-induced peak endothelial [Ca2+]i preceded the peak NO production by 0.6 min at the cellular level, and the magnitudes of NO production and gap formation linearly correlated with that of the peak endothelial [Ca2+]i in each cell, suggesting that the initial levels of endothelial [Ca2+]i determine downstream NO production and gap formation. These results provide direct evidence from intact venules that inflammatory mediator-induced increases in microvessel permeability are associated with the generalized formation of endothelial gaps around all endothelial cells. The spatial differences in the molecular signaling that were initiated by the heterogeneous endothelial Ca2+ response contribute to the heterogeneity in permeability increases along the microvessel wall during inflammation.

Graded modulation of frog microvessel permeability to albumin using ionophore A23187

1990 ◽  
Vol 258 (2) ◽  
pp. H587-H598 ◽  
Author(s):  
F. E. Curry ◽  
W. L. Joyner ◽  
J. C. Rutledge
Keyword(s):  
Endothelial Cells ◽  
Permeability Coefficient ◽  
Ionophore A23187 ◽  
Solvent Drag ◽  
Solute Permeability ◽  
Fibrous Matrix ◽  
Microvessel Permeability ◽  
Quantitative Fluorescence ◽  
Peak Value ◽  
Sustained Increase

We investigated the exchange of water and macromolecules across venular microvessels after permeability was increased. Quantitative fluorescence microscopy was used to measure albumin permeability coefficients in individually perfused microvessels of decerebrate frogs. Control permeability coefficient was 2.3 +/- 0.25 X 10(-7) cm/s. Solvent drag increased the apparent solute permeability coefficient (Ps) by 0.57 +/- 0.05 X 10(-7) cm/s for each cmH2O increase in microvessel pressure. The divalent cation ionophore A23187 (0.1–5 microM) produced a transient increase in Ps to a peak value (within 1–3 min), followed (after 4–8 min) by a sustained increase in permeability (16–34% of peak values). Peak values of Ps were 13 and 80 times control for 0.1 and 5 microM A23187, respectively. Both diffusion and solvent drag contributed to the sustained increase in Ps. The equivalent pore radius of the structures determining diffusion and solvent drag was less than or equal to 25 nm during the sustained increase in permeability, smaller than observed gaps between adjacent endothelial cells. The basement membrane and a fibrous matrix secreted by endothelial cells into the gaps may offer resistance to exchange in the high permeability state.

cGMP modulates basal and activated microvessel permeability independently of [Ca2+]i

1998 ◽  
Vol 274 (6) ◽  
pp. H1865-H1874 ◽  
Author(s):  
P. He ◽  
M. Zeng ◽  
F. E. Curry
Keyword(s):  
Time Course ◽  
Regulatory Mechanisms ◽  
Inflammatory Stimuli ◽  
Cgmp Analog ◽  
Microvessel Permeability ◽  
The Mean ◽  
Mesenteric Microvessels ◽  
Ly 83583 ◽  
Peak Value

To investigate the mechanisms whereby guanosine 3′,5′-cyclic monophosphate (cGMP) modulates microvessel permeability in vivo, we measured changes in microvessel hydraulic conductivity ( L p) and endothelial cytoplasmic Ca2+concentration ([Ca2+]i) in response to the cGMP analogs 8-bromo-cGMP (8-BrcGMP) and 8-( p-chlorophenylthio)cGMP (8-pCPT-cGMP) in the presence and absence of inflammatory stimuli in intact individually perfused microvessels in frog and rat mesenteries. The cGMP analog caused a transient increase in L p and potentiated ATP or bradykinin-induced increases in L p in frog and rat mesenteric microvessels, respectively. The mean peak value of the test L p/control L p after exposure to 8-BrcGMP was 5.3 ± 0.5 in frog microvessels and 2.8 ± 0.4 in rat microvessels. The ATP-induced increase in L p in frog microvessels was further raised by 8-BrcGMP from 7.0 ± 0.9 to 12.4 ± 1.9 times the control. In rat mesenteric microvessels, the bradykinin-induced increase in L p was potentiated by 8-BrcGMP from 4.8 ± 0.4 to 8.3 ± 1.3 times the control and was suppressed by the guanylate cyclase inhibitor LY-83583 to 2.6 ± 0.5 times the control. A similar but larger effect was found when using 8-pCPT-cGMP. In contrast to the actions of increased cGMP on microvessel permeability, cGMP analogs had no effect on basal endothelial [Ca2+]iand did not alter the magnitude and time course of ATP or bradykinin-induced increases in endothelial [Ca2+]i. These results suggested that an elevation of cGMP levels in endothelial cells is a necessary step to increase microvessel permeability in intact microvessels, and this regulatory process occurs downstream from Ca2+ influx, which differs from that reported in large-vessel endothelium in culture and in vascular smooth muscle cells. Experiments carried on microvessels in both frog and rat mesenteries provided a direct comparison of the endothelial cell regulatory mechanisms between species.

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)

scholarly journals Inhibition of mammalian nitric oxide synthases by agmatine, an endogenous polyamine formed by decarboxylation of arginine

1996 ◽  
Vol 316 (1) ◽  
pp. 247-249 ◽  
Author(s):  
Elena GALEA ◽  
S. REGUNATHAN ◽  
Vassily ELIOPOULOS ◽  
Douglas L. FEINSTEIN ◽  
Donald J. REIS
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Enzyme Activity ◽  
Mammalian Cells ◽  
Structural Similarity ◽  
Nitric Oxide Synthases ◽  
No Production ◽  
Brain Macrophages ◽  
Nos Inhibitor ◽  
Nos Isoforms

Agmatine, decarboxylated arginine, is a metabolic product of mammalian cells. Considering the close structural similarity between L-arginine and agmatine, we investigated the interaction of agmatine and nitric oxide synthases (NOSs), which use L-arginine to generate nitric oxide (NO) and citrulline. Brain, macrophages and endothelial cells were respectively used as sources for NOS isoforms I, II and III. Enzyme activity was measured by the production of nitrites or L-citrulline. Agmatine was a competitive NOS inhibitor but not an NO precursor. Ki values were approx. 660 μM (NOS I), 220 μM (NOS II) and 7.5 mM (NOS III). Structurally related polyamines did not inhibit NOS activity. Agmatine, therefore, may be an endogenous regulator of NO production in mammals.

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.

scholarly journals Enhanced permeability responses to inflammation in streptozotocin-induced diabetic rat venules: Rho-mediated alterations of actin cytoskeleton and VE-cadherin

2014 ◽  
Vol 307 (1) ◽  
pp. H44-H53 ◽  
Author(s):  
Dong Yuan ◽  
Sulei Xu ◽  
Pingnian He
Keyword(s):  
Endothelial Cells ◽  
Microvascular Complications ◽  
Diabetic Rats ◽  
Fiber Formation ◽  
Receptor Expression ◽  
Diabetic Patients ◽  
No Production ◽  
Significant Difference ◽  
Microvessel Permeability ◽  
The Impact

Diabetes is a progressive disease that often leads to microvascular complications. This study investigates the impact of diabetes on microvessel permeability under basal and inflammatory conditions. Streptozotocin-induced diabetic rats were used to mimic type 1 diabetes. Parallel experiments were conducted in intact mesenteric venules in normal rats and diabetic rats experiencing hyperglycemia for 2–3 wk. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). The correlated changes in endothelial intracellular Ca2+ concentration ([Ca2+]i), adherens junctions, and cytoskeleton F-actin were examined with fluorescence imaging. Diabetic vessels showed moderately increased basal Lp, but upon platelet-activating factor (PAF) exposure, these vessels showed an ∼10-fold higher Lp increase than the normal vessels. Concomitantly, we observed higher increases in endothelial [Ca2+]i, enhanced stress fiber formation, vascular endothelial-cadherin separation, and larger gap formation between endothelial cells than those occurring in normal vessels. PAF receptor staining showed no significant difference between normal and diabetic vessels. The application of Rho kinase inhibitor Y27632 did not affect PAF-induced increases in endothelial [Ca2+]i but significantly reduced PAF-induced Lp increases by 90% in diabetic vessels. The application of both Y27632 and nitric oxide (NO) synthase inhibitor attenuated PAF-induced Lp increases more than using one inhibitor alone. Our studies indicate that diabetic conditions prime endothelial cells into a phenotype with increased susceptibility to inflammation without altering receptor expression and that the increased Rho activation and NO production play important roles in exaggerated permeability increases when diabetic vessels were exposed to inflammatory mediators, which may account for the exacerbated vascular dysfunction when diabetic patients are exposed to additional inflammation.

Oxygen alters caveolin-1 and nitric oxide synthase-3 functions in ovine fetal and neonatal lung microvascular endothelial cells

2006 ◽  
Vol 291 (5) ◽  
pp. L1079-L1093 ◽  
Author(s):  
Theresa A. John ◽  
Basil O. Ibe ◽  
J. Usha Raj
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Negative Regulator ◽  
Microvascular Endothelial Cells ◽  
No Production ◽  
Caveolin 1 ◽  
Neonatal Lung ◽  
Oxygen Sensitive ◽  
Microvascular Endothelial ◽  
Ovine Fetal

We determined the effect of oxygen [∼100 Torr (normoxia) and ∼30–40 Torr (hypoxia)] on functions of endothelial nitric oxide (NO) synthase (NOS-3) and its negative regulator caveolin-1 in ovine fetal and neonatal lung microvascular endothelial cells (MVECs). Fetal NOS-3 activity, measured as NO production with 0.5–0.9 μM 4-amino-5-methylamino-2,7-difluorofluorescein, was decreased in hypoxia by 14.4% ( P < 0.01), inhibitable by the NOS inhibitor N-nitro-l-arginine, and dependent on extracellular arginine. Caveolar function, assessed as FITC-BSA (160 μg/ml) endocytosis, was decreased in hypoxia by 13.5% in fetal and 22.8% in neonatal MVECs ( P < 0.01). NOS-3 and caveolin-1 were physically associated, as demonstrated by coimmunoprecipitation and colocalization, and functionally associated, as shown by cross-activation of endocytosis, by their specific antibodies and activation of NOS by albumin. Caveolin peptide, containing the sequence for the PKC phosphorylation site of caveolin, and caveolin antiserum against the site increased NO production and endocytosis by 12.3% ( P < 0.05) and 16% ( P < 0.05), respectively, in normoxia and increased endocytosis by 25% ( P < 0.001) in hypoxia. PMA decreased NO production in normoxia and hypoxia by 19.32% ( P < 0.001) and 11.8% ( P < 0.001) and decreased endocytosis in normoxia by 20.35% ( P < 0.001). PKC kinase activity was oxygen sensitive, and threonine phosphorylation was enhanced in hypoxia. Pertussis toxin increased caveolar and NOS functions. These data support our hypothesis that increased Po2 at birth promotes dissociation of caveolin-1 and NOS-3, with an increase in their activities, and that PKC and an oxygen-sensitive cell surface G protein-coupled receptor regulate caveolin-1 and NOS-3 interactions in fetal and neonatal lung MVECs.

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