Although in therapeutic use for more than a century, the mode of cellular action of organic nitrates remains incompletely understood. Despite ample experimental evidence from animal studies to show that nitrates are metabolized to NO in the vascular smooth muscle, direct demonstration of such an activity in human vascular cells is still lacking. Moreover, the role of the endothelium in modulating the pharmacodynamic action of nitrates is far from clear. We therefore aimed to investigate whether or not human endothelial cells are capable of bioactivating these drugs to NO and whether the amounts generated are sufficient to elicit any biological effects. Using cultured human umbilical vein endothelial cells (HUVECs) as an established model system a combination of three different methods was used to address this issue: (1) quantification of NO formation upon endothelial nitrate metabolism using the oxyhaemoglobin technique; (2) evaluation of the second messenger response using radioimmunoassay for cGMP; and (3) assessment of mechanism and extent of potentiation of the anti-aggregatory effect of nitrates in the presence of endothelial cells as a relevant bioassay. We now show that superfusion of cultured human endothelial cells on microcarrier beads with either glyceryl trinitrate (GTN) or isosorbide dinitrate (ISDN; both at 0.1-100 mumol L-1) results in a concentration-dependent formation of NO. NO generation from isosorbide 5-mononitrate (IS-5-N) was below the detection limit. The amounts of NO produced (maximally 2.97 +/- 0.98 pmoles NO min-1 x mg protein with 100 mumol L-1 GTN; n = 8) were similar to those elicited upon challenge of the cells with 100 nM bradykinin. NO formation from either organic nitrate was accompanied, in a concentration-dependent and methylene blue-inhibitable manner, by stimulation of endothelial soluble guanylyl cyclase with consequent increases in the intracellular level of cGMP (maximally 32-fold over basal levels with ISDN), a significant portion of which was released into the extracellular space. Upon continuous 30 min superfusion or repeated application of high concentrations of GTN (100 mumol L-1) nitrate bioactivation to NO was subject to partial tachyphylaxis. Co-incubation of washed human platelets with HUVECs potentiated the anti-aggregatory action of nitrates in a cell number dependent and oxyhaemoglobin-sensitive manner and this effect, too, was accompanied by increases in intraplatelet cGMP levels. The potentiating effect was largely inhibited after blockade of sulfhydryl groups by pre-incubation of HUVECs with N-ethylmaleimide and completely abrogated after pretreatment of cells with the tissue fixative glutaraldehyde. These results demonstrate that human endothelial cells are capable of bioactivating organic nitrates to NO by an enzymatic, apparently thiol-sensitive pathway, in quantities sufficient to influence endothelial and platelet function. Besides the well known vasorelaxant action of organic nitrates, which is mainly due to their metabolism in the smooth muscle compartment, these drugs may therefore be endowed with a hitherto underestimated potential to directly influence endothelial functions via the NO/cGMP pathway. Through specific bioactivation in the endothelium itself organic nitrates can thus mimic and reinforce protective functions normally served by a functional endothelium such as the modulation of blood cell/vessel wall interactions and inhibition of cell proliferation.
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[Show abstract][Hide abstract] ABSTRACT: To assess the dilatory effectiveness of nitric oxide donors in resistance arteries of patients with arterial hypertension in comparison with that in those of normotensive controls.
Endothelium-dependent vasodilation has been demonstrated to be impaired in arterial hypertension. Besides disturbances in endothelial nitric oxide production a reduced vasodilatory effectiveness of nitric oxide might contribute to this phenomenon of endothelial dysfunction. We therefore investigated the dilatory responsiveness of resistance arteries to exogenous nitric oxide by means of administration of the nitric oxide donors glycerol trinitrate (GTN), isosorbide dinitrate (ISDN) and sodium nitroprusside (SNP) in hypertensive patients.
Forearm blood flow was measured by venous occlusion plethysmography at rest and during intra-arterial infusion of nitric oxide donors at increasing doses in 11 patients with arterial hypertension and in 10 age-matched normotensive controls.
Forearm blood flow at rest was comparable in the two groups and was dose-dependently increased by administration of either nitric oxide donor. In patients with arterial hypertension, blood flow responses to infusions of organic nitrates were significantly impaired over the entire dose-response curve compared with those of normotensive controls (220 nmol/min GTN 13.1 +/- 1.3 and 8.6 +/- 0.3 ml/min per 100 ml tissue; 212 nmol/min ISDN 9.9 +/- 0.7 and 5.8 +/- 1.0 ml/min per 100 ml tissue). Blood flow responses to infusion of the nitric oxide donor SNP were also profoundly impaired in the hypertensive patients, the extent of which impairment equalled that found with the organic nitrates. Within the entire set of normotensive and hypertensive subjects, maximal flow responses to either nitric oxide donor were inversely correlated with mean arterial blood pressure.
Dilation of resistance arteries in response to infusion of nitric oxide donors is impaired in hypertensive patients and the degree of this impairment depends critically on the severity of arterial hypertension. The reduced effectiveness of nitric oxide appears to be independent of the class of nitric oxide donor and thus of the mode of intravascular nitric oxide generation. These findings are likely to have important implications not only for our understanding of the pathophysiological mechanisms of endothelial dysfunction but also for nitric oxide donor therapy in arterial hypertension.
Full-text · Article · Aug 1996 · Journal of Hypertension
[Show abstract][Hide abstract] ABSTRACT: Organic nitrates possess important antiplatelet actions that are useful in the treatment of unstable angina and myocardial infarction, but the susceptibility of platelets to nitrate tolerance has not been extensively studied. In normal conscious rats, we showed that continuous infusion of nitroglycerin (NTG) at 10 micrograms/min had no significant effect on mean arterial pressure (MAP) as compared with control, but hemodynamic tolerance could be demonstrated by MAP response to a bolus intravenous (i.v.) NTG challenge. By this criterion, continuous 8-h NTG infusion produced hemodynamic tolerance (a decrease in MAP response of 45.7 +/- 19.9%, p < 0.05), whereas D5W control and S-nitroso-N-acetylpenicillamine (SNAP) infusions did not. During NTG infusion, platelet-rich plasma (PRP) cyclic GMP was increased by 41.4 +/- 13.6% as compared with control and remained increased throughout the infusion (p < 0.05). Bleeding time during a 2-h infusion of NTG was 8.9 +/- 1.2 min as compared to 3.8 +/- 0.4 min in controls (p < 0.05). After 8-h of NTG infusion, the bleeding time was 10.2 +/- 1.4 min versus 4.4 +/- 0.4 min in controls (p < 0.05). NTG also decreased the PRP platelet concentration by 30% in 8 h, whereas D5W had no effect. In vitro experiments showed that platelets in themselves do not produce significant amounts of cyclic GMP. These data indicate that the biochemical and antiaggregation effects of NTG on platelets are not diminished during hemodynamic tolerance and that these effects may be dependent on extraplatelet production of nitric oxide (NO).
No preview · Article · Oct 1996 · Journal of Cardiovascular Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Intravenous infusion of glyceryl trinitrate (GTN) into migraineurs induces an immediate headache followed by migraine. We studied the effect of GTN (0.25 microgram kg-1 min-1) on local cerebrovascular laser Doppler flux (rCBFLDF), artery diameter and NO concentration (selective NO microelectrode) in the pial middle cerebral artery perfusion territory of the anaesthetized cat, at rest and during cortical spreading depression (SD). GTN infusion induced a significant increase in pial artery diameter, rCBFLDF, and NO concentration. Following termination of infusion, NO concentrations remained significantly elevated above controls for 60 min, other parameters returned to baseline within 10 min (p < 0.05, ANOVA, post hoc Dunnett's multiple comparison procedure). Two hours after termination of infusion KCl-evoked SD was initiated. GTN-treated animals exhibited significantly (p < 0.05, Kruskal-Wallis) elevated SD-induced NO release compared to controls. All other parameters remained unaffected. Our results demonstrate that GTN induces a prolonged increase in local NO concentrations and enhances SD-induced NO release.