Endothelial Constitutive Nitric Oxide Synthase Research Articles

Nitric oxide formation and corresponding relaxation of porcine coronary arteries induced by plant phenols: essential structural features.

The high intake of polyphenols is thought to contribute to the beneficial cardiovascular effects of plant-centered diets. A putative mechanism underlying the cardioprotective activity is thought to be a plant phenol-induced increase of nitric oxide formation by the constitutive endothelial nitric oxide synthase. Twenty-eight phenols of different classes commonly occurring in plant foods were examined for their capability of enhancing the endothelial nitric oxide release of isolated porcine coronary arteries by direct real-time measurement of the luminal surface nitric oxide concentration with an amperometric microsensor. Additionally, the relaxing activity of the phenols was measured on porcine coronary rings. Quercetin, myricetin, leucocyanidol, and oligomeric proanthocyanidins induced the highest increases in nitric oxide release (delta[NO] > 8.5 nM ); caffeic acid, fisetin, hyperosid, and isoquercitrin were moderately active (5 nM < delta[NO] < 8.5 nM ); the other phenolic compounds caused only marginal increases of the nitric oxide levels (delta[NO] < 5 nM). The nitric oxide-stimulating activity of the phenols was uniformly positively correlated with their vasorelaxing activity. However, endothelium-dependent vasorelaxations were limited to phenols inducing nitric oxide elevations > 5 nM (= Km value of the soluble guanylate cyclase). Analysis of structure-activity relations revealed that a high nitric oxide activity was confined to a flavan-moiety with free hydroxyl-residues at C3, C3', C4', C5, and C7 and a hydroxyl-, oxo-, or phenolic substituent at C4, whereas the caffeic acid scaffolding emerged as the minimally essential motif for the nitric oxide-dependent vasorelaxation.

Effect of mixed tocopherols on ecNOS, SOD and PKC in leukocytes in human subjects

Epidemiological studies have shown that intake of vitamin E derived from food decreases the risk of cardiovascular disease. However, recent clinical studies revealed no beneficial effect of α-tocopherol alone on cardiac death. Absence of other tocopherols in these latter studies may explain the conflicting results. This study was designed to compare the effects of mixed tocopherols with α-tocopherol alone on endothelial constitutive nitric oxide synthase (ecNOS), superoxide dismutase (SOD) and protein kinase C (PKC) in human leukocytes, which are known to be involved in cardiovascular disease. Sixty-four subjects were randomized into three groups: α-tocopherol, mixed tocopherols, and controls. After 8 weeks of tocopherol supplementation, SOD activity in plasma, ecNOS activation and SOD protein expression in leukocytes were increased, and PKC activation in leukocytes was decreased. Mixed tocopherols had a more potent effect than α-tocopherol alone on ecNOS activation, which may be of importance for the beneficial effects of mixed tocopherols on leukocyte function and cardiovascular disease.

Endothelial nitric oxide synthase gene and the development of diabetic nephropathy

Background: Endothelial nitric oxide synthase gene and the development of diabetic nephropathy Background: Intron 4 insertion/deletion polymorphism of the constitutive endothelial nitric oxide synthase (ecNOS) gene may be related to diabetic nephropathy. Methods: A case-control study was performed in three groups of Japanese patients with Type 2 diabetes mellitus, which including 123 patients with advanced diabetic nephropathy, 107 patients with overt proteinuria and normal serum creatinine level, and a control group of 203 patients with normal renal function despite having diabetes for over 10 years. Additionally, logistic regression analysis was used to assess the findings. Results: When we examined the a-deletion/b-insertion in intron 4 of ecNOS gene, the genotype and allele frequencies were not significantly different between the patients with advanced diabetic nephropathy (a/a 2.4, a/b 21.9, b/b 75.5, ‘a’ 13.4, ‘b’ 86.6%), the patients with overt proteinuria (a/a 2.8, a/b 15.8, b/b 81.4, ‘a’ 10.7, ‘b’ 89.3%) and the control group (a/a 1.4, a/b 21.6, b/b 76.8, ‘a’ 12.8, ‘b’ 87.7%). Logistic regression analysis showed that the ecNOS intron4 a-allele frequency was not the related to nephropathy ( P=0.88). Conclusion: We conclude that there is no association of the ecNOS gene polymorphism with the development of diabetic nephropathy in Japanese patients with type 2 diabetes.

Preoperative glutamine administration induces heat-shock protein 70 expression and attenuates cardiopulmonary bypass-induced inflammatory response by regulating nitric oxide synthase activity.

Heat-shock protein 70 (HSP70) plays a major role in the pathophysiology of inflammation, and the induction of HSP70 before the onset of inflammation can reduce organ damage through a self-protective system. Glutamine is known to be an inducer of HSP70, and its preoperative administration seems useful in attenuating cardiopulmonary bypass (CPB)-induced inflammatory response. Adult male Sprague-Dawley rats (group G, received 100 mg/kg of glutamine via the right jugular vein 3 times per day for 1 week and just before the initiation of CPB; group C served as control) underwent CPB (60 minutes, 100 mL/kg per minute, 34 degrees C) and were killed 3 hours after the termination of CPB. Group G showed significantly lower plasma concentrations of interleukin-6 and interleukin-8 after CPB termination. Myocardial and respiratory damages were significantly attenuated in group G, as evidenced by Langendorff perfusion, respiratory index, and neutrophil adherence. HSP70 expressions in the heart, lung, and liver were detected only in group G before CPB and were markedly stronger in group G 3 hours after CPB termination. Although plasma nitrate+nitrite concentrations were not significantly different between the groups, endothelial-constitutive nitric oxide synthase (NOS) activity was markedly preserved and inducible NOS activity was markedly attenuated in the tissues of group G. These results suggest that preoperative glutamine administration induces HSP70 expression before CPB and attenuates CPB-induced inflammation by regulating NOS activity, which may be a prospective management for conferring tolerance to CPB-induced inflammatory response through a self-protective mechanism.

EcNOS gene polymorphism is associated with endothelium-dependent vasodilation in Type 2 diabetes.

The association between endothelial constitutive nitric oxide synthase (ecNOS) gene polymorphism and vascular endothelial function has not been clarified. We investigated the impact of ecNOS gene polymorphism on endothelial function in 95 patients with Type 2 diabetes (ecNOS genotype: 4b/b, n = 62; 4b/a, n = 30; 4a/a, n = 3). Flow-mediated (endothelium dependent, FMD) and nitroglycerin-induced (endothelium independent, NTG) vasodilations of the right brachial artery were studied using a phase-locked echotracking system. There were no significant differences in clinical characteristics among the ecNOS genotypes. The FMD was significantly lower in the patients with ecNOS4a allele than in those without ecNOS4a allele (P < 0.05). Multiple regression analysis showed that ecNOS4a allele and mean blood pressure were significant independent determinants for reduced FMD in all patients (R(2) = 0.122, P = 0.0025). The ecNOS4a allele was an independent determinant for reduced FMD in smokers but not in nonsmokers. These results suggest that ecNOS4a allele is a genetic risk factor for endothelial dysfunction in diabetic patients, especially in smokers.

Association of the endothelial nitric oxide synthase gene polymorphism with risk of coronary artery disease and myocardial infarction in middle-aged men.

Nitric oxide (NO), formed by endothelial constitutive nitric oxide synthase (eNOS) maintains endothelium-dependent vasodilatation and also mediates antithrombotic actions. The eNOS gene harbours a common polymorphism in intron 4 (4a/b), and some clinical studies have suggested an association of the rare a-allele with coronary artery disease (CAD) and myocardial infarction (MI). However, contradictory results have also been reported. We studied associations of eNOS polymorphism with CAD and MI in two prospective autopsy series comprising altogether 700 Caucasian Finnish men, who died suddenly. In ANCOVA, no significant differences in areas of atherosclerotic lesions and coronary stenosis percentages were found between men carrying the a-allele (ba+aa) compared with those homozygous for the b-allele. Subjects with the a-allele had significantly lower risk of MI (odds ratio 0.44, 95% confidence interval 0.25-0.77, P=0.004) compared with those carrying the bb genotype. Men with the a-allele also tended to have coronary thrombosis less often (odds ratio 0.43, 95% confidence interval 0.18-1.01, P=0.055). The eNOS gene 4a/b polymorphism was not associated with the extent of coronary atherosclerosis, but the a-allele of the variant seems to protect to some degree against the development of MI.

Independent Risk Factor for Moderate to Severe Internal Carotid Artery Stenosis: T786C Mutation of the Endothelial Nitric Oxide Synthase Gene

NO synthesized from L-arginine by the constitutive endothelial NO synthase (eNOS) plays a key role in the atherosclerotic process. We investigated whether common variants in the NOS3 gene (a T786C mutation in the 5' flanking region and the polymorphism on exon 7 that produced the Glu298Arg polymorphism in the protein) are associated with an increased risk of moderate to severe internal carotid artery (ICA) stenosis. We studied 88 patients consecutively operated for ICA stenosis and 133 healthy controls. A T786C mutation in the 5' flanking region and the polymorphism in exon 7 that produces the Glu298Asp polymorphism in the protein were explored by PCR and fluorescent probe analysis. Genotype distribution was significantly different between patients and controls only for T786C, the CC genotype frequency being 26% and 13%, respectively [odds ratio (OR), 2.26; 95% confidence interval (CI), 1.14-4.46; P = 0.018]. Moreover, the CC genotype was significantly more frequent in a subgroup of patients with ulcerative plaques compared with patients with nonulcerative lesions (44% vs 17%; OR, 3.82; 95% CI, 1.79-8.14; P = 0.003). Multiple logistic regression analysis using the most frequent risk factors and the eNOS gene variant showed that the CC genotype is an independent risk factor for ICA stenosis (P = 0.023). C allele homozygosity in position 786 of the eNOS promoter seems to be an independent risk factor for the development of moderate to severe ICA stenosis, especially ulcerative lesions.

The Systemic Inflammatory Response to Cardiac Surgery

The Systemic Inflammatory Response to Cardiac Surgery

Plasma pteridine concentrations in patients with chronic renal failure.

Pteridine metabolism is impaired in the uraemic state. This may affect cardiovascular function and contribute to malnutrition. We wished to clarify further the impact of impaired pteridine metabolism. Using the HPLC method, the plasma concentrations of endogenous pteridines were determined in 64 patients with chronic renal failure (33 on intermittent haemodialysis (HD) treatment vs 31 not yet on renal replacement therapy), and in 18 healthy controls. The patients were classified into three groups on the basis of creatinine clearance (Ccr): group (a), Ccr >60 ml/min; group (b), Ccr=10-60 ml/min; group (c), all patients receiving HD. Total neopterin (NP) and biopterin (BP) levels and the NP/BP ratio (a biomarker for macrophage activity) were significantly higher, whereas tetrahydrobiopterin (BH(4))/dihydrobiopterin (BH(2)) ratio (a biomarker for nitric oxide synthase and phenylalanine hydroxylase activities) was significantly lower in group (c) (118.9+/-11.7 ng/ml, 18.8+/-1.2 ng/ml, 6.79+/-0.53, and 0.26+/-0.06) than in healthy subjects (5.17+/-0.29 ng/ml, 2.83+/-0.19 ng/ml, 1.92+/-0.13, and 1.15+/-0.11; P<0.01). These significant differences were also observed between control and group (b) (12.4+/-2.20 ng/ml, 4.48+/-0.36 ng/ml, 2.81+/-0.48, and 0.74+/-0.08; P<0.01). In groups (a) and (b), significant negative correlations were found between Ccr and the total NP level (r=-0.663, P<0.01), the total BP level (r=-0.492, P<0.01), the BH(2) level (r=-0.677, P<0.01), and the NP/BP ratio (r=-0.493, P<0.01). Conversely, significant positive correlations were found between Ccr and the BH(4)/BH(2) ratio (r=0.602, P<0.01). The reduction of quinoid-type BH(2) to BH(4) is modified in patients with advanced chronic renal failure, before and after the initiation of regular HD treatment. These metabolic alterations may play a role in the impaired macrophage, endothelial constitutive nitric oxide synthase, or phenylalanine hydroxylase (PH) activities observed in such patients.

Hepatocyte growth factor-induced endothelial cell motility is mediated by the upregulation of inducible nitric oxide synthase expression.

Hepatocyte growth factor (HGF) is an angiogenic mitogen which stimulates migration in various cell types and has been shown to induce the production of nitric oxide (NO) in epithelial cells. Conflicting data exist on the effect of NO on endothelial cell migration. The aim of this study was to investigate a possible role for NO in HGF-stimulated endothelial cell motility. The study was performed primarily using an endothelial cell line derived from adult human saphenous vein. Transient transfection experiments were additionally performed using an adult human coronary artery endothelial cell line. Nitric oxide synthase expression was examined by western blot analysis. Time-lapse digital image microscopy was used to measure cell motility. A DNA construct was used in transient transfections to over-express inducible nitric oxide synthase (iNOS) as an N-terminal fusion to enhanced green fluorescent protein (EGFP). HGF upregulated the expression of iNOS but not constitutive endothelial nitric oxide synthase (eNOS). Treatment of cells with the specific iNOS inhibitor 1400 W revealed that functional iNOS was required for HGF-stimulated endothelial cell motility. HGF-induced iNOS expression was partially abrogated in the presence of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002, but not the Src kinase inhibitor, PP1. Endothelial cell motility increased significantly (P<0.0001) in the presence of the exogenous NO donor spermine-NO and cells expressing the iNOS-EGFP fusion protein exhibited significantly greater (P=0.0038) motility than those expressing EGFP alone. These combined data show that elevated NO production is sufficient to stimulate endothelial cell motility and link HGF and NO, both previously implicated in modulating motility, in a common signalling pathway.

The cardiovascular response in sepsis: proposed mechanisms of the beneficial effect of adrenomedullin and its binding protein (Review)

Sepsis and its complications are leading causes of morbidity and mortality. A better understanding of the mechanisms responsible for the shift from the early, hyperdynamic phase of sepsis to the late hypodynamic phase could lead to novel therapies that might improve the outcome of the septic patient. Adrenomedullin is a vasodilatory peptide which shows sustained elevation starting early in sepsis and is important in initiating the hyperdynamic response. As sepsis progresses, however, the vascular response to adrenomedullin is blunted and this decreased sensitivity is important in producing the shift to the late, hypodynamic phase. The decline in the vascular response to adrenomedullin is related to a sepsis-induced decrease in the binding protein for adrenomedullin (i.e., adrenomedullin binding protein-1) rather than a change in gene expression of the components of adrenomedullin receptors. Treatment of septic animals with the combination of adrenomedullin and its binding protein prevents the transition to the late phase of sepsis, maintains cardiovascular stability, and reduces sepsis-induced mortality. We propose that the mechanisms responsible for the beneficial effect of adrenomedullin and adrenomedullin binding protein-1 in sepsis are associated with downregulation of proinflammatory cytokines (TNF-alpha, IL-1beta, IL-6), maintainence of endothelial constitutive nitric oxide synthase, and reduction of vascular endothelial cell apoptosis.

Atorvastatin inhibition of cytokine‐inducible nitric oxide synthase expression in native endothelial cells in situ

Animal experimental studies have demonstrated that inducible nitric oxide synthase (iNOS) expression correlates with neointima formation and is prevented by HMG-CoA reductase inhibitors (statins). In the present study we have investigated the underlying mechanism of action of these drugs in isolated segments of the rat aorta. Western blot analysis and immunohistochemistry revealed that tumour necrosis factor alpha (TNFalpha) plus interferon-gamma (IFNgamma) synergistically induce iNOS gene expression in the endothelium but not in the smooth muscle of these segments while constitutive endothelial NO synthase (eNOS) abundance was markedly reduced. Pre-treatment with 1 - 10 microM atorvastatin, cerivastatin or pravastatin decreased TNFalpha plus IFNgamma stimulated iNOS expression in the endothelium irrespective of the presence of the HMG-CoA reductase product mevalonate (400 microM). Electrophoretic mobility shift assay experiments confirmed that the combination of TNFalpha plus IFNgamma causes activation of the transcription factors STAT-1 and NF-kappaB in native endothelial cells. Neutralization of these transcription factors by employing the corresponding decoy oligonucleotides confirmed their involvement in TNFalpha plus IFNgamma stimulated iNOS expression. Translocation of both transcription factors was attenuated by atorvastatin, and this effect was insensitive to exogenous mevalonate. The present findings thus demonstrate a specific HMG-CoA reductase-independent inhibitory effect of statins, namely atorvastatin, on cytokine-stimulated transcription factor activation in native endothelial cells in situ and the subsequent expression of a gene product implicated in vascular inflammation. This effect may be therapeutically relevant and in addition provide an explanation for the reported rapid onset of action of these drugs in humans.

Aged garlic extract enhances production of nitric oxide

Nitric oxide (NO) controls several physiological functions of the cardiovascular system. Three kinds of NO synthases (NOSs), neuronal constitutive NOS (ncNOS), inducible NOS (iNOS) and endothelial constitutive NOS (ecNOS), were responsible for NO biosynthesis. This study investigated the effect of aged garlic extract (AGE) on NO production by measuring the NO metabolites nitrite and nitrate in the plasma of mice. AGE (2.86 g/kg, p.o.) temporarily increased NO production by 30–40% from 15 to 60 min after administration. The time course of the fluctuation in NO levels in the AGE-treated group was clearly different to that in a group of mice treated with lipopolysaccharides, a typical iNOS inducer. Arginine (63 mg/kg, p.o.) at the equivalent dose of AGE did not increase NO production. However diphenyleneiodonium chloride (1 mg/kg, i.p.), a selective cNOS inhibitor, administered prior to AGE, overcame the effect of AGE. These results indicate that AGE increased NO production by activating cNOS, but not iNOS. The arginine contained in AGE was not responsible for the effect. AGE may be a useful tool for the prevention of cardiovascular disease.

Placental imbalance of vasoactive factors does not affect pregnancy outcome in patients treated with Cyclosporine A after transplantation

Endothelin-1 (ET-1) and nitric oxide (NO) have been suggested to have a focal role in the regulation of placental and fetal growth. Cyclosporine A (CsA) has been shown to strongly modulate ET-1 and NO synthesis and thus has the potential to affect fetal growth and maternal state. Eleven CsA-treated female kidney transplant recipients were recruited. Fourteen healthy pregnant women served as controls. Placental expression of ET-1 and tissue factor (TF) was evaluated by in situ hybridization, and NO synthase (NOS) was evaluated by staining with the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH)-diaphorase and in situ hybridization. Kidney transplant recipients showed a marked reduction in NADPH-diaphorase staining, as well as endothelial constitutive NOS (ecNOS) messenger RNA, whereas inducible NOS expression was unchanged. Normal placenta showed a strong positive ET-1 signal along the endothelium of uteroplacental arteries within the basal plate, which increased markedly in decidua of transplant recipients. Thus, transplant recipients showed a remarkable alteration in ET-1/ecNOS balance without alteration in fetal growth or maternal renal function. Next, we explored the state of placental endothelial cell activation downstream from vasoactive factors by evaluating TF gene expression. Transplant recipients did not show modification of TF transcript compared with healthy pregnant women. CsA potently affected the placental ET-1/ecNOS vasoactive balance. Nevertheless, newborns from transplant recipient mothers were appropriate for gestational age, and transplant recipients did not show systemic hypertension or impending renal damage. It is suggested that CsA may blunt the activation of endothelial cells and priming of endothelial-derived substances, which possibly lie downstream from the cited vasoactive agents. © 2002 by the National Kidney Foundation, Inc.

Expression of constitutive endothelial and inducible nitric oxide synthase in the sciatic nerve of Lewis rats with experimental autoimmune neuritis

This study examined the expression of constitutive endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) in the sciatic nerve of Lewis rats with experimental autoimmune neuritis (EAN). Western blot analysis showed that both eNOS and iNOS expressions in the sciatic nerves of rats increased significantly during the peak stage of EAN, but declined thereafter. Only minimal amounts of these enzymes were identified in normal rat sciatic nerves. Immunohistochemical studies showed that eNOS was increased in vascular endothelial cells and Schwann cells, but not in inflammatory cells, during the peak stage of EAN. However, iNOS was found mainly in inflammatory macrophages in sciatic nerve EAN lesions. These findings suggest that, depending on the stage of peripheral nervous system autoimmune disease, the increased expressions of both eNOS and iNOS might be involved in either the production of detrimental effects during the induction stage of EAN or in the recovery from EAN paralysis.

Hypertension after renal transplantation and polymorphism of genes involved in essential hypertension: ACE, AGT, AT1 R and ecNOS.

Arterial hypertension (HT), secondary to cyclosporine A (CsA) used as main immunosuppressive treatment in renal transplantation (RTx), is very frequent (70%), usually severe and explained mostly by vasoconstriction of the glomerular afferent arteriole with secondary sodium and water retention. In a retrospective study, we have analyzed 294 consecutive recipients receiving a first renal cadaveric allograft and all treated with CsA (the majority with triple therapy). We studied, by molecular biology, the polymorphism of genes previously implicated in essential HT such as: angiotensin-converting enzyme (ACE: II, ID and DD), angiotensinogen (AGT: MM, MT and TT), angiotensin II type 1 receptor (AT1-R: AA, AC and CC) and endothelial constitutive nitric oxide synthase (ecNOS: aa, ab and bb), and correlated the data to the prevalence and severity of post-Tx HT. This cohort included 195 (66%) males and 99 females with a mean age of 42 years at time of Tx. The presence and severity of post-Tx HT were indicated by initial persistent blood pressure over 140/90 mmHg with the need for at least one anti-hypertensive drug and by the number of anti-HT medications required to achieve its control. The distribution of the specific alleles and genotypes for ACE, AGT, AT1-R, and ecNOS was not different in transplant recipients compared to 181 controls. At 5 years post-Tx, the prevalence of HT was 72% (169 out of 235) among functioning grafts. There was no significant difference for ACE, AGT, AT1R and ecNOS genotypes distribution between hypertensive vs non-HT recipients. The number of anti-hypertensive drugs prescribed was not different among ACE, AGT, and AT1-R genotypes. However, the a allele and the non-bb genotype (aa + ab) for ecNOS were significantly (p = 0.001) associated with a less severe HT, needing fewer anti-HT drugs. At 10 years post-Tx, the HT prevalence remained high 78% (67 out of 86) among functioning Tx. However, the limited numbers did not allow further correlation. This study produced mainly negative results except for ecNOS-a allele, which seems to protect against severe hypertension. The explanation remains speculative but probably relates to the known cyclosporine-induced upregulation of ecNOS gene and enzyme activity.

Bleeding duodenal ulcer and association with polymorphism of endothelial constitutive nitric oxide synthase gene.

Nitric oxide (NO) exerts both protective and proinflammatory actions in the gastrointestinal tract. Enhanced gastric NO synthase (NOS) activity has been shown in duodenal ulcer patients. Recently, intron-4 polymorphism of the endothelial constitutive (ec) NOS gene has been associated with some pathological conditions. Our aim was to determine the genotype and allele frequencies of the ecNOS4 polymorphism in peptic ulcer patients. The distribution of the polymorphism ecNOS4a/b was studied in 188 ulcer patients and 120 healthy controls, from genomic DNA. Genotypes ab, bb, and aa and allele frequency were similar in both peptic ulcer patients and controls, and no differences were found when patients and controls were analyzed according to the presence of several etiological factors. However, alelle "a" carrier status was associated with decreased risk of bleeding in duodenal ulcer patients (OR = 0.49; 95% CI = 0.25-0.95; P = 0.03). In conclusion, this ecNOS4 polymorphism gene could be related to susceptibility of duodenal ulcer patients to bleeding.

Pathogenesis of portal hypertensive gastropathy: A clinical and experimental review

Portal hypertensive gastropathy (PHG) is recognized as a clinical entity in portal hypertension, but the pathogenesis of PHG is still unclear. Therefore, we reviewed the current state of knowledge concerning the portal hypertensive gastric mucosa and hypothesized the pathogenesis of PHG. Elevated portal pressure can induce changes of local hemodynamics, thus causing congestion in the upper stomach and gastric tissue damage. These changes may then activate cytokines and growth factors, such as tumor necrosis factor alpha, which are substances that activate endothelial constitutive nitric oxide synthase and endothelin 1 in the portal hypertensive gastric mucosa. Overexpressed nitric oxide synthase produces an excess of nitric oxide, which induces hyperdynamic circulation and peroxynitrite overproduction. The overproduction of peroxynitrite, together with endothelin overproduction may cause an increased susceptibility of gastric mucosa to damage. When combined with the characteristics of impaired mucosal defense and healing, these factors may together produce PHG in patients with portal hypertension.

Nitric oxide and apoptosis during human head and neck squamous cell carcinoma development

Purpose: Apoptosis index (AI), Bcl-2, and Bax have shown prognostic significance in head and neck squamous cell carcinoma (HNSCCa). Other areas of research have implicated nitric oxide (NO) or its various intermediate species in both proapoptotic and antiapoptotic processes. We have previously shown that NO-generating enzymes are significantly increased during the stepwise progression to HNSCCa. The aim of this study was to explore the interrelationship of NO and a known consequence of NO-related oxidative stress, apoptosis, during this step-wise process. Materials and Methods: Formalin fixed-paraffin embedded tissue samples of 10 normal oral mucosa, 15 reactive/dysplastic lesions, and 17 HNSCCa lesions studied previously were subjected to the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP labeling (TUNEL) assay as well as immunohistochemical staining against Bcl-2, Bax, and p53. Patient charts were reviewed and clinical data were compared. The study pathologist (G.K.H) reviewed these slides blinded to patient identifiers or clinical data. The number of immunopositive cell nuclei or staining intensity was graded, noting the pattern of immunostaining. These staining characteristics were compared with the results of immunostaining previously obtained for endothelial constitutive NO synthase (ecNOS) and nitrotyrosine. Results: Compared with normal oral mucosa, the AI, Bcl-2, Bax, Bcl-2/Bax intensity and frequency ratios, and mutant p53 intensity significantly changed in reactive/dysplastic and HNSCCa lesions (P <.001 for all). Correlations between the staining characteristics of the antigens studied are presented. Furthermore, perilesional inflammatory cells showed staining in the TUNEL assay. Conclusions: In a set of tissue samples previously well characterized, these new findings implicate a link between NO and the induction of apoptotic cell death in HNSCCa development. (Am J Otolaryngol 2002;23:4-11. Copyright © 2002 by W.B. Saunders Company)

Enhancement of nitric oxide production by methylecgonidine in cultured neonatal rat cardiomyocytes.

1. In the present experiments, we investigated the effects of methylecgonidine (MEG) on nitric oxide (NO) production in cultured neonatal rat cardiomyocytes. Incubation of cultured cardiomyocytes with carbachol or MEG for 48 h significantly enhanced NO production. No release was increased from 1.48+/-0.13 microM (mg protein)(-1) for control to 5.73+/-0.19 microM (mg protein)(-1) for 1 microM carbachol treated cells (P<0.001). In addition, incubation with 1 microM MEG enhanced NO production to 5.55+/-0.28 microM (mg protein)(-1). The effects of MEG on NO production were concentration-dependent. The muscarinic antagonist atropine prevented the enhancement of NO production induced by carbachol or MEG. Compared to MEG-induced NO production, cocaine was much less potent. 2. The enhancement of NO production by carbachol or MEG was even greater in cultured cardiomyocytes transfected with the M(2) cDNA. After 48-h incubation with 1 microM carbachol or 1 microM MEG, NO production was increased by 6.5 and 6.7 fold, respectively, in cardiomyocytes overexpressing M(2) receptors. Coincubation with atropine or N(G)-nitro-L-arginine methyl ester abolished the enhancement of NO production. In contrast, NO production enhanced by carbachol or MEG in M(1)- or M(3)-transfected cardiomyocytes was similar to the level in non-transfected cells. 3. Western blot analysis showed that the protein levels of M(1), M(2), and M(3) were significantly increased in cardiomyocytes transfected with the receptor cDNAs, but MEG had no effect on the expressions. It is interesting that both carbachol and MEG caused a significant increase in constitutive endothelial NO synthase (eNOS) only in M(2)-transfected cardiomyocytes, not in non-transfected, M(1)- or M(3)-transfected cells. Again, atropine blocked the MEG-produced induction of eNOS. 4. Our data demonstrate that MEG significantly enhanced NO production in cultured cardiomyocytes and that the enhancement of NO production may result from MEG stimulation of muscarinic M(2) receptors.