Ca
            <sup>2+</sup>
            -Independent Activation of the Endothelial Nitric Oxide Synthase in Response to Tyrosine Phosphatase Inhibitors and Fluid Shear Stress

Fleming, Ingrid; Bauersachs, Johann; Fißlthaler, Beate; Busse, Rudi

doi:10.1161/01.res.82.6.686

Cited by 244 publications

(226 citation statements)

References 45 publications

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“…Furthermore, by using single bead magnetic trap loading similar to this focal adhesion translocation study did not induce a significant change in intracellular calcium compared with unforced control cells (unpublished data). These findings are consistent with previous studies indicating that calcium signaling is not an essential component to focal adhesion dynamics (13) and with data from Sawada and Sheetz (37) showing that substrate strain activates focal adhesion formation in Triton-X cytoskeletons, even in the absence of cell membranes, cytosolic-free ions, and other intracellular biomolecules.…”

Section: Discussionsupporting

confidence: 93%

Force-induced focal adhesion translocation: effects of force amplitude and frequency

Mack

Kaazempur-Mofrad²,

Karcher³

et al. 2004

American Journal of Physiology-Cell Physiology

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. Force-induced focal adhesion translocation: effects of force amplitude and frequency. Am J Physiol Cell Physiol 287: C954 -C962, 2004. First published June 9, 2004 10.1152/ajpcell. 00567.2003.-Vascular endothelial cells rapidly transduce local mechanical forces into biological signals through numerous processes including the activation of focal adhesion sites. To examine the mechanosensing capabilities of these adhesion sites, focal adhesion translocation was monitored over the course of 5 min with GFPpaxillin while applying nN-level magnetic trap shear forces to the cell apex via integrin-linked magnetic beads. A nongraded steady-load threshold for mechanotransduction was established between 0.90 and 1.45 nN. Activation was greatest near the point of forcing (Ͻ7.5 m), indicating that shear forces imposed on the apical cell membrane transmit nonuniformly to the basal cell surface and that focal adhesion sites may function as individual mechanosensors responding to local levels of force. Results from a continuum, viscoelastic finite element model of magnetocytometry that represented experimental focal adhesion attachments provided support for a nonuniform force transmission to basal surface focal adhesion sites. To further understand the role of force transmission on focal adhesion activation and dynamics, sinusoidally varying forces were applied at 0.1, 1.0, 10, and 50 Hz with a 1.45 nN offset and a 2.25 nN maximum. At 10 and 50 Hz, focal adhesion activation did not vary with spatial location, as observed for steady loading, whereas the response was minimized at 1.0 Hz. Furthermore, applying the tyrosine kinase inhibitors genistein and PP2, a specific Src family kinase inhibitor, showed tyrosine kinase signaling has a role in force-induced translocation. These results highlight the mutual importance of force transmission and biochemical signaling in focal adhesion mechanotransduction. mechanotransduction; endothelial cell; paxillin; viscoelastic model MECHANOTRANSDUCTION IS AN essential function of the cell, controlling its growth, proliferation, protein synthesis, and gene expression (8,18). Extensive data exist documenting the cellular responses to external force (15, 41, 44), but less is known about how force affects rapid biological signaling. Although integrins/focal adhesion sites (42), cytoskeleton constituents, G proteins (6), ion channels, intercellular junction proteins, and membrane biomolecules have all been identified as potential mechanosensors (6,16,42,43), we know little about the force level and frequency-dependent thresholds required to initiate mechanotransduction or the role of intracellular force transmission on mechanosensor activation. Biological readouts used to study mechanotransduction range from long-term gene expression and cell morphology changes (8, 26) to rapid variations in intracellular ion concentration and protein activity (8,26,42). Morphological and gene expression comparisons provide a robust marker of mechanotransduction, but the response is slow on the scale of hours, a...

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Section: Discussionsupporting

confidence: 93%

Force-induced focal adhesion translocation: effects of force amplitude and frequency

Mack

Kaazempur-Mofrad²,

Karcher³

et al. 2004

American Journal of Physiology-Cell Physiology

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“…Phosphorylation of Thr 495 is generally associated with a decrease in enzyme activity [41,42]. While endothelial NO production can be modulated by tyrosine kinase inhibitors and tyrosine phosphatase inhibitors [43,44], little is known about which tyrosine residues are phosphorylated [45]. Thus changes in threonine or tyrosine phosphorylation of NOS3 could also explain our findings.…”

Section: Discussionmentioning

confidence: 67%

Nitric oxide generation mediated by ?-adrenoceptors is impaired in platelets from patients with Type 2 diabetes mellitus

Queen

Goubareva

et al. 2003

Diabetologia

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Aims/hypothesis. Type 2 diabetic patients have been shown to have reduced basal platelet nitric oxide synthase activity, which is a possible contributor to the vascular complications seen in the disease. We investigated platelet nitric oxide generation stimulated by β-adrenoceptors and adenylyl cyclase in Type 2 diabetic patients and control subjects. Methods. Platelets isolated from blood taken from nine Type 2 diabetic patients and nine healthy control subjects of similar age were treated with isoproterenol 1 µmol/l, forskolin 1 µmol/l or vehicle. Platelet nitric oxide synthase activity was measured by L-[ 3 H]-arginine to L-[ 3 H]-citrulline conversion, cyclic GMP content by radioimmunoassay, and nitric oxide synthase type 3 expression by western blotting. Results. Basal platelet nitric oxide synthase activity was lower in diabetic patients than in control subjects (0.01±0.02 pmol L-citrulline/10 8 platelets, compared with 0.12±0.05; p<0.05), although no corresponding difference was seen in basal platelet cyclic GMP (0.61±0.39 and 0.13±0.22 pmol cyclic GMP/10 8 platelets respectively; p=0.37). In control subjects isoproterenol 1 µmol/l and forskolin 1 µmol/l increased platelet nitric oxide synthase activity (to 0.27±0.08 and 0.27±0.07 pmol L-citrulline/10 8 platelets respectively; p<0.05 for each in comparison with basal) and cyclic GMP (to 1.84±0.41 and 1.86±0.48; p<0.05 for each in comparison with basal). This effect was not achieved in diabetic patients. Isoproterenol-and forskolin-stimulated cyclic GMP correlated inversely with plasma glucose and HbA 1c . Platelet nitric oxide synthase type 3 expression was not different in control and diabetic subjects and was not changed by acute exposure of platelets to isoproterenol. Conclusions/interpretation. Nitric oxide generation stimulated by β-adrenoceptors and adenylyl cyclase is impaired in platelets of people with Type 2 diabetes mellitus, with no corresponding change in nitric oxide synthase type 3 expression. It is possible that this impairment contributes to the thrombotic and atherosclerotic complications of Type 2 diabetes. [Diabetologia (2003[Diabetologia ( ) 46:1474[Diabetologia ( -1482

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“…Shear induced NO release in vitro and flow-dependent vasodilation in vivo can be blocked with NOS inhibitors. Interestingly, shearinduced NO release appears to "independent" of fluctuations of calcium since shear causes a rapid burst of calcium release that does not parallel the sustained release of NO; chelation of intracellular calcium does not influence the rate of NO production elicited by shear and calmodulin antagonists can block bradykinin induced NO release but not shear induced release (Kuchan & Frangos 1994, Fleming et al 1998. These data collectively suggest a fundamental difference in the signal transduction mechanisms for agonist versus shear-or growth factorinduced activation of eNOS.…”

Section: Physiological Activation Of Enos and No Releasementioning

confidence: 96%

Regulation of endothelial derived nitric oxide in health and disease

Sessa

2005

Mem. Inst. Oswaldo Cruz

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In the past decade the importance of the vascular endothelium as a multifunctional regulator of vascular smooth muscle physiology and pathophysiology has been appreciated. Indeed, the endothelium responds to hemodynamic stimuli (pressure, shear stress, and wall strain) and locally manufactured mediators (such as bradykinin, prostaglandins, and angiotensin) and in turn can release factors that can influence the adhesion and aggregation of circulating cells to the endothelium and the tone of vascular smooth muscle. In many diseases, including atherosclerosis, diabetes or cirrhosis endothelial dysfunction manifested as an impairment nitric oxide (NO) production or bioactivity may be an early hallmark of disease and a treatable entity. In this chapter, the importance of NO as a mediator of vascular function and potential mechanisms of endothelial NO synthase (eNOS) activation in disease will be discussed. Regulation of vascular tone by endogenous NOeNOS is the NOS isoform responsible for producing the classical endothelium-derived relaxing factor as originally described by (Furchgott & Zawadski 1981). Evidence for the importance of eNOS derived NO in the regulation of vascular tone is based on experiments in animals and in humans demonstrating that L-arginine based inhibitors of NOS increase blood or perfusion pressure and vascular resistance and reduce blood flow in vivo and in vitro. More recently, this has been unequivocally confirmed using mice with targeted disruption of the eNOS gene locus. eNOS knockout mice (-/-) are mildly hypertensive relative to wild-type littermate control mice (+/+) of the same generation. Importantly, the pressor effect of nitro-L-arginine, a NOS inhibi- tor, is attenuated in the -/-mice and endothelium-dependent relaxation in response to acetylcholine is abrogated in isolated vessels (Huang et al. 1995, Shesely et al. 1996. This fundamental finding is direct "proof of-principal" for the major contribution of NO in vasomotor control in large blood vessels. Physiological activation of eNOS and NO releaseTypically, endothelial cells release NO in response to autacoids that mobilize intracellular calcium such as thrombin, VEGF or ADP. The proposed mechanism for eNOS activation is that the released calcium will bind to calmodulin (CaM) and the calcium/CaM complex will bind to the CaM site in the enzyme promote NO synthesis. However, the most physiological agonist for NO release is fluid shear stress. Shear stress in vitro or shear rate in vivo, is the tangential vector of force elicited by the flow of blood over the endothelial cell surface. Exposure of endothelial cells to shear stress results in a burst of NO release, followed by a sustained phase. In vivo, increasing shear rate due to high blood flow will cause flow dependent dilations of certain vascular beds while decreasing shear rate will promote vasoconstriction. Shear induced NO release in vitro and flow-dependent vasodilation in vivo can be blocked with NOS inhibitors. Interestingly, shearinduced NO release appears to "independen...

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Ca ²⁺ -Independent Activation of the Endothelial Nitric Oxide Synthase in Response to Tyrosine Phosphatase Inhibitors and Fluid Shear Stress

Cited by 244 publications

References 45 publications

Force-induced focal adhesion translocation: effects of force amplitude and frequency

Force-induced focal adhesion translocation: effects of force amplitude and frequency

Nitric oxide generation mediated by ?-adrenoceptors is impaired in platelets from patients with Type 2 diabetes mellitus

Regulation of endothelial derived nitric oxide in health and disease

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Ca 2+ -Independent Activation of the Endothelial Nitric Oxide Synthase in Response to Tyrosine Phosphatase Inhibitors and Fluid Shear Stress

Cited by 244 publications

References 45 publications

Force-induced focal adhesion translocation: effects of force amplitude and frequency

Force-induced focal adhesion translocation: effects of force amplitude and frequency

Nitric oxide generation mediated by ?-adrenoceptors is impaired in platelets from patients with Type 2 diabetes mellitus

Regulation of endothelial derived nitric oxide in health and disease

Contact Info

Product

Resources

About

Ca ²⁺ -Independent Activation of the Endothelial Nitric Oxide Synthase in Response to Tyrosine Phosphatase Inhibitors and Fluid Shear Stress