Endothelial Nitric Oxide Generation Research Articles

Platelet dysfunction in type 2 diabetes.

Insulin resistance is a uniform finding in type 2 diabetes, as are abnormalities in the microvascular and macrovascular circulations. These complications are associated with dysfunction of platelets and the neurovascular unit. Platelets are essential for hemostasis, and knowledge of their function is basic to understanding the pathophysiology of vascular disease in diabetes. Intact healthy vascular endothelium is central to the normal functioning of smooth muscle contractility as well as its normal interaction with platelets. What is not clear is the role of hyperglycemia in the functional and organic microvascular deficiencies and platelet hyperactivity in individuals with diabetes. The entire coagulation cascade is dysfunctional in diabetes. Increased levels of fibrinogen and plasminogen activator inhibitor 1 favor both thrombosis and defective dissolution of clots once formed. Platelets in type 2 diabetic individuals adhere to vascular endothelium and aggregate more readily than those in healthy people. Loss of sensitivity to the normal restraints exercised by prostacyclin (PGI(2)) and nitric oxide (NO) generated by the vascular endothelium presents as the major defect in platelet function. Insulin is a natural antagonist of platelet hyperactivity. It sensitizes the platelet to PGI(2) and enhances endothelial generation of PGI(2) and NO. Thus, the defects in insulin action in diabetes create a milieu of disordered platelet activity conducive to macrovascular and microvascular events.

Nitric oxide and hypertension: not just an endothelium derived relaxing factor!

The importance of endothelial nitric oxide (NO) generation in sustaining a tonic systemic vasodilatation is well established. Inhibiting NO production produces hypertension in animals and in humans and not surprisingly there has been considerable interest in establishing whether deficiencies of endothelial NO pathway activity are implicated in the aetiology of essential hypertension. The results of these investigations have been inconsistent with some suggestion that observed deficiencies of both basal and stimulated endothelial NO generation in hypertensive subjects may be an effect rather than the cause of raised arterial pressure. It is increasingly recognised that neuronal production of NO also influences cardiovascular homeostasis through its action as a neuromodulator within the autonomic nervous system. Overall NO has been shown to have sympatho-inhibitory and vagotonic effects, acting by both central and peripheral mechanisms. Sympathetic overactivity, coupled with the permissive role of a depressed level of baroreflex mediated cardiac vagal control, may play a significant role in the genesis of human hypertension. Early work in hypertensive rats suggests that neuronal NO production is impaired at a number of key central sites concerned with autonomic cardiovascular regulation. This data is consistent with the pattern of autonomic dysfunction observed in human hypertension. The possibility that neuronal rather than endothelial production of NO might play a significant role in the aetiology of essential hypertension is a promising area for future human research.

Prevalence and pathogenesis of sleep apnea and lung disease in acromegaly.

Respiratory disorders are common and important complications in acromegaly. Patients suffering from acromegaly display a 1.6-3.3 fold increase in mortality rate, which is due to respiratory disorders in 25% of cases. In these patients, mortality for lung disease is 2-3 fold higher than in the general population. Every portion of the respiratory system may be involved. Deformities of facial bones, edema and hypertrophy of the mucosae and pharyngeal and laryngeal cartilages, enlargement of the tongue and inspiratory collapse of the hypopharinx, all may contribute to respiratory alterations. Nasal polyps, "hormonal rhinitis", changes of the voice and snoring are common occurrences. Though rarely, a laryngocele may ensue. Pneumomegaly is frequently observed and, as suggested by functional studies, might be due to an increased number rather than volume of the alveoli. An obstructive respiratory syndrome caused by mucosal thickening of the upper airways and bronchi is observed in 25% of female and 70% of male patients. The sleep apnea syndrome (SAS) affects 60-70% of acromegalic patients. SAS may be of obstructive, central or mixed type. Obstructive SAS is the prevailing form in acromegaly. It is due to intermittent obstruction of upper airways with preserved activity of the respiratory center, as testified by the remarkable thoracic and abdominal respiratory efforts. The pathogenesis of the central type of SAS is more complex. Narrowing of the upper airways may induce reflex inhibition of the respiratory center. Moreover, increased GH levels and, possibly, defects in the somatostatinergic pathways, may increase the ventilatory response of the respiratory center to carbon dioxide, thereby leading to respiratory arrest. In the mixed type of SAS, the phenomena underlying the other two forms coexist. Oxygen desaturation concomitant with the apneic episodes accounts for the frequent nocturnal wakening and diurnal drowsiness. Among the clinical correlates of SAS, arterial hypertension is of particular interest due to the close correlation existing between the two disorders. Sleep deprivation related to SAS seems per se to favor the appearance of hypertension. Moreover, short lasting hypoxemia may induce prolonged elevations of blood pressure, mediated by decreased endothelial generation of nitric oxide. Thus, since cardiovascular events are the main cause of mortality in patients with acromegaly, it is reasonable to hypothesize that SAS is involved in the reduced life span of these patients.

Ca2+- and Phosphatidylinositol 3-Kinase-dependent Nitric Oxide Generation in Lung Endothelial Cells in Situ with Ischemia

Endothelial cells generate nitric oxide (NO) in response to agonist stimulation or increased shear stress. In this study, we evaluated the effects of abrupt cessation of shear stress on pulmonary endothelial NO generation and its relationship to changes in intracellular Ca(2+). In situ endothelial generation of NO and changes in intracellular Ca(2+) in isolated, intact rat lungs were evaluated using fluorescence microscopy with diaminofluorescein diacetate, an NO probe, and Fluo-3, a Ca(2+) probe. The onset of increased NO generation in endothelial cells of subpleural microvessels in situ occurred between 30 and 90 s after onset of ischemia and was preceded by an increase in intracellular Ca(2+) due to both influx of extracellular Ca(2+) and release from intracellular stores. Flow cessation-induced NO generation in endothelial cells in situ was Ca(2+)-, calmodulin-, and PI3-kinase-dependent. The similarity of endothelial cell response (increased NO generation) to either increased flow or cessation of flow suggests that cells respond to an imposed alteration from a state of adaptation. This response to flow cessation may constitute a compensatory vasodilatatory mechanism and may play a role in signaling for cell proliferation and vascular remodeling.

Role of Endothelial Nitric Oxide Generation and Peroxynitrite Formation in Reperfusion Injury After Focal Cerebral Ischemia

Reperfusion injury is one of the factors that unfavorably affects stroke outcome and shortens the window of opportunity for thrombolysis. Surges of nitric oxide (NO) and superoxide generation on reperfusion have been demonstrated. Concomitant generation of these radicals can lead to formation of the strong oxidant peroxynitrite during reperfusion. We have examined the role of NO generation and peroxynitrite formation on reperfusion injury in a mouse model of middle cerebral artery occlusion (2 hours) and reperfusion (22 hours). The infarct volume was assessed by 2,3,5-triphenyl tetrazolium chloride staining; blood-brain barrier permeability was evaluated by Evans blue extravasation. Nitrotyrosine formation and matrix metalloproteinase-9 expression were detected by immunohistochemistry. Infarct volume was significantly decreased (47%) in animals treated with the nonselective nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine (L-NA) at reperfusion. The specific inhibitor of neuronal NOS, 7-nitroindazole (7-NI), given at reperfusion, showed no protection, although preischemic treatment with 7-NI decreased infarct volume by 40%. Interestingly, prereperfusion administration of both NOS inhibitors decreased tyrosine nitration (a marker of peroxynitrite toxicity) in the ischemic area. L-NA treatment also significantly reduced vascular damage, as indicated by decreased Evans blue extravasation and matrix metalloproteinase-9 expression. These data support the hypothesis that in addition to the detrimental action of NO formed by neuronal NOS during ischemia, NO generation at reperfusion plays a significant role in reperfusion injury, possibly through peroxynitrite formation. Contrary to L-NA, failure of 7-NI to protect against reperfusion injury suggests that the source of NO is the cerebrovascular compartment.

Partial inhibition of nitric oxide synthase primes the stimulated pathway of vWF-secretion in man

Increased release of von Willebrand factor (vWF) has been linked to the pathogenesis of atherosclerosis. For this complex disease, impairment of endothelium-derived, nitric oxide production and impaired vascular relaxation has also been reported. Since endothelially produced nitric oxide (NO) is known to inhibit secretion of the Weibel–Palade bodies in animals, we hypothesized that NO could mitigate vWF secretion. In a randomized, placebo controlled cross-over trial, eight male volunteers received N-monomethyl- l-arginine (LNMMA) to block endothelial NO production or placebo, and vWF release was stimulated by infusing desmopressin in three cumulative doses (0.05, 0.15, 0.4 μg/kg) in both periods. At a threshold dose of 0.l5 μg/kg desmopressin, concomitant partial blockade of NO production resulted in 20% higher levels of vWF ( P<0.04). However, maximal vWF release after 0.4 μg/kg desmopressin was unaffected by L-NMMA (Δ7% between periods, P=0.88). These data show the dampening effect of NO production on vWF release in response to threshold concentrations of secretagogues. This may in part explain the higher vWF levels in cardiovascular diseases associated with impaired endothelial NO generation.

Hemodialysis and L-arginine, but not D-arginine, correct renal failure-associated endothelial dysfunction

In end-stage renal failure (ESRF) symptomatic hemodialysis-related hypotension may prevent effective provision of renal replacement therapy. Endogenous inhibitors of nitric oxide synthase accumulate in ESRF and are cleared by dialysis. We, therefore, hypothesised that removal of these inhibitors by hemodialysis would increase endothelial nitric oxide generation and promote venodilation. In vivo responses of norepinephrine preconstricted dorsal hand veins to locally active doses of acetylcholine (an activator of nitric oxide synthase) and glyceryl trinitrate (GTN; a nitric oxide donor) were examined in patients undergoing maintenance hemodialysis for ESRF and in healthy age- and sex-matched controls. Patient studies were undertaken before and after dialysis. Studies before dialysis were repeated with co-infusion of either L-arginine or its inactive enantiomer D-arginine. Venodilation in response to acetylcholine was impaired before, and corrected by, dialysis whereas venodilation to GTN was similar before and after dialysis. Venodilation in response to acetylcholine before dialysis was restored by co-infusion of L- but not D-arginine. Therefore, patients with ESRF undergoing hemodialysis have impaired acetylcholine-mediated venodilation consistent with the accumulation in ESRF of functionally important inhibitors of nitric oxide synthase that are cleared by dialysis.

Renal endothelial function in humans.

Animal experiments have shown extensively that the endothelial generation of nitric oxide participates in the regulation of renal haemodynamics and function, probably as a factor modulating the effects of endogenous vasoconstrictors. Although the endothelial function of the systemic vasculature is assessed through the vasodilatory effect of acetylcholine, only L-arginine infusion is available for studies of the influence of nitric oxide on the renal circulation in humans. In addition to peripheral vessels, L-arginine is a potent renal vasodilator, having no effect on the glomerular filtration rate; and the renal relaxation induced by L-arginine is markedly blunted in patients with essential hypertension. A few studies showing the renal vasoconstrictor effect of an inhibitor of nitric oxide synthesis have been reported.

Impaired insulin-induced attenuation of noradrenaline-mediated vasoconstriction in insulin-resistant obese Zucker rats.

1. Insulin resistance is associated with hypertension but the underlying mechanism is unclear. We tested the hypothesis that insulin-induced vasodilatation is impaired in insulin-resistant obese Zucker rats. We studied mesenteric artery (approximately 220 microns diameter) function before the development of hypertension in 3-month old obese Zucker rats and age-matched lean rats. 2. In vessels from lean rats, insulin at concentrations of 50, 500 and 5000 m-units/l attenuated the constriction in response to noradrenaline (50 m-units/l: 8 +/- 3%, P < 0.05; 500 m-units/l: 13 +/- 3%, P < 0.02; 5000 m-units/l: 13 +/- 2%, P < 0.02). 3. Vessels from obese rats failed to show any such response to insulin (2 +/- 6% increase in maximal tension with 5000 m-units/l; not significant), both in the presence and absence of L-arginine (3 mmol/l). 4. Vessels from obese rats showed slight but significant impairment in the vasodilator response to acetylcholine (5 x 10(-8)-10(-4) mol/l) (obese: 64.1 +/- 3.7% relaxation; lean: 77.3 +/- 3.7% relaxation; P < 0.05); however, relaxation in response to A23187 was not significantly different between the phenotypes (obese: 81.3 +/- 10.6% relaxation; lean: 79.1 +/- 9.7% relaxation; not significant). 5. Systolic blood pressure was not significantly different in lean (126 +/- 8 mmHg) and obese (127 +/- 7 mmHg) rats at the time of study (not significant). 6. We conclude that insulin-induced attenuation of noradrenaline-mediated vasoconstriction is impaired in the obese Zucker rat and that this defect precedes and therefore could contribute to the development of hypertension in this insulin-resistant model. The defect in insulin action could reside in the endothelial generation of nitric oxide, as endothelial function is also abnormal.

Potential cellular signaling mechanisms mediating upregulation of endothelial nitric oxide production by estrogen.

Experimental and clinical studies have provided ample evidence that estrogens exert a significant antiatherosclerotic effect and reduce morbidity and mortality due to cardiovascular diseases. The exact cellular mechanism of this vasculoprotective action of estrogen is not known, but recent work in our and other laboratories suggests that upregulation of endothelial nitric oxide (NO) production may significantly contribute to the mechanism. The vascular endothelium of female animals and humans produces more NO than that of males. Estrogen treatment significantly increases endothelial NO generation in ovariectomized animals and in postmenopausal women. Reduced endothelial NO production in the aorta of estrogen-receptor-deficient mice indicates that the nuclear estrogen receptor mediates the effect of estrogen. The most probable mechanism of estrogen-induced upregulation of endothelial NO production is the transcriptional stimulation of NOS III gene expression. However, the following alternative mechanisms may be involved as well: (1) inhibition of cytokine-induced downregulation of NOS III gene expression, (2) posttranslational modification of NOS III protein, (3) increased cofactor or L-arginine availability, (4) nongenomic activation of second messengers (e.g., Ca2+, cAMP) and tyrosine kinase, and (5) modulation of NO degrading systems (e.g., reactive oxygen radical generation and antioxidants). This paper reviews current data supporting these potential mechanisms.

Control of regional blood flow by endothelium-derived nitric oxide.

The regional hemodynamic consequences of inhibiting vascular endothelial nitric oxide generation with NG-monomethyl-L-arginine (L-NMMA) were studied in conscious Long-Evans rats. Experiments were carried out in groups of chronically instrumented rats with intravascular catheters and pulsed Doppler probes to monitor regional blood flow. L-NMMA (0.3-300 mg/kg) caused a dose-dependent, long-lasting (5-90 minutes), and enantiomerically specific increase in mean blood pressure and also caused bradycardia. The increase in blood pressure was accompanied by a dose-dependent and long-lasting vasoconstriction in the internal carotid, mesenteric, renal, and hindquarters vascular beds that could be attenuated, in a concentration-dependent manner, by L-arginine but not by D-arginine. In contrast, L-arginine did not affect the pressor or vasoconstrictor effects of vasopressin. These results indicate that nitric oxide production by vascular endothelial cells contributes to the maintenance of blood pressure and to the control of the resting tone of different vascular beds in the conscious rat.