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Nitric oxide (NO) is known to counteract apoptosis by S-nitrosylation of protein thiol groups. NO is generated and stored in erythrocytes, which may undergo eryptosis, a suicidal cell death similar to apoptosis of nucleated cells. Eryptosis is triggered by increased cytosolic Ca2+ activity and/or ceramide and characterized by cell shrinkage and phosphatidylserine exposure at the cell surface. The present study explored whether nitric oxide could interfere with the machinery underlying eryptosis. To this end, erythrocyte phosphatidylserine exposure (annexin V-binding) and cell volume (forward scatter) were determined by flow cytometry. The Ca2+ ionophore ionomycin (0.1 μM) increased cytosolic Ca2+ activity, triggered annexin binding, and decreased forward scatter. The annexin binding and decrease of forward scatter but not the increase of cytosolic Ca2+ activity were reversed by the NO-donor nitroprusside (1 μM) and papanonoate (100 μM). Higher concentrations of nitroprusside (0.1 and 1 mM) stimulated eryptosis. Glucose depletion, exposure to C6-ceramide (3 μM), hypertonic (addition of 550 mM sucrose), and isotonic (replacement of Cl− with gluconate) cell shrinkage all triggered annexin V binding, effects all reversed by nitroprusside (1 μM). Dibutyryl–cGMP (1 mM) blunted the ionomycin- but not the ceramide-induced annexin V binding. Ionomycin decreased protein nitrosylation and thioredoxin activity, effects reversed by the NO-donor papanonoate. Clearance of erythrocytes from circulating blood was significantly faster in eNOS knockout mice than in their wild-type littermates. In conclusion, nitric oxide participates in the regulation of erythrocyte survival, an effect partially mimicked by cGMP and paralleled by alterations of protein nitrosylation and thioredoxin activity. [ABSTRACT FROM AUTHOR]

In this study, we investigated the effects of prolonged administration of the selective COX-2 inhibitors celecoxib and rofecoxib and the non-selective NSAID naproxen on the initiation and progression of atherosclerosis. ApoE−/− mice, as well as corresponding wild-type mice, were fed either a normal chow or a high fat Western diet with or without addition of the respective drugs over a period of 16 weeks. Thereafter, aortic lesion size, plasma lipid levels, and COX-2 expression in the plaques were determined. The results showed that neither the COX-2 selective inhibitors nor naproxen had a significant impact on the initiation and progression of atherosclerosis in diet-fed ApoE−/− mice, although both celecoxib and rofecoxib showed a tendency to reduce plaque size. This slight effect may be due to selective inhibition of COX-2 activity because the COX-2 expression was not altered in the plaque. Plasma lipid levels were also not significantly influenced by these drugs. Interestingly, in ApoE−/− mice that have been fed with normal chow, we found an increased incidence of plaque formation after treatment with celecoxib and rofecoxib, indicating that coxibs may promote the initiation of atherosclerosis. This effect was probably masked in diet-fed mice by the more pronounced effects of the high cholesterol diet. In conclusion, the reduction in diet-induced plaque size in animals fed a high fat diet and the promotion of atherosclerosis in mice on a normal diet indicate a dual role of the coxibs. In advanced stages of atherosclerosis, they may exert antithrombotic properties due to their COX-2 inhibiting activity, whereas in very early stages they may favor the initiation of atherogenesis. However, because these results were only observed in ApoE−/− and not in wild-type animals, coxibs may increase the risk of thrombosis in patients with a predisposition for thrombotic complications. [ABSTRACT FROM AUTHOR]

In the present study we determined whether the endothelium-derived hyperpolarizing factor (EDHF), in addition to its acute effects on vascular tone, activates intracellular signalling pathways other than those associated with Ca2+-dependent K+ channels. EDHF was generated by rhythmic distension of porcine coronary arteries under conditions of combined nitric oxide (NO) synthase/cyclo-oxygenase blockade, and the EDHF-containing luminal incubate was applied to cultured human coronary endothelial or smooth muscle cells. In both cell types, the luminal incubate activated tyrosine kinases, the mitogen-activated protein (MAP) kinases, extracellular signal regulated kinases 1 and 2 (Erk1/2) and p38, as well as protein kinase B/Akt. The constituent responsible for Erk1/2 phosphorylation was identified as a cytochrome P450 (CYP) metabolite, as Erk1/2 activation was attenuated by pretreating the EDHF donor with the CYP 2C inhibitor sulfaphenazole as well as by CYP 2C antisense oligonucleotides. Erk1/2 phosphorylation in detector cells was also observed following the transfer of supernatant from cultured endothelial cells treated with the CYP inducer β-naphthoflavone. The CYP 2C product 11,12-epoxyeicosatrienoic acid (11,12-EET) also activated tyrosine kinases, Erk1/2 and p38 MAP kinase. Overexpression of CYP 2C8 in native porcine coronary artery endothelial cells resulted in an increase in endothelial 11,12-EET production and Erk1/2 phosphorylation compared to that detected in untreated cells or cells transfected with an antisense CYP 2C8. Endothelial cell number was unaffected by transfection with LacZ or CYP 2C8 antisense but was significantly enhanced in cells overexpressing CYP 2C8. These observations indicate that EDHF/11,12-EET is not simply a vasodilator and that its continuous release under pulsatile conditions in vivo may affect vascular cell signalling and proliferation. [ABSTRACT FROM AUTHOR]

Presents a study on endothelium-derived hyperpolarizing factor (EDHF) synthase. The induction of cytochrome P450 (CYP) 2C8/34 in native porcine coronary artery endothelial cells by beta-naphthoflavone enhancing the formation of 11,12-epoxyeicosatrienoic acid as well as EDHF-mediated hyperpolarization and relaxation; Implications.

Presents research which demonstrated that the serine/threonine protein kinase Akt/PKB mediates the activation of endothelial nitric oxide synthase (eNOS). Role of NO produced by eNOS in cardiovascular homeostasis; Viscous drag, or shear stress, and the formation of nitric oxide; Impact of shear-stress-mediated phosphorylation of eNOS; Factors in serine phosphorylation and the activation of eNOS; Impact of mimicking the phosphorylation of Ser 1177; Regulation of the activation of eNOS.

We studied the effect of endothelium-derived relaxing factor (EDRF) on norepinephrine-induced contractile responses and on the tissue guanosine-3′,5′-phosphate (cGMP) concentration of isolated rabbit arteries with an increasing endothelium to smooth muscle cell ratio (aorta, femoral and mesenteric arteries). After suppression of EDRF formation (either by N-nitro- l-arginine or, in mesenteric arteries, by saponin), contractions elicited by cumulative doses of norepinephrine were unaltered in aorta but were enhanced by 22.5% in femoral arteries and by 44.3% in mesenteric arteries (at the highest norepinephrine concentration). The cGMP concentration (pmol/mg protein) of unstimulated, endotheliumintact vessels decreased after suppression of EDRF formation from 1.09±0.24 to 0.74±0.28 in aortic, from 2.86±0.4 to 0.61±0.19 in femoral and from 6.3±0.9 to 0.7±0.15 in mesenteric arterial segments. The basal cGMP concentration did not differ in endothelium-denuded segments of these arteries, suggesting a similar basal activity of soluble guanylate cyclase (sGC). A higher sensitivity of sGC may have contributed to the higher cGMP concentration observed in the smaller arteries, since in the presence of sodium nitroprusside the cGMP concentration of endothelium-denuded segments increased 1.8-fold in aortic, 2.9-fold in femoral and 2.4 fold in mesenteric arterial segments. However, these differences in sGC activation cannot be solely responsible for the high basal cGMP concentration in endotheliumintact mesenteric arteries. The greater ratio of endothelium to smooth muscle cell layers in the smaller arteries might result in a higher EDRF concentration in the vascular wall and subsequently in a higher cGMP concentration. In conclusion, these data support the view of a greater importance of EDRF-mediated vascular control in small arteries than in large conduit arteries. [ABSTRACT FROM AUTHOR]

The effects of a recently described inhibitor of endothelial NO synthesis, N-nitro- l-arginine ( l-NNA), on the vasomotor responses to endothelium-dependent and independent vasodilators, and on the release of endothelium-derived relaxing factor (EDRF), were studied in the isolated saline-perfused rabbit heart. Infusion of l-NNA (30 μM) resulted in a 52±12% increase in basal coronary perfusion pressure. The vasomotor responses to 1 μM acetylcholine (ACh) and serotonin after l-NNA became biphasic, showing a small transient dilation followed by a pronounced vasoconstriction. In contrast, the dilation observed with sodium nitroprusside was not affected by l-NNA. None of the above-mentioned effects was elicited by the Stereo-isomer d-NNA. Similarly, an increase in the basal coronary perfusion pressure by endothelin-1 (0.3 nM) to the same level as observed with l-NNA did not alter the vasomotor responses to ACh and sodium nitroprusside. The increase in cyclic GMP (cGMP) in platelets passing through the coronary vascular bed was used as an index of EDRF release. Platelet cGMP amounted to 0.50±0.10 pmol/mg protein after passage through the coronary bed of the unstimulated heart. When platelets were injected during an ACh infusion (1 μM), a 2.7 fold increase in cGMP was observed ( P<0.01). After a 30-min infusion with l-NNA, the cGMP content of platelets passing through the unstimulated heart was reduced by 62%. Likewise, the ACh-induced increase in platelet cGMP was totally blocked. These results show that l-NNA inhibits EDRF release, and is thus a potent and selective inhibitor of EDRF-mediated dilation in the isolated rabbit heart. [ABSTRACT FROM AUTHOR]

We studied the role of the membrane potential in the control of the intracellular free calcium concentration ([Ca]) and release of the two autacoids endothelium-derived relaxing factor (EDRF = nitric oxide) and prostaglandin I in endothelial cells. ATP (3 μmol/l) and bradykinin (1 nmol/l) evoked rapid increases (sixfold) in [Ca] in cultured endothelial cells. [Ca] remained elevated over several minutes. When the cells were depolarized, either by K (70-90 mmol/l) or by preincubation with the blocker of K channels tetraethylammonium (3 mmol/l), the initial peak of [Ca] remained unaffected but [Ca] returned significantly faster to resting levels, indicating a reduction in Ca influx. In native, freshly isolated endothelial cells, K abolished increases in [Ca] induced by acetylcholine (3 μmol/l). Release of EDRF in response to bradykinin (cultured cells) and acetylcholine (native cells) was inhibited by K (by 70%), whereas release of prostaglandin I was not significantly reduced. Preincubation of cultured endothelial cells with the receptor-independent stimulus thimerosal (5 μmol/l, 40 min) evoked a long-lasting release of EDRF and small elevations of [Ca] (twofold) after washout of the drug. Depolarization with K decreased thimerosal-induced EDRF release and [Ca] in a reversible manner. In patch-clamped endothelial cells, bradykinin (1 nmol/l) induced transient hyperpolarizations that were significantly prolonged by BRL 34915 (1 μmol/l), an activator of K channels. BRL 34915 also elicited increases in [Ca], particularly in thimerosal-stimulated endothelial cells. These effects were abolished by K. We conclude that the initial rise in [Ca] in response to receptor-binding agonists, caused by mobilization of Ca from intracellular stores, activates K channels, thereby inducing hyperpolarization. This hyperpolarization provides the driving force for transmembrane Ca influx into endothelial cells and is thus an important signal for synthesis and release of EDRF. [ABSTRACT FROM AUTHOR]

We studied the cellular mechanism involved in the desensitization of cultured endothelial cells to bradykinin. Bradykinin (10 nmol/l) evoked a rise in the intracellular free calcium concentration ([Ca]), measured with the fluorescent probe indo-1, from 137±30 (±SEM) to 623±101 nmol/l. Cells were desensitized to bradykinin by repetitive stimulation with the peptide over 10 min, after which they no longer responded to bradykinin. However, purinergic stimulation with ATP (10 μmol/l) elicited the same increase in [Ca] in endothelial cells desensitized to bradykinin as in cells never exposed to bradykinin. The initial peak of [Ca] after stimulation with bradykinin or ATP was not affected by removal of extracellular calcium ions, indicating mobilization of Ca from intracellular stores. Since GTP-binding proteins (G-proteins) are probably involved in the receptor-mediated stimulation of endothelial cells, we also tested the effects of sodium fluoride (NaF), a reported direct stimulator of G-proteins, on endothelial [Ca]. NaF (5 mmol/l) increased [Ca] to 412±88 nmol/l in control cells and was equally effective in cells desensitized to bradykinin. We conclude that the homologous desensitization to bradykinin does not occur at the level of intracellular signal transduction but at the level of membrane receptors. [ABSTRACT FROM AUTHOR]

In addition to nitric oxide (NO) and prostacyclin (PGI) another endothelium-derived factor, which hyperpolarizes vascular smooth muscle cell via activation of K channels, contributes to the vasorelaxant effect of bradykinin in different vascular beds. Preliminary findings suggest that this endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation is attenuated by both volatile and intravenous anesthetics. Since EDHF may play an important role in the coronary microcirculation, we investigated the effects of isoflurane (2 vol.% equivalent to ∼ 250 μM), etomidate (30 and 100 μM), phenobarbital (100 μM) and thiopental (30 and 100 μM) on the EDHF-mediated dilator response to bradykinin and on the endothelium-independent dilatation evoked by sodium nitroprusside (SNP) in the isolated salineperfused rat heart (Langendorff preparation). None of the anesthetics tested affected the dilator response to bradykinin or SNP under basal conditions. However, following inhibition of NO and PGIZ formation with N-nitro-L-arginine (100 μM) and diclofenac (1 μM) respectively, isoflurane, etomidate and thiopental, but not phenobarbital, significantly attenuated the NO/PGI-independent, i.e. EDHF-mediated dilator response to bradykinin, while the vasorelaxant effect of SNP remained unaffected. Isoflurane, etomidate and thiopental, but not phenobarbital, display cytochrome P450-inhibiting properties, suggesting that these anesthetics impair the cytochrome P450-dependent synthesis of EDHF in the coronary microcirculation. [ABSTRACT FROM AUTHOR]

The vasodilator effects of C92-4609 (4-hydroxymethyl-furoxan-3-carboxamide, CAS 1609), C92-4678(4-phenyl-furoxan-3-carboxylic acid (pyridyl-3-yl-methyl)-amide), C92-4679 (3-phenyl-furoxan4-carboxylic acid (pyridyl-3-yl-methyl)-amide) and C93-4759 (3-hydroxymethyl-furoxan-4-carboxamide) were studied in the isolated rabbit femoral artery and jugular vein. All furoxans were potent vasodilators in the femoral artery (EC 0.1-50 μM), while they were less potent in the jugular vein by at least one order of magnitude. Apart from C92-4679, the vasodilatory potency of the furoxans correlated well with their nitric oxide (NO)-releasing capacity which was estimated both by stimulation of purified soluble guanylyl cyclase activity and electron spin resonance spectroscopy with a trapping agent for NO. The hypothesis that furoxans stimulate soluble guanylyl cyclase in the smooth muscle by spontaneously releasing NO was supported by the marked attenuation of their vasodilator effect in the presence of oxyhaemoglobin (10 μM) or following treatment with methylene blue (30 μM). In contrast to earlier findings, NO release from these furoxans was not thiol-dependent, as demonstrated for C92-4609, the relaxant effect of which in the femoral artery was not altered in the presence of N-acetyl- l-cysteine (1 MM). Moreover the Kchannel inhibitor, tetrabutylammonium (3 mM), but not the Kchannel inhibitor, glibenclamide (3 μM), significantly attenuated the dilator response to C92-4679 in the femoral artery. Pretreatment of these segments with the cytochrome P450 inhibitor, SKF525a (30 μM), also reduced the C92-4679-induced relaxation in this vascular bed. The vasorelaxant activity of C92-4679 thus appears to be based both on the spontaneous release of NO which is accelerated due to biotransformation within the vessel wall, and a direct interaction with K channels in the smooth muscle. In the rabbit jugular vein, the dilator response to C92-4609 was not affected following exposure to glyceryl trinitrate (1 mM) for 1 hour or pretreatment with the same concentration of C92-4609 or its isomer, C93-4759. This lack of tolerance development together with their pharmacological potency may render the new generation of furoxans useful drugs in the treatment of coronary heart disease. [ABSTRACT FROM AUTHOR]

The molecular mechanism(s) underlying the vasodilator activity of CAS 936 (3-(cis-2,6-dimethylpiperidino)-N-(4-methoxybenzoyl)-sydnonimine) and its metabolites 3-(cis-2,6-dimethylpiperidino)-sydnonimine (C87 3754) and N-(cis-2,6-dimethylpiperidino)-N-nitroso-2-aminoacetonitril (C873786) was investigated. These compounds were tested for their relaxant activity in isolated rabbit arterial segments, activation of purified soluble guanylyl cyclase and release of nitric oxide (NO) in vitro and in vivo. C873754 and C873786 inhibited the noradrenalin-induced contraction and increased the cyclic GMP content of endothelium-denuded rabbit aortic and femoral segments, whereas CAS 936 was without effect. Similarly, both metabolites, but not CAS 936, activated purified soluble guanylyl cyclase (EC about 30 μM) and released NO in buffered aqueous solutions, as detected by electron spin resonance (esr) spectrometry. Both in vitro and in vivo an accumulation of NO was detected by esr spectrometry in vascular tissues exposed to the metabolites of CAS 936, whereas a significant release of NO from CAS 936 was only detected in the isolated rabbit liver, but not in vascular tissue. It is conceivable, therefore, that the metabolites of CAS 936 appearing in the systemic circulation after hepatic biotransformation induce vasodilatation by release of NO and activation of soluble guanylyl cyclase in vascular smooth muscle. Moreover, the activation of soluble guanylyl cyclase in vitro by the metabolites of CAS 936 was significantly enhanced by co-incubation with certain particulate fractions from bovine aortic endothelial and smooth muscle cells. Thus, an enzymic release of NO from these metabolites in addition to their spontaneous decomposition may play a significant role for their vasodilator activity in vivo. [ABSTRACT FROM AUTHOR]

The influence of endothelium-derived nitric oxide (EDNO) on relaxation induced by the nitrovasodilators, sodium nitroprusside and sodium nitrite was assessed in phenylephrine-stimulated hamster thoracic aortas, a preparation that displays significant basal release of EDNO. Removal of the endothelium or treatment with the NO synthase inhibitors, N-nitro- l-arginine (L-NAG, 10-30 µM) or N-methyl- l-arginine (L-NMMA; 100 µM) increased the potency and, except for sodium nitroprusside in endothelium-denuded segments, also increased the efficacy of the nitrovasodilators. Removal of the endothelium had no effect on relaxations induced by isoproterenol, an indication that these effects were specific for the nitrovasodilators. Removal of the endothelium, treatment of endothelium-intact preparations with L-NAG or L-NMMA, or exposure of these vessels to the guanylate cyclase inhibitor, methylene blue (10 µM) increased reactivity of the aortas to the guanosine 3′:5′-cyclic monophosphate (cGMP) analogue, 8-Br cGMP. Measurement of cGMP revealed that endothelium-intact segments had a 6.5 fold higher level of cGMP than endothelium-denuded preparations and that sodium nitroprusside increased cGMP in both preparations by similar amounts in a concentration-dependent fashion. Exposure of endothelium-denuded or L-NAG-treated segments to sodium nitroprusside, to mimic the effects of basally released EDNO, depressed sodium nitrite and 8-Br cGMP reactivity in a manner similar to endothelium-intact segments. These data indicate that EDNO increases cGMP levels in vascular smooth muscle and that the elevated cGMP levels depress nitrovasodilator and 8-Br cGMP reactivities. [ABSTRACT FROM AUTHOR]

The effects of angiotensin-converting enzyme (ACE) inhibitors on intracellular calcium concentration ([Ca]) were examined under resting conditions and after stimulation with bradykinin in cultured human umbilical vein endothelial cells. The ACE inhibitors ramiprilat and enalaprilat (0.3 μM) enhanced the increase in [Ca] elicited by bradykinin (3 nM) and also caused an increase in resting [Ca] when given alone. This increase in resting [Ca] was long-lasting and accompanied by an increased formation of nitric oxide, as assessed by a N-nitro- l-arginine-sensitive cyclic GMP accumulation in the cells. Both increases in resting [Ca] and nitric oxide production by ACE inhibitors were inhibited by preincubation of the cells with the B-receptor antagonist Hoe 140. These data indicate that ACE inhibitors are able to unmask a release of bradykinin from cultured human endothelial cells. This endothelium-derived bradykinin can exert an autocrine function by stimulating endothelial B-receptors with a subsequent increase in [Ca] and nitric oxide formation. [ABSTRACT FROM AUTHOR]

Ca-calmodulin dependent phosphorylation of myosin is essential for the induction of platelet shape change and subsequent reactions. Therefore, we studied the effects of the calmodulin antagonists fendiline and calmidazolium on the thrombin-induced aggregation, secretion of ATP, and increases in the intracellular free calcium concentration ([Ca]) in washed human platelets in the absence and presence of extracellular Ca. In Ca free medium, fendiline (10-100 μM) and calmidazolium (3-30 μM) concentration-dependently inhibited aggregation. The effect of fendiline could be partly reversed by extracellular Ca and higher thrombin concentrations. Furthermore, aggregations induced by the calcium ionophore ionomycin and by the protein kinase C-activator 4-β-phorbol 12-myristate 13-acetate were inhibited by fendiline, although to a smaller degree than the thrombin-induced aggregation. Thrombin-induced secretion of ATP was attenuated by low concentrations of fendiline (1-30 μM) and calmidazolium (1 μM) but enhanced by higher concentrations (10-30 and 3-10 μM, respectively), independently of extracellular Ca. Fendiline (1-10 μM) did not affect [Ca] in resting and thrombin-stimulated platelets. At higher concentrations (30-100 μM), it induced increases in [Ca] in unstimulated platelets and attenuated the response to thrombin in Ca free medium, whereas thrombin-induced Ca influx was markedly enhanced. Similar results were obtained with calmidazolium (1-3 μM). These stimulating effects on ATP secretion and on [Ca] of fendiline and calmidazolium may be attributed to interactions with platelet membranes by which the permeability of small cations is increased. In contrast to these actions that would be normally considered platelet-activating, our study demonstrates that the two calmodulin antagonists effectively antagonize [Ca] mediated induction of platelet aggregation. [ABSTRACT FROM AUTHOR]

Ca influx into stimulated endothelial cells is attenuated by depolarization. We hypothesized that Ca influx is driven by the membrane potential and may be enhanced by hyperpolarizing drugs like activators of K channels. Therefore we studied the effects of pinacidil, cromakalim, and cicletanine on membrane currents and on the intracellular free calcium concentration ([Ca]i) in cultured endothelial cells from porcine aorta. In patch-clamped cells, pinacidil (1 μmol/l) and cromakalim (1 μmol/l) elicited outward currents carried by K and significantly prolonged the Ca-dependent K currents induced by bradykinin and ATP. Peak currents in response to bradykinin were not affected. In cells loaded with the fluorescent Ca indicator indo-1 and prestimulated with thimerosal, pinacidil (0.1-1 μmol/l elicited long-lasting increases in [Ca) from 100 ± 10 to 550 ± 110 nmol/l. These effects were completely abolished in a medium containing 90 mmol/l K. Similar results were obtained with cromakalim. Likewise, in cells stimulated with bradykinin, pinacidil raised [Ca] when applied during the decline of [Ca] after the initial peak. Cicletanine elicited K currents in resting and attenuated K currents in bradykinin-stimulated cells. It elevated [Ca] even in the absence of extracellular Ca and in K-rich medium. Hence, the effects of cicletanine cannot be explained by direct actions on K channels. However, our studies demonstrate that pinacidil and cromakalim elevate [Ca] secondary to their activation of K channels by inducing hyperpolarization and augmenting the driving force for potential-dependent Ca influx. In this way, the two drugs may promote Ca-dependent formation of endothelium-derived relaxing factor. [ABSTRACT FROM AUTHOR]

We studied the effects of the l-arginine analogue N-nitro- l-arginine ( l-NNA), in comparison with its d-isomer ( d-NNA), on endothelium-dependent dilations of rabbit femoral arteries (RFA) and on the release of endothelium-derived relaxant factor (EDRF) from native and cultured endothelial cells. In addition, we examined the effects of l- and d-NNA on the l-arginine- and NADPH-dependent synthesis of nitric oxide (NO) in the cytosol of porcine aortic endothelial cells. l-NNA enhanced the noradrenaline-induced contraction of endothelium-intact, but not of endothelium-denuded segments of RFA, indicating an inhibition of basal EDRF release. l-NNA also inhibited significantly the endothelium-dependent dilations to acetylcholine (ACh). Both effects of l-NNA were attenuated by l-arginine. l-NNA rapidly inhibited the release of EDRF from cultured and native endothelial cells stimulated with thimerosal or ACh. l-NNA concentration-dependently and reversibly antagonized the l-arginine- and NADPH-dependent activation of a purified soluble guanylate cyclase (GC) by cytosol from.Sreshly harvested porcine aortic endothelial cells, suggesting a direct competition between l-NNA a l-arginine at the level of endothelial NO-synthesis. d-NNA was ineffective in all instances. These results prove l-NNA to be a stereospecific inhibitor of the cytosolic NO formation from l-arginine in endothelial cells. Therefore, l-NNA will be a useful tool to elucidate the molecular mechanism of mammalian NO synthesis. [ABSTRACT FROM AUTHOR]

Release of nitric oxide (NO) from endothelial cells critically depends on a sustained increase in intracellular free calcium maintained by a transmembrane calcium influx into the cells. Therefore, we studied whether the free cytosolic calcium concentration directly affects the activity of the NO-forming enzyme(s) present in the cytosol from freshly harvested porcine aortic endothelial cells. NO was quantified by activation of a purified soluble guanylate cyclase coincubated with the cytosol. In the presence of 1 mM L-arginine, 0.1 mM NADPH and 0.1 mM EGTA, endothelial cytosol (0.2 mg of cytosolic protein per ml) stimulated the activity of guanylate cyclase 5.0 + 0.5-fold (from 31 + 9 to 153 + 15 nmol cyclic GMP formed per min per mg guanylate cyclase). Calcium chloride increased this stimulation further in a concentration-dependent fashion by up to 136 + 15% (with 2 μM free calcium; EC 0.3 μM). The calcium-dependent and -independent activation of guanylate cyclase was enhanced by superoxide dismutase (0.3 μM) and was inhibited by the stereospecifically acting inhibitor of L-arginine-dependent NO formation N-nitro- L-arginine (1 mM) and by LY 83583 (1 μM), a generator of superoxide anions. Our findings suggest a calcium-dependent and -independent synthesis of NO from L-arginine by native porcine aortic endothelial cells. [ABSTRACT FROM AUTHOR]

Tolerance to the cyclic GMP-mediated vasodilator action of nitroglycerin develops with prolonged exposure and may be mediated either by formation of less nitric oxide from nitroglycerin or by desensitization of soluble guanylate cyclase to activation with nitric oxide. In the latter case, smooth muscle cells tolerant to nitroglycerin should show cross-tolerance to nitric oxide released from sydnonimines and endothelial cells (endothelium-derived relaxing factor). Therefore cultured smooth muscle cells from rabbit aorta were pretreated for 1 h with vehicle or high concentrations (0.55 mM) of nitroglycerin or the sydnonimine SIN-1. The formation of cyclic GMP induced by subsequent small doses of nitroglycerin, sydnonimine SIN-1 and endothelium-derived relaxing factor (released from cultured endothelial cells) was compared with the changes in activation of soluble guanylate cyclase, cyclic GMP formation and vasodilation in response to the same stimuli in similarly pretreated segments from rabbit thoracic aortae. Both cultured and native smooth muscle cells remained responsive to stimulation with sydnonimine SIN-1 and endothelium-derived relaxing factor after pretreatment with nitroglycerin, vehicle, or sydnonimine SIN-1, even though they were tolerant to nitroglycerin after pretreatment with nitroglycerin. In contrast, activation of soluble guanylate cyclase by nitroglycerin and sydnonimine SIN-1 was attenuated in homogenates of nitrate-tolerant aortae. The findings suggest that nitroglycerin tolerance in intact cells does not involve desensitization of soluble guanylate cyclase, because in intact cells nitrate tolerance can be overcome by direct activators of soluble guanylate cyclase. [ABSTRACT FROM AUTHOR]

Acetylcholine elicits an endothelium-dependent hyperpolarization of vascular smooth muscle cells. The experiments reported here tested the hypothesis that this hyperpolarization is mediated by the endothelium-derived relaxant factor (EDRF) identified as nitric oxide. Membrane potential was recorded with standard glass microelectrodes in smooth muscle cells in segments of rabbit femoral arteries. In endothelium-intact vessels, smooth muscle cells (resting potential: −67.0 ± 1.3 mV) hyperpolarized significantly ( P < 0.001) by 5.7 ± 0.9 mV in response to acetylcholine (1 μM). Inhibition of EDRF, either in the presence of hemoglobin or by pretreatment with gossypol, attenuated the relaxation elicited by acetylcholine in endothelium-intact segments precontracted with 0.1 μM noradrenaline but had no significant effect on either the control membrane potential (−62.2 ± 1.9 mV and −68.5 ± 2.1 mV, respectively) or the hyperpolarization in response to acetylcholine (5.0 ± 1.6 mV and 5.8 ± 1.6 mV, respectively). In contrast, in vessel segments with the endothelium removed, the hyperpolarization in response to acetylcholine was abolished although the control membrane potential (−68.0 ± 5.1 mV) was not significantly different from that in endotheliumintact vessels. Sodium nitroprusside, an endothelium-dependent vasodilator and exogenous analog of EDRF, also had no significant effect on membrane potential. The lack of response to acetylcholine was not merely the result of nonspecific damage to the smooth muscle cells: vessel segments without endothelium were still able to hyperpolarize in response to various other vasodilators. These results suggest that the endothelium-dependent hyperpolarization of vascular smooth muscle cells in response to acetylcholine is not mediated by EDRF. [ABSTRACT FROM AUTHOR]

In normotension the endothelium produces mainly nitric oxide (NO) and prostacyclin, and the vasodilator and growth inhibitory influence predominates. Hypertension, however, is associated with a shift towards enhanced constriction and vascular hypertrophy. These effects are associated with an apparent decrease in the production of bioactive NO and concomitant increase in the generation of oxygen-derived free radicals, such as superoxide anions (O2-). While the enzymatic source of endothelial O2-has been debated intensely over the past few years, it may well turn out that the endothelial NO synthase is itself an important producer of O2-. Because the redox state of endothelial cells and, for example, the activation of redoxsensitive transcription factors is regulated by the balance between NO and O2-production, endothelial NO synthase may well be the most crucial enzyme determining the anti- or prohypertensive and eventually proatherogenic state of the vascular wall. [ABSTRACT FROM AUTHOR]

Coronary vascular endothelial cells control vascular tone by modulating the local concentration of circulating vasoactive substances (e.g. adenine nucleotides, biogenic amines and bradykinin) and by synthesising and releasing the vasoactive autacoids nitric oxide (NO) and prostacyclin (PGI). The fluid shear stress exerted by the streaming blood is the physiologically most important stimulus for a continuous endothelial NO production, which counteracts neuro- and myogenic constriction. This shear stress-dependent NO release represents a highly effective local system for maintaining adequate blood flow to the myocardial tissue. At the transcriptional level endothelium-derived NO modulates the regulation of a number of genes (e.g. monocyte chemoattractant protein-1, P-selectin and vascular cell adhesion molecule-1) most probably by direct and/or indirect interaction with transcription factors. In addition to NO and PGI, the coronary vascular endothelium is also able to release a factor which causes hyperpolarisation of the underlying smooth muscle. This so-called endothelium-derived hyperpolarising factor (EDHF) displays the characteristics of a cytochrome P450-derived arachidonic acid metabolite. However, since NO is able to attenuate production of this factor, EDHF may contribute to the regulation of vascular tone essentially in situations associated with an apparent dysfunction of the endothelium. [ABSTRACT FROM AUTHOR]