Endothelial dysfunction in hypertension

The endothelium modulates the tone of the underlying vascular smooth muscle by releasing relaxing factors, including prostacyclin, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). In most types of hypertension, endothelium-dependent relaxations are impaired because of a reduced production and/or action of endothelium-derived NO and EDHF. In essential hypertension, endothelium-dependent relaxations are reduced because of a concomitant release of vasoconstrictor prostanoids (endoperoxides and thromboxane A2). These prostanoids may be produced in the vascular smooth muscle rather than in the endothelium. The endothelial dysfunction observed in hypertension is likely to be a consequence rather than a cause of the disease, representing premature aging of the blood vessels due to the chronic exposure to the high blood pressure. The endothelial dysfunction can be improved by antihypertensive therapy, favoring the prevention of the occurrence of vascular complications in hypertension.     

Endothelium and high blood pressure

Due to its strategic anatomical position, the endothelium is constantly exposed to the different risk factors for atherosclerosis. During the last decade it has become clear that hypertension profoundly affects endothelial function. Depending on the form of hypertension, endothelium-dependent relaxation is impaired in most vascular beds. In spontaneous hypertension, the production of nitric oxide, which in endothelial cells is formed from L-arginine via the constitutively expressed enzyme endothelial nitric oxide synthase, represents the main mediator of endothelium-dependent vasodilation and seems to be enhanced. On the other hand, the release of endothelium-dependent contracting factors such as prostaglandin H2 and thromboxane A2 have been demonstrated in this model of hypertension. Similar results have been obtained in the forearm circulation of patients with essential hypertension. In contrast, in models of salt-sensitive hypertension no release of vasoconstrictor prostanoids can be found indicating a decreased production of nitric oxide. Thus, in spontaneous hypertension an increased production of nitric oxide seems to occur, which is ineffective due to either the simultaneous release of endothelium-dependent vasoconstrictors and/or inactivation of nitric oxide, or due to anatomical changes such as hypertension-induced intimal thickness which inhibits its action on vascular smooth muscle cells. In summary, in hypertension, endothelium-dependent vasodilation is blunted and the endothelial L-arginine nitric oxide pathway is altered. These changes seem to represent a consequence rather than a cause of hypertension.     

Reproductive health law: where next, after Cairo and Beijing?

OBJECTIVE: Diabetes selectively injures receptor-mediated endothelium-dependent relaxation. In this study, we investigated the effect of elevated glucose concentrations on intracellular calcium (Ca2+i) signal transduction in response to stimulants of EDRF/nitric oxide release in cultured bovine aortic endothelial cells. METHODS: [Ca2+i] was measured in cell suspensions using Fura-2 and fluorescence spectroscopy while nitric oxide production was evaluated using radioimmunoassay of cGMP production. RESULTS: After 24 h exposure to 25 mM glucose in Ham's F-12 media, the increase in endothelial cell [Ca2+i] in response to 100 nM bradykinin was attenuated by 40% while the response to ionomycin was unaltered. When RMPI medium was used, no reduction in response to bradykinin was observed at 25 mM glucose, but a significant reduction in [Ca2+i] signal was observed after exposure to 35 mM glucose for a similar time period. Defective [Ca2+i] signaling was also seen in cells using MEM medium. [Ca2+i] signal responses to ionomycin and NaF, a G-protein activator of extracellular calcium entry via calcium channels, were unaltered by elevated glucose exposure. The defect in [Ca2+i] signal was not mimicked by either mannose or sucrose, but was prevented by co-incubation with cytochalasin B to inhibit glucose uptake. Neither superoxide dismutase nor catalase nor the extracellular hydroxyl radical scavenger, mannitol, blocked the reduction in the bradykinin-induced increase of [Ca2+i] in elevated glucose-exposed cells; however, the reduction was completely blocked by the cell-permeable hydroxyl radical scavenger, dimethylthiourea. Bradykinin-stimulated (but not ionomycin-stimulated) cGMP production within endothelial cells or in RFL-6 detector cells was attenuated by elevated glucose exposure. CONCLUSIONS: Hyperglycemia may contribute to defective endothelium-dependent relaxation in diabetes via an attenuated increase in Ca2+i signal transduction for the release of nitric oxide by endothelial cells. This defect possibly arises as a consequence of hydroxyl radicals formed intracellularly.     

15-Hydroxyeicosatetraenoic acid and diabetic endothelial dysfunction in rabbit aorta

We examined the effects of diabetes on eicosanoid metabolism and endothelium-dependent relaxation in isolated aorta from alloxan-induced diabetic rabbits and that from normal rabbits incubated in increased concentrations (44 mM) of glucose in vitro for 6 h. Immunoreactive 15-hydroxyeicosatetraenoic acid (HETE) was assayed in the incubation media of isolated aortic segments. Basal and acetylcholine (ACh)-stimulated release of 15-HETE was significantly greater in aorta of diabetic animals as compared with those of normal rabbits. Incubation of aortic segments from normal rabbits in increased concentrations of glucose caused a significant increase in basal and ACh-stimulated release of 15-HETE; and the release was significantly greater in aortic segments with endothelium than in segments without endothelium. Basal and ACh-stimulated release of 15-HETE was inhibited by indomethacin, a cyclooxygenase inhibitor. 15-HETE caused contractions of aortic rings that were inhibited by the prostaglandin H2 (PGH2) thromboxane A2 (TXA2) receptor blocker SQ-29548, but not by the TXA2 synthase inhibitor carbethoxyhexyl imidazole or indomethacin. Treatment of aortic rings with subthreshold concentrations of 15-HETE impaired ACh-induced relaxation; this was prevented by treatment with SQ-29548. Thus, abnormal release of endothelium-derived 15-HETE may play a role in endothelial cell dysfunction and increased vasoconstriction in diabetes by a mechanism that involves interaction with PGH2/TXA2 receptors.     

Functional and morphological damage of endothelium in rabbit ear artery following irradiation with cobalt60

1. The relaxant actions of acetylcholine and A23187 were examined in the rabbit central ear artery at different intervals following exposure to different doses of radiation with a cobalt60 unit. The artery was irradiated with a dose of 10 Gy, 20 Gy and 45 Gy. Radiation caused dose- and time-dependent impairment of the endothelium-dependent relaxations. The impaired endothelium-dependent relaxations occurred as early as 1 week postirradiation and persisted throughout the experimental period (10 weeks). 2. The endothelium-independent response to sodium nitroprusside was well preserved up to 6 weeks after irradiation. The contractile response to noradrenaline was unaltered by irradiation throughout the experimental period, but in contrast to control vessels, an increase in the sensitivity to noradrenaline in the presence of the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine was not observed in the irradiated vessels. 3. The impaired endothelium-dependent relaxations in the irradiated vessels were not improved by pretreatment with the NOS substrate L-arginine, the cyclo-oxygenase inhibitor indomethacin or the free radical scavengers superoxide dismutase and catalase. 4. Scanning electron microscopy indicated morphologically intact endothelial cells within the first 4 weeks after irradiation. 5. Western blot analysis showed a significant decrease in the expression of endothelial NOS (eNOS) in the irradiated vessels. 6. These data indicate that endothelial cell function is specifically impaired in the irradiated vessels before morphological endothelial cell damage can be detected. This impairment may be related to diminished eNOS expression.     

Long-term treatment in vivo with NOX-101, a scavenger of nitric oxide, prevents diabetes-induced endothelial dysfunction

Substantial evidence exists that diabetes results in impaired endothelial dysfunction suggesting diminished nitric oxide production from diabetic endothelium. It is not known what factors contribute to the development of this defect. In this study, we tested whether chronic treatment in vivo with NOX-101, a water-soluble nitric oxide scavenger, prevents endothelial dysfunction in diabetes. Sprague-Dawley rats were made diabetic by an intravenous injection of streptozotocin. A subgroup of control or diabetic animals received twice daily subcutaneous injections of 80 mg/kg NOX-101 beginning at 48 h after streptozotocin was injected and throughout 8 weeks of diabetes. Body weights and glucose concentrations were monitored weekly. At the end of 8 weeks, blood glucose and glycosylated haemoglobin was raised in diabetic rats but serum insulin concentrations were reduced. Treatment with NOX-101 did not alter glucose or insulin concentrations in control or diabetic rats; however, total glycosylated haemoglobin was partially reduced compared with untreated rats. In a subgroup of 2-week diabetic and age-matched rats fasted for 24 h, NOX-101 abolished total urinary nitrate plus nitrite (an index of nitric oxide production in vivo). In isolated tissue baths, relaxation to the endothelium-dependent vasodilator, acetylcholine, was impaired in diabetic aortic rings and relaxation to nitroglycerin was unaltered. Treatment of control rats with NOX-101 did not alter maximum relaxation to acetylcholine but shifted the response curve slightly to the right. In contrast in diabetic rats, NOX-101 prevented the impairment in endothelium-dependent relaxation but had no effect on relaxation induced by nitroglycerin. These data suggest the possibility that diabetes-induced endothelial dysfunction in diabetes results, in part, from a paradoxical increase in nitric oxide production during the course of the disease. This suggests a novel pathway of vascular complications.     

Endothelial dysfunction in spontaneously hypertensive rats: consequences of chronic treatment with losartan or captopril

BACKGROUND: Hypertension is associated with endothelial dysfunction characterized by decreased endothelium-dependent relaxations and increased endothelium-dependent contractions. Angiotensin converting enzyme inhibitors and thromboxane A2 receptor antagonists decreased the endothelium dysfunction in hypertensive animals. OBJECTIVE: To investigate the effects of prolonged treatment with losartan on endothelium-dependent and -independent relaxations and contractions in aortic rings from spontaneously hypertensive rats (SHR). MATERIAL AND METHODS: Male SHR aged 16 weeks were treated for 12 consecutive weeks either with 10 mg/kg losartan per day or with 60 mg/kg captopril per day administered via their drinking water. The systolic blood pressure was evaluated basally and during week 12. At the end of the treatment period, the vascular reactivity in aortic rings was studies. A group of rats treated with captopril was studies as a reference group. RESULTS: Losartan and captopril reduced the blood pressure significantly and comparably. Both drugs enhanced acetylcholine-induced relaxations and reduced the maximal contractile response to acetylcholine in the presence of NG-nitro-L arginine methyl ester (L-NAME). Contractile responses to phenylephrine, endothelin-l and U46619 were not affected by these treatments. Increased relaxing responses to superoxide dismutase were observed only in captopril-treated rats. Losartan reduced the contractile response to angiotensin II. By contrast this contractile response was elevated in rats treated with captopril. CONCLUSIONS: Prolonged antihypertensive treatments with losartan and captopril decreased the endothelial dysfunction in aortic rings from SHR not only by enhancing NO-dependent relaxations but also by reducing the contractions in response to an endothelium-derived contracting factor. The results further confirm that an endothelium-derived contracting factor plays a role in vascular dysfunction in SHR and the relationships between this factor and angiotensin II.     

Different populations of progesterone receptor-steroid complexes in binding to specific DNA sequences: effects of salts on kinetics and specificity

The endothelium plays an obligatory role in a number of relaxations of isolated arteries. These endothelium-dependent relaxations are due to the release by the endothelial cells of potent vasodilator substances [endothelium-derived relaxing factors (EDRF)]. The best characterized EDRF is nitric oxide (NO). Nitric oxide is formed by the metabolism of L-arginine by the constitutive NO synthase of endothelial cells. In arterial smooth muscle, the relaxations evoked by EDRF are explained best by the stimulation by NO of soluble guanylate cyclase that leads to the accumulation of cyclic GMP. The endothelial cells also release an unidentified substance that causes hyperpolarization of the cell membrane (endothelium-derived hyperpolarizing factor, EDHF). The release of EDRF from the endothelium can be mediated by both pertussis toxin-sensitive (alpha2-adrenergic activation, serotonin, thrombin, aggregating platelets) and insensitive (adenosine diphosphate, bradykinin) G-proteins. In blood vessels from animals with regenerated endothelium, and/or atherosclerosis, there is a selective loss of the pertussis-toxin sensitive mechanism of EDRF-release which favors the occurrence of vasospasm, thrombosis and cellular growth.     

Differential role of vasoactive prostanoids in porcine and human isolated pulmonary arteries in response to endothelium-dependent relaxants

The pig is increasingly being used in medical research, both as a model of the human cardiovascular system, and as a possible source of organs for xenotransplantation. However, little is known about the comparative functions of the vascular endothelium between porcine and human arteries. We have therefore compared the effects of two endothelium-dependent vasorelaxants, acetylcholine (ACh) and the Ca2+-ATPase inhibitor, cyclopiazonic acid (CPA) on the porcine and human isolated pulmonary artery using isometric tension recording. ACh and CPA produced endothelium-dependent relaxations of both the human and porcine pulmonary arteries. In the porcine pulmonary artery, the cyclo-oxygenase inhibitor, flurbiprofen had no effect on relaxations to ACh (Emax: control 67.8+/-8.8% versus 72.4+/-9.5% (n=11)) or CPA (Emax: control 79.6+/-5.0% versus 94.0+/-10.6% (n=7)). The nitric oxide synthase inhibitor, L-NAME converted relaxations to both ACh and CPA into contractile responses (maximum response: ACh 30.0+/-11.1% (n = 10); CPA 80.4+/-26.2% (n = 8) of U46619-induced tone). These contractile responses in the presence of L-NAME were abolished by flurbiprofen. In the human pulmonary artery, L-NAME and flurbiprofen partly attenuated relaxations to ACh (Emax: control: 45.1+/-12.1%; flurbiprofen: 33.4+/-13.5%; L-NAME: 10.1+/-7.2%) and CPA (Emax: control: 78.1+/-5.5%; flurbiprofen: 69.6+/-7.2%; L-NAME 37.9+/-10.7% of U46619-induced tone). These responses were abolished by the combination of both inhibitors. We have demonstrated that while the release of nitric oxide is important in responses to endothelium-dependent vasorelaxants in both human and porcine pulmonary arteries, in the human arteries, there is an important role for vasorelaxant prostanoids whilst in the porcine arteries, vasoconstrictor prostanoids are released.     

Regional differences in endothelial function in horse lungs: possible role in blood flow distribution?

We investigated regional differences of in vitro responses of pulmonary arteries (6-mm OD) from the dorsocaudal (top) and cranioventral (bottom) lung regions to endothelium-dependent vasodilators (methacholine, bradykinin, and calcium ionophore A-23187). Methacholine relaxed endothelium-intact top vessels; however, in bottom vessels, a small relaxation preceded a profound contraction. In top vessels, removal of endothelial cells converted relaxation to contraction, and in bottom vessels it abolished relaxation and enhanced contraction. Bradykinin and A-23187 were more potent and caused greater endothelium-mediated relaxation in top than in bottom arteries. The endothelium-independent vasodilator sodium nitroprusside caused similar relaxations in all rings. Nomega-nitro-L-arginine and NG-monomethyl-L-arginine and methylene blue abolished relaxation of top and bottom arteries to methacholine; meclofenamate had little effect. We conclude that regional differences in endothelium-mediated relaxation are caused by differences in the magnitude of the endothelial release of nitric oxide. Similar differences in endothelium-dependent flow-mediated vasodilation and endothelial nitric oxide release may result in preferential perfusion of caudodorsal lung regions.     

Endothelial dysfunction and metabolic control in streptozotocin-induced diabetic rats

1. The aim of this work was to study the influence of the metabolic control, estimated by the levels of glycosylated haemoglobin in total blood samples (HbA1c), in developing vascular endothelial dysfunction in streptozotocin-induced diabetic rats. Four groups of animals with different levels of insulin treatment were established, by determining HbA1c values in 5.5 to 7.4%, 7.5 to 9.4%, 9.5 to 12% and > 12%, respectively. 2. The parameters analysed were: (1) the endothelium-dependent relaxations to acetylcholine (ACh) in isolated aorta and mesenteric microvessels; (2) the vasodilator responses to exogenous nitric oxide (NO) in aorta: and (3) the existence of oxidative stress by studying the influence of the free radical scavenger superoxide dismutase (SOD) on the vasodilator responses to both ACh and NO. 3. In both isolated aortic segments and mesenteric microvessels, the endothelium-mediated concentration-dependent relaxant responses elicited by ACh were significantly decreased when the vessels were obtained from diabetic animals but only with HbA1c values higher than 7.5%. There was a high correlation between HbA1c levels and the impairment of ACh-induced relaxations, measured by pD2 values. 4. The concentration-dependent vasorelaxant responses to NO in endothelium-denuded aortic segments were significantly reduced only in vessels from diabetic animals with HbA1c values higher than 7.5%. Again, a very high correlation was found between the HbA1c values and pD2 for NO-evoked responses. 5. In the presence of SOD, the responses to ACh or NO were only increased in the segments from diabetic rats with HbA1c levels higher than 7.5%, but not in those from non-diabetic or diabetic rats with a good metabolic control (HbA1c levels <7.5%). 6. These results suggest the existence of: (1) a close relation between the degree of endothelial dysfunction and the metabolic control of diabetes, estimated by the levels of HbA1c; and (2) an increased production of superoxide anions in the vascular wall of the diabetic rats, which is also related to the metabolic control of the disease.     

Tyrosine nitration as a mechanism of selective inactivation of prostacyclin synthase by peroxynitrite

Vascular tone critically depends on the endothelial release of nitric oxide and prostacyclin. Superoxide anions counteract these relaxations by trapping nitric oxide under formation of peroxynitrite. As we have recently reported, peroxynitrite is able to inhibit prostacyclin formation in aortic microsomes (Zou et al., 1996). Here we show that peroxynitrite also blocks purified prostacyclin synthase with an IC50 value of about 50 nM and with a similar sensitivity also inhibits the enzyme activity in the EaHy 926 endothelial cell line. Thromboxane synthase, having the same heme-thiolate (P450) structure and a closely-related mechanism was unaffected by peroxynitrite. Anti-nitrotyrosine antibodies reacted positive by a Western blot after treatment of the purified enzyme with 1 microM peroxynitrite. Tetranitromethane also inhibited the enzyme activity which, like the inhibition by peroxynitrite, could be partially prevented in the presence of the substrate analog U46619. The simultaneous generation of superoxide and nitric oxide proved to be as efficient as a bolus of peroxynitrite which supports a possible inactivation of prostacyclin synthase under in vivo conditions. This substantiates an often suggested crucial role of superoxide in the pathophysiology of the cardiovascular system.     

Neurogenic contraction and relaxation of human penile deep dorsal vein

1. The aim of the present study was to characterize neurogenic and pharmacological responses of human penile deep dorsal vein and to determine whether the responses are mediated by nitric oxide from neural or endothelial origin. 2. Ring segments of human penile deep dorsal vein were obtained from 22 multiorgan donors during procurement of organs for transplantation. The rings were suspended in organ bath chambers for isometric recording of tension. We then studied the contractile and relaxant responses to electrical field stimulation and to vasoactive agents. 3. Electrical field stimulation (0.5-2 Hz) and noradrenaline (3 x 10(-10)-3 x 10(-5) M) caused frequency- and concentration-dependent contractions that were of greater magnitude in veins denuded of endothelium. The inhibitor of nitric oxide synthesis NG-nitro-L-arginine methyl ester hydrochloride (L-NAME, l0(-4) M) increased the adrenergic responses only in rings with endothelium. 4. In preparations contracted with noradrenaline in the presence of guanethidine (10(-6) M) and atropine (10(-6) M), electrical stimulation induced frequency-dependent relaxations. This neurogenic relaxation was prevented by L-NAME, methylene blue (3 x 10(-5) M) and tetrodotoxin (10(-6) M), but was unaffected by removal of endothelium. 5. Acetylcholine (10(-8)-3 x 10(-5) M) and substance P (3 x 10(-11) -3 x 10(-7) M) induced endothelium-dependent relaxations. In contrast, sodium nitroprusside (10(-9)-3 x 10(-5) M) and papaverine (10(-8) 3 x 10(-5) M) caused endothelium-independent relaxations. 6. The results provide functional evidence that the human penile deep dorsal vein is an active component of the penile vascular resistance through the release of nitric oxide from both neural and endothelial origin. Dysfunction in any of these sources of nitric oxide should be considered in some forms of impotence.     

Impaired endothelium-dependent relaxation after coronary reperfusion injury: evidence for G-protein dysfunction

This study was done to determine whether abnormal receptor-dependent release of endothelium-derived relaxing factor (EDRF) might be caused by G-protein dysfunction. Dogs were exposed to global myocardial ischemia (45 minutes, induced by aortic cross-clamping) followed by reperfusion (60 minutes) while on cardiopulmonary bypass, and coronary arteries were then studied in vitro in organ chamber experiments. After reperfusion, endothelium-dependent relaxation to the receptor-dependent agonists adenosine diphosphate and acetyl-choline was significantly impaired as well as to sodium fluoride, which acts on a pertussis toxin-sensitive G-protein. In contrast, endothelium-dependent relaxations to the receptor-independent agonists A23187 and phospholipase C were normal. Furthermore, endothelium-dependent relaxation to poly-L-arginine (molecular weight, 139,200), which appears to induce endothelium-dependent relaxation of the canine coronary artery by a nonnitric oxide pathway, was unaffected by ischemia and reperfusion. These experiments suggest that global myocardial ischemia and reperfusion selectively impair receptor-mediated release of EDRF (nitric oxide) but that the ability of the endothelial cell to produce EDRF or generate endothelium-dependent relaxation to nonnitric oxide-dependent agonists remains intact. We hypothesize that coronary reperfusion injury leads to G-protein dysfunction in the endothelium.     

Nitric oxide- and superoxide-mediated toxicity in cerebral endothelial cells

Nitric oxide and superoxide are free radicals that appear to contribute to the pathogenesis of a number of brain disorders, and cerebral endothelial cells are a potential target of these agents. Because of the capacity for these two agents to combine, it has been suggested that nitric oxide might either enhance or inhibit the toxic effects of superoxide. To establish the effect of the generation of superoxide and nitric oxide alone and in combination, cerebral endothelial cells were exposed to sodium nitroprusside, a source of nitric oxide, and/or paraquat, a source of superoxide. Paraquat enhanced the toxicity of sodium nitroprusside, as did diethyldithiocarbamate, an inhibitor of superoxide dismutase, which supports the hypothesis that enhanced levels of superoxide can combine with nitric oxide to form a more toxic product. Also, the toxicity of paraquat could be partially inhibited by blocking endogenous nitric oxide synthesis using N(G)-monomethyl-L-arginine. When ascorbate was administered along with sodium nitroprusside to increase nitric oxide generation, as little as 5 microM sodium nitroprusside was toxic when superoxide dismutase was inhibited. Whereas concentrations of 50 to 500 microM sodium nitroprusside and 0.4 mM ascorbate caused approximately 100% toxicity, there was no measurable toxicity when these doses were accompanied by 2 mM glutathione or 50 U/ml of catalase; this suggests that peroxides may also contribute to nitric oxide toxicity. These results suggest that the simultaneous generation of nitric oxide and superoxide is synergistic, resulting in enhanced toxicity.     

Impairment of endothelial function induced by glyc-oxidized lipoprotein a [Lp(a)]

Diabetic patients develop endothelial dysfunction early in the course of the disease. Atherogenic lipoproteins such as LDL and Lp(a) are important risk factors for endothelial dysfunction and undergo nonenzymatic glycation in hyperglycaemia. Here we assessed whether glycation of Lp(a) potentiates its damaging influence on endothelial function. Human Lp(a) was glycated by dialyzation for 7 days against buffer containing 200 mmol/l glucose, or sham-treated without glucose and oxidized by incubation with Cu++. The degree of glycation accounted to 32 +/- 4%, and glycation rendered Lp(a) more susceptible to oxidative modification when exposed to Cu++. Isolated rings of rabbit aorta were superfused with physiological salt solution, and isometric tension was recorded. Incubation of the aortic rings with sham-treated or with 30 microg/ml glycated Lp(a), not oxidized, had no influence on acetylcholine-induced, endothelium-dependent relaxation. Exposure of the aortic rings to 30 microg/ml oxidized non-glycated (ox) Lp(a) caused a significant inhibition (19% at 1 microM acetylcholine) of the endothelium-dependent relaxation. Incubation of aortic rings with 30 microg/ml oxidized glycated (glyc-ox) Lp(a) attenuated endothelium-dependent relaxation more potently than oxLp(a) (by 34% at 1 microM acetylcholine). The presence of diethyl-dithio-carbamate (DDC), an inhibitor of the endogenous superoxide dismutase (SOD), potentiated the inhibition of relaxation induced by oxLp(a) and by glyc-oxLp(a) [38% inhibition at 1 microM acetylcholine for oxLp(a), and 49% inhibition at 1 microM acetylcholine for glyc-oxLp(a)]. Co-incubation with the O2- scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid disodium salt (TIRON) prevented the inhibition of relaxation by the oxidized lipoproteins, suggesting that enhanced NO-inactivation by O2- could be the underlying mechanism for the impairment of endothelium-dependent dilations by ox- and glyc-oxLp(a). The concentration of lysophosphatidycholine, a lipoprotein oxidation product and stimulus for O2- formation, was significantly enhanced in oxLp(a) and in glyc-oxLp(a) compared to native lipoproteins. Conclusion: Glycation enhances the endothelium-damaging influence of oxLp(a), presumably by enhancing oxidative stress. The likely mechanism for attenuation of endothelium-dependent dilations is increased formation of O2-, resulting in inactivation of nitric oxide. This mechanism may play an important role in diabetic patients and may contribute to disturbed organ perfusion.     

Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors

The mechanism of superoxide generation by endothelial nitric oxide synthase (eNOS) was investigated by the electron spin resonance spin-trapping technique using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. In the absence of calcium/calmodulin, eNOS produces low amounts of superoxide. Upon activating eNOS electron transfer reactions by calcium/calmodulin binding, superoxide formation is increased. Heme-iron ligands, cyanide, imidazole, and the phenyl(diazene)-derived radical inhibit superoxide generation. No inhibition is observed after addition of L-arginine, NG-hydroxy-L-arginine, L-thiocitrulline, and L-NG-monomethyl arginine to activated eNOS. These results demonstrate that superoxide is generated from the oxygenase domain by dissociation of the ferrous-dioxygen complex and that occupation of the L-arginine binding site does not inhibit this process. However, the concomitant addition of L-arginine and tetrahydrobiopterin (BH4) abolishes superoxide generation by eNOS. Under these conditions, L-citrulline production is close to maximal. Our data indicate that BH4 fully couples L-arginine oxidation to NADPH consumption and prevents dissociation of the ferrous-dioxygen complex. Under these conditions, eNOS does not generate superoxide. The presence of flavins, at concentrations commonly employed in NOS assay systems, enhances superoxide generation from the reductase domain. Our data indicate that modulation of BH4 concentration may regulate the ratio of superoxide to nitric oxide generated by eNOS.     

Effect of low density lipoprotein on adenosine receptor-mediated coronary vasorelaxation in vitro

We investigated the effect of low density lipoprotein (LDL) on vasorelaxations and nitric oxide generation induced by the adenosine analogs, 5'-(N-ethylcarboxamide)adenosine, 2-p-(2-carboxyethyl)phenylethyl-amino-5'N-ethylcarboxamidoadenosine and/or 2-chloroadenosine in porcine coronary artery rings in vitro. Preincubation of tissues with native LDL (100 and 200 microg/ml) for 4 hr in the absence or presence of copper sulfate (5 microM) selectively attenuated the endothelium-dependent relaxations elicited by 5'-(N-ethylcarboxamide)adenosine and 2-p-(2-carboxyethyl)phenylethyl-amino-5'N-ethylcarboxamideoadenosine+ ++ without altering the response to 2-chloroadenosine which produced endothelium-independent relaxation. The 4-hr exposure of tissues to native LDL (100 microg/ml) also inhibited the production of nitrite induced by 5'-(N-ethylcarboxamide)adenosine in endothelium-intact rings. These effects were associated with enhanced oxidation of the lipoprotein. The inhibitory action of LDL on tissue relaxations and nitrite generation as well as the oxidation of the lipoprotein were all prevented by high density lipoprotein (100 microg/ml). In contrast, a relatively short period (20 min) of tissue incubation with native LDL produced no alterations of the relaxations and nitrite production evoked by 5'-(N-ethylcarboxamide)adenosine and 2-p-(2-carboxyethyl)phenylethyl-amino-5'N-ethylcarboxamidoadenosine. Under this condition, the oxidation of LDL was not also significantly altered. In conclusion, the results indicate that in coronary artery LDL, with oxidative modification, causes attenuation of nitric oxide-mediated endothelial responses induced by adenosine receptors activation, and this effect is prevented by high density lipoprotein. Such modulation may be of importance in hypercholesterolemia and in the development of atherosclerosis.     

Attenuation of contractions to acetylcholine in canine bronchi by an endogenous nitric oxide-like substance

1. The involvement was assessed of an endogenous nitric oxide-like substance in contractions of canine bronchi to acetylcholine. 2. Canine third order bronchial rings, in some of which the epithelium was removed mechanically, were suspended in organ chambers and isometric tension was recorded. In some experiments, the content of guanosine 3',5'-cyclic monophosphate (cyclic GMP) of the bronchi was also measured. 3. Acetylcholine induced concentration-dependent contractions. The contractions were potentiated by nitro-L-arginine (an inhibitor of the synthesis of nitric oxide), oxyhaemoglobin (a scavenger of nitric oxide), and methylene blue (an inhibitor of soluble guanylate cyclase). The magnitude of the potentiation to acetylcholine-induced contractions by these inhibitors were not significantly different between tissues with and without epithelium. 4. Acetylcholine induced a concentration-dependent increase in intracellular content of cyclic GMP, which was similar in bronchi with and without epithelium. These increases were abolished by nitro-L-arginine and methylene blue. 5. During contractions to acetylcholine, exogenous nitric oxide relaxed the canine bronchi. The relaxations were not affected by nitro-L-arginine, but were augmented by superoxide dismutase plus catalase, and were abolished by methylene blue. 6. These observations suggest that, during contraction evoked by acetylcholine, the production of an endogenous nitric oxide-like substance increases and in turn attenuates the response of the airways to the muscarinic agonist. However, the endogenous nitric oxide-like substance does not play a major role in the epithelium-dependent attenuation of the contraction to acetylcholine in canine bronchi.     

Superoxide generation from endothelial nitric-oxide synthase. A Ca2+/calmodulin-dependent and tetrahydrobiopterin regulatory process

It has been previously shown that besides synthesizing nitric oxide (NO), neuronal and inducible NO synthase (NOS) generates superoxide (O-2) under conditions of L-arginine depletion. However, there is controversy regarding whether endothelial NOS (eNOS) can also produce O-2. Moreover, the mechanism and control of this process are not fully understood. Therefore, we performed electron paramagnetic resonance spin-trapping experiments to directly measure and characterize the O-2 generation from purified eNOS. With the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), prominent signals of O-2 adduct, DMPO-OOH, were detected from eNOS in the absence of added tetrahydrobiopterin (BH4), and these were quenched by superoxide dismutase. This O-2 formation required Ca2+/calmodulin and was blocked by the specific NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) but not its non-inhibitory enantiomer D-NAME. A parallel process of Ca2+/calmodulin-dependent NADPH oxidation was observed which was also inhibited by L-NAME but not D-NAME. Pretreatment of the enzyme with the heme blockers cyanide or imidazole also prevented O-2 generation. BH4 exerted dose-dependent inhibition of the O-2 signals generated by eNOS. Conversely, in the absence of BH4 L-arginine did not decrease this O-2 generation. Thus, eNOS can also catalyze O-2 formation, and this appears to occur primarily at the heme center of its oxygenase domain. O-2 synthesis from eNOS requires Ca2+/calmodulin and is primarily regulated by BH4 rather than L-arginine.