Gender-related differences in the effects of nitric oxide donors on neuroleptic-induced catalepsy in mice

It has been suggested that nigrostriatal dopaminergic transmission is modulated by nitric oxide (NO). Since there is evidence that gonadal hormones can affect extrapyramidal motor behavior in mammals, we investigated the effects of isosorbide dinitrate (ISD), linsidomine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP), three pharmacologically different NO donors, on neuroleptic-induced catalepsy in 60- to 80-day-old male and female albino mice. Catalepsy was induced with haloperidol (1 mg/kg, ip) and measured at 30-min intervals by means of a bar test. Drugs (or appropriate vehicle) were injected ip 30 min before haloperidol, with each animal being used only once. ISD (5, 20 and 50 mg/kg) caused a dose-dependent inhibition of catalepsy in male mice (maximal effect 120 min after haloperidol: 64% inhibition). In the females only at the highest dose of ISD was an attenuation of catalepsy observed, which was mild and short lasting. SIN-1 (10 and 50 mg/kg) did not significantly affect catalepsy in female mice, while a significant attenuation was observed in males at the dose of 50 mg/kg (maximal inhibition: 60%). SNAP (20 mg/kg) significantly attenuated catalepsy in males 120 min after haloperidol (44% inhibition), but had no significant effect on females. These results basically agree with literature data showing that NO facilitates central dopaminergic transmission, although the mechanisms are not fully understood. They also reveal the existence of gender-related differences in this nitrergic modulation in mice, with females being less affected than males.

Nitric oxide release from the S-nitrosothiol zinc phthalocyanine complex by flash photolysis

The photogeneration of nitric oxide (NO) using laser flash photolysis was investigated for S-nitroso-glutathione (GSNO) and S-nitroso-N-acetylcysteine (NacySNO) at pH 6.4 (PBS/HCl) and 7.4 (PBS). Irradiation of S-nitrosothiol with light (lambda = 355 nm followed by absorption spectroscopy) resulted in the homolytic decomposition of NacySNO and GSNO to generate radicals (GS· and NacyS·) and NO. The release of NO from donor compounds measured with an ISO-Nometer apparatus was larger at pH 7.4 than pH 6.4. NacySNO was also incorporated into dipalmitoyl-phosphatidylcholine liposomes in the presence and absence of zinc phthalocyanine (ZnPC), a well-known photosensitizer useful for photodynamic therapy. Liposomes are usually used as carriers for hydrophobic compounds such as ZnPC. Inclusion of ZnPC resulted in a decrease in NO liberation in liposomal medium. However, there was a synergistic action of both photosensitizers and S-nitrosothiols resulting in the formation of other reactive species such as peroxynitrite, which is a potent oxidizing agent. These data show that NO release depends on pH and the medium, as well as on the laser energy applied to the system. Changes in the absorption spectrum were monitored as a function of light exposure.

Effect of acute nitric oxide synthase inhibition in the modulation of heart rate in rats

Acute nitric oxide synthase inhibition with N G-nitro-L-arginine methyl ester (L-NAME) on chronotropic and pressor responses was studied in anesthetized intact rats and rats submitted to partial and complete autonomic blockade. Blood pressure and heart rate were monitored intra-arterially. Intravenous L-NAME injection (7.5 mg/kg) elicited the same hypertensive response in intact rats and in rats with partial (ganglionic and parasympathetic blockade) and complete autonomic blockade (38 ± 3, 55 ± 6, 54 ± 5, 45 ± 5 mmHg, respectively; N = 9, P = NS). L-NAME-induced bradycardia at the time when blood pressure reached the peak plateau was similar in intact rats and in rats with partial autonomic blockade (43 ± 8, 38 ± 5, 46 ± 6 bpm, respectively; N = 9, P = NS). Rats with combined autonomic blockade showed a tachycardic response to L-NAME (10 ± 3 bpm, P<0.05 vs intact animals, N = 9). Increasing doses of L-NAME (5.0, 7.5 and 10 mg/kg, N = 9) caused a similar increase in blood pressure (45 ± 5, 38 ± 3, 44 ± 9 mmHg, respectively; P = NS) and heart rate (31 ± 4, 34 ± 3, 35 ± 4 bpm, respectively; P = NS). Addition of L-NAME (500 µM) to isolated atria from rats killed by cervical dislocation and rats previously subjected to complete autonomic blockade did not affect spontaneous beating or contractile strength (N = 9). In vivo results showed that L-NAME promoted a tachycardic response in rats with complete autonomic blockade, whereas the in vitro experiments showed no effect on intrinsic heart rate, suggesting that humoral mechanisms may be involved in the L-NAME-induced cardiac response.

Role of nitric oxide of the median preoptic nucleus (MnPO) in the alterations of salivary flow, arterial pressure and heart rate induced by injection of pilocarpine into the MnPO and intraperitoneally

We investigated the effect of L-NAME, a nitric oxide (NO) inhibitor and sodium nitroprusside (SNP), an NO-donating agent, on pilocarpine-induced alterations in salivary flow, mean arterial blood pressure (MAP) and heart rate (HR) in rats. Male Holtzman rats (250-300 g) were implanted with a stainless steel cannula directly into the median preoptic nucleus (MnPO). Pilocarpine (10, 20, 40, 80, 160 µg) injected into the MnPO induced an increase in salivary secretion (P<0.01). Pilocarpine (1, 2, 4, 8, 16 mg/kg) ip also increased salivary secretion (P<0.01). Injection of L-NAME (40 µg) into the MnPO prior to pilocarpine (10, 20, 40, 80, 160 µg) injected into the MnPO or ip (1, 2, 4, 8, 16 mg/kg) increased salivary secretion (P<0.01). SNP (30 µg) injected into the MnPO or ip prior to pilocarpine attenuated salivary secretion (P<0.01). Pilocarpine (40 µg) injection into the MnPO increased MAP and decreased HR (P<0.01). Pilocarpine (4 mg/kg body weight) ip produced a decrease in MAP and an increase in HR (P<0.01). Injection of L-NAME (40 µg) into the MnPO prior to pilocarpine potentiated the increase in MAP and reduced HR (P<0.01). SNP (30 µg) injected into the MnPO prior to pilocarpine attenuated (100%) the effect of pilocarpine on MAP, with no effect on HR. Administration of L-NAME (40 µg) into the MnPO potentiated the effect of pilocarpine injected ip. SNP (30 µg) injected into the MnPO attenuated the effect of ip pilocarpine on MAP and HR. The present study suggests that in the rat MnPO 1) NO is important for the effects of pilocarpine on salivary flow, and 2) pilocarpine interferes with blood pressure and HR (side effects of pilocarpine), that is attenuated by NO.

4-Aminopyridine inhibits the neuromuscular effects of nitric oxide and 8-Br-cGMP

The effects induced by nitric oxide (NO) in different tissues depend on direct and/or indirect interactions with K+ channels. The indirect interaction of NO is produced by activation of guanylyl cyclase which increases the intracellular cGMP. Since NO, cGMP and 4-aminopyridine alone induce tetanic fade and increase amplitude of muscular contractions in isolated rat neuromuscular preparations, the present study was undertaken to determine whether or not the neuromuscular effects of NO and 8-Br-cGMP can be modified by 4-aminopyridine. Using the phrenic nerve and diaphragm muscle isolated from male Wistar rats (200-250 g), we observed that L-arginine (4.7 mM) and 8-Br-cGMP (18 µM), in contrast to D-arginine, induced an increase in the amplitude of muscle contraction (10.5 ± 0.7%, N = 10 and 8.0 ± 0.7%, N = 10) and tetanic fade (15 ± 2.0%, N = 8 and 11.6 ± 1.7%, N = 8) at 0.2 and 50 Hz, respectively. N G-nitro-L-arginine (4 mM, N = 8 and 8 mM, N = 8) antagonized the effects of L-arginine. 4-Aminopyridine (1 and 10 µM) caused a dose-dependent increase in the amplitude of muscle contraction (15 ± 1.8%, N = 9 and 40 ± 3.1%, N = 10) and tetanic fade (17.7 ± 3.3%, N = 8 and 37.4 ± 1.3%, N = 8). 4-Aminopyridine (1 µM, N = 8) did not cause any change in muscle contraction amplitude or tetanic fade of preparations previously paralyzed with d-tubocurarine or stimulated directly. The effects induced by 4-aminopyridine alone were similar to those observed when the drug was administered in combination with L-arginine or 8-Br-cGMP. The data suggest that the blockage of K+ channels produced by 4-aminopyridine inhibits the neuromuscular effects induced by NO and 8-Br-cGMP. Therefore, the presynaptic effects induced by NO seem to depend on indirect interactions with K+ channels.

Do endogenous opioids and nitric oxide participate in the anticonvulsant action of dipyrone?

It was previously reported that systemic administration of dipyrone inhibited the tonic component of generalized tonic-clonic seizures in both the electroshock and the audiogenic seizure models. The aim of the present study was to investigate the mechanisms involved in the anticonvulsant action of dipyrone by assessing the role of nitric oxide and opioids in the electroshock (female 60- to 90-day-old Wistar rats, N = 5-11) and audiogenic seizure (female 60- to 90-day-old Wistar audiogenic rats, N = 5-11) models of epilepsy. Naloxone (5 mg/kg, sc) significantly reversed the anticonvulsant effect of dipyrone in rats submitted to the induction of audiogenic seizures (ANOVA/Bonferroni's test), suggesting the involvement of opioid peptides in this action. In the electroshock model no reversal of the anticonvulsant effect of dipyrone by naloxone (5 mg/kg, sc) was demonstrable. The acute (120 mg/kg, ip) and chronic (25 mg/kg, ip, twice a day/4 days) administration of L-NOARG did not reverse the anticonvulsant action of dipyrone in the audiogenic seizure model, suggesting that the nitric oxide pathway does not participate in such effect. Indomethacin (10, 20 and 30 mg/kg, ip) used for comparison had no anticonvulsant effect in the audiogenic seizure model. In conclusion, opioid peptides but not nitric oxide seem to be involved in the anticonvulsant action of dipyrone in audiogenic seizures.

The L-arginine/nitric oxide/cyclic-GMP pathway apparently mediates the peripheral antihyperalgesic action of fentanyl in rats

There are only a few studies on the molecular mechanisms underlying the peripheral antihyperalgesic effect of opioids. The aim of this study was to investigate the molecular bases of the peripheral antihyperalgesic effect of fentanyl in a model of prostaglandin-induced chemical hyperalgesia. Prostaglandin E2 (1.4 nmol) injected into one hind paw of male Wistar rats (200-250 g, N = 6 in each experimental or control group) pretreated with indomethacin (2.5 mg/kg) potentiated the nocifensive response to formalin (1%) injection made 60 min later. Drugs applied locally 30 min after prostaglandin E2 induced the following effects: fentanyl (0.1-1.0 nmol) caused a dose-dependent reversal of the hyperalgesic state, naloxone (2 nmol) co-injected with fentanyl (1 nmol) completely reversed the antihyperalgesic effect, Nomega-nitro-L-arginine (NOARG, 0.05-0.2 µmol) in combination with fentanyl (1.0 nmol) caused a dose-dependent inhibition of the antihyperalgesic effect of fentanyl, co-administration of L-arginine (0.5 µmol) with NOARG (0.2 µmol) plus fentanyl (1.0 nmol) fully restored the antihyperalgesic effect, and the cyclic-GMP phosphodiesterase inhibitor UK-114,542-27 (5-[2-ethoxy-5-(morpholinylacetyl) phenyl]-1,6-dihydro-1-methyl-3-propyl-7H-pyrazolo [4,3-d]-pyrimidin-7-one methanesulfonate monohydrate; 0.5-2.0 µmol) potentiated a subeffective dose of fentanyl (0.1 nmol) in a dose-dependent manner. However, UK-114,542-27 (2.0 µmol) injected alone did not produce this antihyperalgesic effect. Systemically administered fentanyl (1.0 nmol, sc) did not cause antinociception. Taken together, these results support the view that fentanyl reverses prostaglandin E2-induced hyperalgesia, probably by activating an opioid receptor at the periphery, and furthermore the L-arginine/nitric oxide/cyclic-GMP pathway may mediate this peripheral effect of fentanyl.

Hypotrophy of conduit artery walls of the offspring of nitric oxide-defective rats

The objective of the present study was to investigate the structure of the arterial walls of the offspring stemming from nitric oxide (NO)-defective hypertensive parents. The parents were treated with N G-nitro-L-arginine methyl ester (40 mg kg-1 day-1) for 5 weeks. Blood pressure was measured noninvasively in six 30-day-old rats and nine age-matched controls. The cardiovascular system was perfused with glutaraldehyde at 120 mmHg. The thoracic aorta and carotid artery were processed for electron microscopy, and geometry was determined by light microscopy. Endothelial cells, smooth muscle cells (SMC) and extracellular matrix (ECM) were determined by the point counting method in electron micrographs of the carotid artery. The blood pressure of experimental offspring was 150.0 ± 2.3 vs 104.6 ± 2.1 mmHg (P < 0.01) for the controls and their heart/body weight ratio of 3.9 ± 0.1 vs 4.4 ± 0.2 (P < 0.05) for the controls indicated cardiac hypotrophy. The wall thickness (tunica intima and media) of the thoracic aorta and carotid artery of experimental offspring was decreased to 78.9% (P < 0.01) and 83.8% (P < 0.01), respectively, compared to controls, as confirmed by a respective cross-sectional area of 85.3% (P < 0.01) and 84.1% (P < 0.01). The wall thickness/inner diameter ratio was reduced to 75% (P < 0.01) in the thoracic artery and to 81.5% (P < 0.01) in the carotid artery. No change in endothelial cell volume density or ECM was observed in the tunica intima of the carotid artery, and SMC volume density was lower in the tunica media (37.6 ± 0.9 vs 44.7 ± 1.1% for controls, P < 0.01), indicating compromised SMC development. Interference with arginine metabolism, a decrease in NO, and other factors are possible mechanisms underlying the structural alterations of the cardiovascular system of offspring from NO-defective hypertensive rats.

Expression of neuronal nitric oxide synthase in the developing superficial layers of the rat superior colliculus

We investigated the level of expression of neuronal nitric oxide synthase (nNOS) in the retinorecipient layers of the rat superior colliculus during early postnatal development. Male and female Lister rats ranging in age between the day of birth (P0) and the fourth postnatal week were used in the present study. Two biochemical methods were used, i.e., in vitro measurement of NOS specific activity by the conversion of [³H]-arginine to [³H]-citrulline, and analysis of Western blotting immunoreactive bands from superior colliculus homogenates. As revealed by Western blotting, very weak immunoreactive bands were observed as early as P0-2, and their intensity increased progressively at least until P21. The analysis of specific activity of NOS showed similar results. There was a progressive increase in enzymatic activity until near the end of the second postnatal week, and a nonsignificant tendency to an increase until the end of the third week was also observed. Thus, these results indicated an increase in the amount of nNOS during the first weeks after birth. Our results confirm and extend previous reports using histochemistry for NADPH-diaphorase and immunocytochemistry for nNOS, which showed a progressive increase in the number of stained cells in the superficial layers during the first two postnatal weeks, reaching an adult pattern at the end of the third week. Furthermore, our results suggested that nNOS is present in an active form in the rat superior colliculus during the period of refinement of the retinocollicular pathway.

Cytokine profile and nitric oxide levels in sera from patients with brucellosis

The aims of this study were to investigate the serum levels of some cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin 1ß (IL-1ß), IL-2R, IL-6, and IL-8] and nitric oxide (NO) levels in patients with untreated brucellosis and to test the correlation of these parameters with each other. The study was conducted on 67 subjects, 37 patients with brucellosis and 30 healthy individuals with no history of Brucella infection. Brucellosis was identified by a positive blood culture and/or increased Brucella antibodies in serological tests in addition to compatible clinical symptoms. Cytokine profile analysis was performed by the immulite chemiluminescent enzyme immunometric assay whose inter- and intra-assay coefficients of variance were 2.6-3.6 and 4.4-8.5%, respectively. The levels of nitrites/nitrates, which are representative of NO levels, were measured by the Griess method. Patients with brucellosis had significantly elevated serum levels of nitrites/nitrates, IL-2R, IL-6 and IL-8 (mean ± SD, 102.8 ± 23.8 µmol/l, 806.1 ± 58.5 U/ml, 21.1 ± 2.3 pg/ml, and 8.8 ± 1.6 pg/ml, respectively) compared to healthy controls, whereas TNF-alpha and IL-1ß levels were unchanged. No statistically significant correlation was detected between any of the studied cytokine levels and nitrate/nitrite concentrations according to Pearson's linear correlation test. We conclude that only IL-6, IL-8 and IL-2R are elevated in brucellosis and the extent of elevation depends on the severity and clinical pattern of the disease. Moderate elevation in serum NO was comparable to that observed in previous studies. This explains the absence or very rare occurrence of septic shock in brucellosis.

Acetylcholine and bradykinin enhance hypotension and affect the function of remodeled conduit arteries in SHR and SHR treated with nitric oxide donors

Discrepancy was found between enhanced hypotension and attenuated relaxation of conduit arteries in response to acetylcholine (ACh) and bradykinin (BK) in nitric oxide (NO)-deficient hypertension. The question is whether a similar phenomenon occurs in spontaneously hypertensive rats (SHR) with a different pathogenesis. Wistar rats, SHR, and SHR treated with NO donors [molsidomine (50 mg/kg) or pentaerythritol tetranitrate (100 mg/kg), twice a day, by gavage] were studied. After 6 weeks of treatment systolic blood pressure (BP) was increased significantly in experimental groups. Under anesthesia, the carotid artery was cannulated for BP recording and the jugular vein for drug administration. The iliac artery was used for in vitro studies and determination of geometry. Compared to control, SHR showed a significantly enhanced (P < 0.01) hypotensive response to ACh (1 and 10 µg, 87.9 ± 6.9 and 108.1 ± 5.1 vs 35.9 ± 4.7 and 64.0 ± 3.3 mmHg), and BK (100 µg, 106.7 ± 8.3 vs 53.3 ± 5.2 mmHg). SHR receiving NO donors yielded similar results. In contrast, maximum relaxation of the iliac artery in response to ACh was attenuated in SHR (12.1 ± 3.6 vs 74.2 ± 8.6% in controls, P < 0.01). Iliac artery inner diameter also increased (680 ± 46 vs 828 ± 28 µm in controls, P < 0.01). Wall thickness, wall cross-section area, wall thickness/inner diameter ratio increased significantly (P < 0.01). No differences were found in this respect among SHR and SHR treated with NO donors. These findings demonstrated enhanced hypotension and attenuated relaxation of the conduit artery in response to NO activators in SHR and in SHR treated with NO donors, a response similar to that found in NO-deficient hypertension.

Activation of neural cholecystokinin-1 receptors induces relaxation of the isolated rat duodenum which is reduced by nitric oxide synthase inhibitors

Cholecystokinin (CCK) influences gastrointestinal motility, by acting on central and peripheral receptors. The aim of the present study was to determine whether CCK has any effect on isolated duodenum longitudinal muscle activity and to characterize the mechanisms involved. Isolated segments of the rat proximal duodenum were mounted for the recording of isometric contractions of longitudinal muscle in the presence of atropine and guanethidine. CCK-8S (EC50: 39; 95% CI: 4.1-152 nM) and cerulein (EC50: 58; 95% CI: 18-281 nM) induced a concentration-dependent and tetrodotoxin-sensitive relaxation. Nomeganitro-L-arginine (L-NOARG) reduced CCK-8S- and cerulein-induced relaxation (IC50: 5.2; 95% CI: 2.5-18 µM) in a concentration-dependent manner. The magnitude of 300 nM CCK-8S-induced relaxation was reduced by 100 µM L-NOARG from 73 ± 5.1 to 19 ± 3.5% in an L-arginine but not D-arginine preventable manner. The CCK-1 receptor antagonists proglumide, lorglumide and devazepide, but not the CCK-2 receptor antagonist L-365,260, antagonized CCK-8S-induced relaxation in a concentration-dependent manner. These findings suggest that CCK-8S and cerulein activate intrinsic nitrergic nerves acting on CCK-1 receptors in order to cause relaxation of the rat duodenum longitudinal muscle.

Methylmercury intoxication activates nitric oxide synthase in chick retinal cell culture

The visual system is a potential target for methylmercury (MeHg) intoxication. Nevertheless, there are few studies about the cellular mechanisms of toxicity induced by MeHg in retinal cells. Various reports have indicated a critical role for nitric oxide synthase (NOS) activation in modulating MeHg neurotoxicity in cerebellar and cortical regions. The aim of the present study is to describe the effects of MeHg on cell viability and NOS activation in chick retinal cell cultures. For this purpose, primary cultures were prepared from 7-day-old chick embryos: retinas were aseptically dissected and dissociated and cells were grown at 37ºC for 7-8 days. Cultures were exposed to MeHg (10 µM, 100 µM, and 1 mM) for 2, 4, and 6 h. Cell viability was measured by MTT method and NOS activity by monitoring the conversion of L-[H³]-arginine to L-[H³]-citrulline. The incubation of cultured retina cells with 10 and 100 µM MeHg promoted an increase of NOS activity compared to control (P < 0.05). Maximum values (P < 0.05) were reached after 4 h of MeHg incubation: increases of 81.6 ± 5.3 and 91.3 ± 3.7%, respectively (data are reported as mean ± SEM for 4 replicates). MeHg also promoted a concentration- and time-dependent decrease in cell viability, with the highest toxicity (a reduction of about 80% in cell viability) being observed at the concentration of 1 mM and after 4-6 h of incubation. The present study demonstrates for the first time the modulation of MeHg neurotoxicity in retinal cells by the nitrergic system.

Inducible nitric oxide synthase and tumor necrosis factor-alpha in delayed gastric emptying and gastrointestinal transit induced by lipopolysaccharide in mice

Gastrointestinal motility disturbances during endotoxemia are probably caused by lipopolysaccharide (LPS)-induced factors: candidates include nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), interleukin-1ß, and interleukin-6. Flow cytometry was used to determine the effects of LPS and these factors on gastric emptying (evaluated indirectly by determining percent gastric retention; %GR) and gastrointestinal transit (GIT) in male BALB/c mice (23-28 g). NO (300 µg/mouse, N = 8) and TNF-alpha (2 µg/mouse, N = 7) increased (P < 0.01) GR and delayed GIT, mimicking the effect of LPS (50 µg/mouse). During early endotoxemia (1.5 h after LPS), inhibition of inducible NO synthase (iNOS) by a selective inhibitor, 1400 W (150 µg/mouse, N = 11), but not antibody neutralization of TNF-alpha (200 µg/mouse, N = 11), reversed the increase of GR (%GR 78.8 ± 3.3 vs 47.2 ± 7.5%) and the delay of GIT (geometric center 3.7 ± 0.4 vs 5.6 ± 0.2). During late endotoxemia (8 h after LPS), both iNOS inhibition (N = 9) and TNF-alpha neutralization (N = 9) reversed the increase of GR (%GR 33.7 ± 2.0 vs 19.1 ± 2.6% (1400 W) and 20.1 ± 2.0% (anti-TNF-alpha)), but only TNF-alpha neutralization reversed the delay of GIT (geometric center 3.9 ± 0.4 vs 5.9 ± 0.2). These findings suggest that iNOS, but not TNF-alpha, is associated with delayed gastric emptying and GIT during early endotoxemia and that during late endotoxemia, both factors are associated with delayed gastric emptying, but only TNF-alpha is associated with delayed GIT.

Association of the 894G>T polymorphism of the endothelial constitutive nitric oxide synthase gene with unstable angina

The 894G>T polymorphism of the endothelial constitutive nitric oxide synthase gene consists of the substitution of a guanine base by a thymine at the 894th nucleotide of the gene. An association of this polymorphism with acute coronary syndromes has been described, only when in combination with other polymorphisms of this gene. The aim of the present study was to search for an association between this polymorphism and unstable angina in a southern Brazilian population. In a case-control study, 156 patients (group 1 (N = 83): unstable angina, group 2 (N = 73): stable angina) were genotyped by PCR and digestion of the product. Univariate analysis demonstrated that the minimal luminal diameter and the degree of stenosis of the culprit lesion differed between groups (P = 0.006 and 0.005, respectively). In addition, the frequencies of the T allele and of the T allele carriers (combined TT and TG genotypes) were significantly higher in the group with unstable angina (41.6 vs 28.8%; P = 0.025, Pearson chi-square test, and 73.5 vs 45.2%; P = 0.001, Pearson chi-square test, respectively). Multivariate logistic regression showed that the frequency of the T allele carriers was the only variable with a predictive value for unstable angina, when controlled for the other variables (6.1 (95% CI = 2.55-14.43); P < 0.001). Thus, in a homogenous group of patients, the endothelial constitutive nitric oxide synthase 894G>T polymorphism was associated with unstable angina. We suggest that this polymorphism may be a genetic risk factor for unstable angina.