scholarly journals Subcellular localization and characterization of nitric oxide synthase(s) in endothelial cells: physiological implications

1994 ◽  
Vol 299 (1) ◽  
pp. 247-252 ◽  
Author(s):  
M Hecker ◽  
A Mülsch ◽  
E Bassenge ◽  
U Förstermann ◽  
R Busse
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Enzyme Activity ◽  
Plasma Membrane ◽  
Immunoblot Analysis ◽  
Subcellular Fractionation ◽  
Continuous Exposure ◽  
Ph Optimum

Endothelial cells (EC) contain a constitutive Ca2+/calmodulin-dependent nitric oxide (NO) synthase (cNOS) which plays an important role in the local control of vascular tone. We compared the subcellular distribution of this enzyme in cultured and freshly isolated pig EC by determination of specific cNOS activity and immunoblot analysis. Similar studies were also performed with cultured and freshly isolated bovine and cultured human EC. Enzyme activity was predominantly (> 70%) associated with the particulate fraction of all EC types tested and was highest in freshly isolated porcine EC. Both specific cNOS activity and immunoreactivity were substantially higher (> 3-fold) in the microsomal as compared with the soluble fraction of all EC types tested. In freshly isolated pig EC, these two fractions also differed in terms of their Ca(2+)-dependency, pH optimum and inhibitor specificity. EC may thus contain either two different cNOS isoenzymes or a single enzyme, the conformation of which differs between the soluble and membrane-bound state. Moreover, detailed subcellular fractionation of freshly isolated pig EC revealed that the distribution of cNOS activity closely resembled that of the plasma membrane marker 5′-nucleotidase, suggesting that most, if not all, of the cNOS activity in these cells is associated with the plasma membrane. This localization might render the enzyme more susceptible to activation by physical stimuli, such as a shear stress-induced change in the fluidity of the plasma membrane. Moreover, the continuous exposure to shear stress in vivo may also upregulate cNOS expression in EC, since specific enzyme activity, immunoreactivity and basal NO release were significantly higher in freshly isolated EC as compared with cultured EC.

Studies on the subcellular localization and role of glutathione-insulin transhydrogenase in rat liver

1983 ◽  
Vol 3 (4) ◽  
pp. 323-329 ◽  
Author(s):  
Balvinder K. Chowdhary ◽  
Geoffrey D. Smith ◽  
Robert Mahler ◽  
Timothy J. Peters
Keyword(s):  
Endoplasmic Reticulum ◽  
Enzyme Activity ◽  
Plasma Membrane ◽  
Subcellular Fractionation ◽  
Low Density ◽  
Specific Enzyme ◽  
Specific Enzyme Activity ◽  
Discrete Population

125I-insulin was shown to be internalized in vivo to a discrete population of low-density membranes (ligandosomes), distinct from the Golgi, endoplasmic reticulum, plasma membrane, and lysosomes. However, analytical subcellular fractionation shows that glutathione-insulin transhydrogenase is localized to the endoplasmic reticulum. Measurement of the specific enzyme activity of glutathione-insulin transhydrogenase showed no differences between normal, diabetic, and hyperinsulinaemic rats. These results suggest that glutathione-insulin transhydrogenase is not directly involved in the subceltular processing of receptor-bound internalized insulin.

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.

scholarly journals Nitric Oxide–Dependent and Nitric Oxide–Independent Transcriptional Responses to High Shear Stress in Endothelial Cells

Hypertension ◽  
2005 ◽  
Vol 45 (4) ◽  
pp. 672-680 ◽  
Author(s):  
Branko Braam ◽  
Remmert de Roos ◽  
Hans Bluyssen ◽  
Patrick Kemmeren ◽  
Frank Holstege ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
High Shear ◽  
High Shear Stress ◽  
Transcriptional Responses

Expression of endothelial nitric oxide synthase in developing rat kidney

2005 ◽  
Vol 288 (4) ◽  
pp. F694-F702 ◽  
Author(s):  
Ki-Hwan Han ◽  
Jung-Mi Lim ◽  
Wan-Young Kim ◽  
Hyang Kim ◽  
Kirsten M. Madsen ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Kidney Development ◽  
Early Stage ◽  
Rat Kidney ◽  
Immunoblot Analysis ◽  
Renal Hemodynamics ◽  
Vascular Bundles ◽  
Peritubular Capillaries ◽  
Endothelial Nitric Oxide

Endothelium-derived nitric oxide (NO) is synthesized within the developing kidney and may play a crucial role in the regulation of renal hemodynamics. The purpose of this study was to establish the expression and intrarenal localization of the NO-synthesizing enzyme endothelial NO synthase (eNOS) during kidney development. Rat kidneys from 14 ( E14)-, 16 ( E16)-, 18 ( E18)-, and 20-day-old ( E20) fetuses and 1 ( P1)-, 3 ( P3)-, 7 ( P7)-, 14 ( P14)-, and 21-day-old ( P21) pups were processed for immunocytochemical and immunoblot analysis. In fetal kidneys, expression of eNOS was first observed in the endothelial cells of the undifferentiated intrarenal capillary network at E14. At E16, strong eNOS immunoreactivity was observed in the endothelial cells of renal vesicles, S-shaped bodies (stage II glomeruli), and stage III glomeruli at the corticomedullary junction. At E18- 20, early-stage developing glomeruli located in the subcapsular region showed less strong eNOS immunoreactivity than those of E16. The eNOS-positive immature glomeruli were observed in the nephrogenic zone until 7 days after birth. In fetal kidneys, eNOS was also expressed in the medulla in the endothelial cells of the capillaries surrounding medullary collecting ducts. After birth, eNOS immunostaining gradually increased in the developing vascular bundles and peritubular capillaries in the medulla and was highest at P21. Surprisingly, eNOS was also expressed in proximal tubules, in the endocytic vacuolar apparatus, only at P1. The strong expression of eNOS in the early stages of developing glomeruli and vasculature suggests that eNOS may play a role in regulating renal hemodynamics of the immature kidney.

scholarly journals Membrane Estrogen Receptor-Dependent Extracellular Signal-Regulated Kinase Pathway Mediates Acute Activation of Endothelial Nitric Oxide Synthase by Estrogen in Uterine Artery Endothelial Cells

Endocrinology ◽  
2004 ◽  
Vol 145 (1) ◽  
pp. 113-125 ◽  
Author(s):  
Dong-bao Chen ◽  
Ian M. Bird ◽  
Jing Zheng ◽  
Ronald R. Magness
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Estrogen Receptor ◽  
Plasma Membrane ◽  
Uterine Artery ◽  
Fluorescein Isothiocyanate ◽  
Estrogen Receptor Α ◽  
Dependent Manner ◽  
Enos Phosphorylation

Abstract Rapid uterine vasodilatation after estrogen administration is believed to be mediated by endothelial production of nitric oxide (NO) via endothelial NO synthase (eNOS). However, the mechanism(s) by which estrogen activates eNOS in uterine artery endothelial cells (UAEC) is unknown. In this study, we observed that estradiol-17β (E2) and E2-BSA rapidly (&lt;2 min) increased total NOx production in UAEC in vitro. This was associated with rapid eNOS phosphorylation and activation but was unaltered by pretreatment with actinomycin-D. estrogen receptor-α protein was detectable in isolated plasma membrane proteins by immunoblotting, and E2-BSA-fluorescein isothiocyanate binding was evident on the plasma membrane of UAEC. E2 did not mobilize intracellular Ca2+, but E2 and ionomycin in combination induced greater eNOS phosphorylation than either E2 or ionomycin alone. E2 did not stimulate rapid Akt phosphorylation. E2 stimulated rapid ERK2/1 activation in a time- and dose-dependent manner, with maximal responses observed at 5–10 min with E2 (10 nm to 1 μm) treatment. Acute activation of eNOS and NOx production by E2 could be inhibited by PD98059 but not by LY294002. When E2-BSA was applied, similar responses in NOx production, eNOS, and ERK2/1 activation to those of E2 were achieved. In addition, E2 and E2-BSA-induced ERK2/1 activation and ICI 182,780 could inhibit NOx production by E2. Thus, acute activation of eNOS to produce NO in UAEC by estrogen is at least partially through an ERK pathway, possibly via estrogen receptor localized on the plasma membrane. This pathway may provide a novel mechanism for NO-mediated rapid uterine vasodilatation by estrogen.

Shear Stress Modulates the Expression of Thrombospondin-1 and CD36 in Endothelial Cells in vitro and during Shear Stress-Induced Angiogenesis in vivo

2006 ◽  
Vol 19 (1) ◽  
pp. 205873920601900 ◽  
Author(s):  
M. Bongrazio ◽  
L. DA Silva-Azevedo ◽  
E.C. Bergmann ◽  
O. Baum ◽  
B. Hinz ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Western Blot ◽  
Dependent Manner ◽  
Vascular Networks ◽  
Thrombospondin 1 ◽  
Matrix Bound ◽  
Stress Dependent

Binding of thrombospondin-1 (TSP-1) to the CD36 receptor inhibits angiogenesis and induces apoptosis in endothelial cells (EC). Conversely, matrix-bound TSP-1 supports vessel formation. In this study we analyzed the shear stress-dependent expression of TSP-1 and CD36 in endothelial cells in vitro and in vivo to reveal its putative role in the blood flow-induced remodelling of vascular networks. Shear stress was applied to EC using a cone-and-plate apparatus and gene expression was analyzed by RT-PCR, Northern and Western blot. Angiogenesis in skeletal muscles of prazosin-fed (50 mg/1 drinking water; 4 d) mice was assessed by measuring capillary-to-fiber (C/F) ratios. Protein expression in whole muscle homogenates (WMH) or BS-1 lectin-enriched EC fractions (ECF) was analyzed by Western blot. Shear stress down-regulated TSP-1 and CD36 expression in vitro in a force- and time-dependent manner sustained for at least 72 h and reversible by restoration of no-flow conditions. In vivo, shear stress-driven increase of C/F in prazosin-fed mice was associated with reduced expression of TSP-1 and CD36 in ECF, while TSP-1 expression in WMH was increased. Down-regulation of endothelial TSP-1/CD36 by shear stress suggests a mechanism for inhibition of apoptosis in perfused vessels and pruning in the absence of flow. The increase of extra-endothelial (e.g. matrix-bound) TSP-1 could support a splitting type of vessel growth.

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.

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