Effects of acute treatment with dexamethasone on hemodynamic and histopathological changes in rats

We assessed the time-dependent effects of intraperitoneal (i.p.) and intravenous (i.v.) application of dexamethasone (Dexa) on the mean arterial blood pressure (MAP), heart rate (HR) and total blood volume (TBV). We evaluated also the relation between the effects and immunoreactivities of transforming growth factor-beta (TGF-β), epithelial nitric oxide synthase (eNOS), interleukin-1 beta (IL1-β) and vascular endothelial growth factor (VEGF) in rat brain, lung and kidney tissues. Rats were anesthetized and while still breathing spontaneously, a tracheotomy and femoral vein and artery catheterizations were performed. To determine TBV using the hemodilution method, 2 ml albumin-electrolyte solutions were applied by i.v. injection. Group 1 (control group) received a 1 ml bolus injection of physiologic saline, Group 2 received 15 mg/kg and Group 3 received 75 mg/kg Dexa i.p. The hematocrit was measured at 10, 20, 60 and 120 min. For each animal, the values of MAP, HR and TBV were measured within 2 h. For immunohistochemical evaluation, anti-TGF-β, anti-eNOS, anti-IL1-β and anti-VEGF primary antibodies were tested using the avidin-biotin-peroxidase method. TBV was decreased in Group 1 and the increase in MAP was statistically significant. HR values increased slightly. None of the values changed significantly in Group 2. Although TBV was unchanged in Group 3, the decrease in MAP was statistically significant. HR values increased, but the increase was not statistically significant. Mild IL1-β immunoreactivity and moderate TGF-β, eNOS and VEGF immunoreactivities were observed in the brain, lung and kidney samples in Group 1. Increased eNOS immunoreactivity in the kidney samples were observed in Group 2. eNOS immunoreactivity was as strong in the brain and the kidney samples in Group 3. Decreased VEGF immunoreactivity was observed in the lung and kidney tissues in Group 3. Significantly decreased TGF-β immunoreactivity was observed in all tissue samples in Group 3. The decreased MAP values in Group 3 differed from those in Groups 1 and 2. Despite increased eNOS immunoreactivity, especially in brain and kidney, the decrease in VEGF immunoreactivity in Group 3, especially lung and kidney, were consistent with a drop in blood pressure.     

Effects of acute treatment with dexamethasone on hemodynamic and histopathological changes in rats

Abstract

We assessed the time-dependent effects of intraperitoneal (i.p.) and intravenous (i.v.) application of dexamethasone (Dexa) on the mean arterial blood pressure (MAP), heart rate (HR) and total blood volume (TBV). We evaluated also the relation between the effects and immunoreactivities of transforming growth factor-beta (TGF-β), epithelial nitric oxide synthase (eNOS), interleukin-1 beta (IL1-β) and vascular endothelial growth factor (VEGF) in rat brain, lung and kidney tissues. Rats were anesthetized and while still breathing spontaneously, a tracheotomy and femoral vein and artery catheterizations were performed. To determine TBV using the hemodilution method, 2 ml albumin-electrolyte solutions were applied by i.v. injection. Group 1 (control group) received a 1 ml bolus injection of physiologic saline, Group 2 received 15 mg/kg and Group 3 received 75 mg/kg Dexa i.p. The hematocrit was measured at 10, 20, 60 and 120 min. For each animal, the values of MAP, HR and TBV were measured within 2 h. For immunohistochemical evaluation, anti-TGF-β, anti-eNOS, anti-IL1-β and anti-VEGF primary antibodies were tested using the avidin-biotin-peroxidase method. TBV was decreased in Group 1 and the increase in MAP was statistically significant. HR values increased slightly. None of the values changed significantly in Group 2. Although TBV was unchanged in Group 3, the decrease in MAP was statistically significant. HR values increased, but the increase was not statistically significant. Mild IL1-β immunoreactivity and moderate TGF-β, eNOS and VEGF immunoreactivities were observed in the brain, lung and kidney samples in Group 1. Increased eNOS immunoreactivity in the kidney samples were observed in Group 2. eNOS immunoreactivity was as strong in the brain and the kidney samples in Group 3. Decreased VEGF immunoreactivity was observed in the lung and kidney tissues in Group 3. Significantly decreased TGF-β immunoreactivity was observed in all tissue samples in Group 3. The decreased MAP values in Group 3 differed from those in Groups 1 and 2. Despite increased eNOS immunoreactivity, especially in brain and kidney, the decrease in VEGF immunoreactivity in Group 3, especially lung and kidney, were consistent with a drop in blood pressure.     

Angiotensin II type 1 receptor blocker preserves tolerance to ischemia-reperfusion injury in Dahl salt-sensitive rat heart

Oxidative stress is involved in the tolerance to ischemia-reperfusion (I/R) injury. Because angiotensin II type 1 receptor blockers (ARBs) inhibit oxidative stress, there is concern that ARBs abolish the tolerance to I/R injury. Dahl salt-sensitive (DS) hypertensive and salt-resistant (DR) normotensive rats received an antioxidant, 2-mercaptopropionylglycine (MPG), or an ARB, losartan, for 7 days. Losartan and MPG significantly inhibited oxidative stress as determined by tissue malondialdehyde + 4-hydroxynoneal and increased expression of inducible nitric oxide synthase (iNOS) in the DS rat heart. However, losartan but not MPG activated endothelial nitric oxide synthase (eNOS) as assessed by phosphorylation of eNOS on Ser1177. Infarct size after 30-min left coronary artery occlusion followed by 2-h reperfusion was comparable between DS and DR rat hearts. Although MPG and losartan had no effect on infarct size in the DR rat heart, MPG but not losartan significantly increased infarct size in the DS rat heart. A selective iNOS inhibitor, 1400W, increased infarct size in the DS rat heart, but it had no effect on infarct size in the losartan-treated DS rat heart. However, a nonselective NOS inhibitor, Nomega-nitro-l-arginine methyl ester, increased infarct size in the losartan-treated DS rat heart. These results suggest that losartan preserves the tolerance to I/R injury by activating eNOS despite elimination of redox-sensitive upregulation of iNOS and iNOS-dependent cardioprotection in the DS rat heart.     

Control of nitric oxide synthase expression by transforming growth factor-β: Implications for homeostasis

Production of nitric oxide (NO) can be stimulated by inflammatory cytokines and bacterial lipopolysaccharide (LPS) in mammalian cells via an inducible nitric oxide synthase (iNOS). Conversely, the transforming growth factor-βs (TGF-βs) suppress NO production by reducing iNOS expression. Production of NO leads to disparate consequences, some beneficial and some damaging to the host, depending on the cell and context in which iNOS is induced. The TGF-βs counter these NO-mediated processes in macrophages, cardiac myocytes, smooth muscle cells, bone marrow cells, and retinal pigment epithelial cells. Autocrine or paracrine production of TGF-β may thus serve as a physiological counterbalance for iNOS expression, a mechanism which may be subverted by pathogens and tumors for their own survival. A greater understanding of the mechanisms and consequences of NO and TGF-β production may lead to effective therapeutic strategies in various diseases.     

Genistein prevents isoproterenol-induced cardiac hypertrophy in rats

Genistein, an isoflavone and a rich constituent of soy, possesses important regulatory effects on nitric oxide (NO) synthesis and oxidative stress. Transient and low release of NO by endothelial nitric oxide synthase (eNOS) has been shown to be beneficial, while high and sustained release by inducible nitric oxide synthase (iNOS) may be detrimental in pathological cardiac hypertrophy. The present study was designed to evaluate whether genistein could prevent isoproterenol-induced cardiac hypertrophy in male Wistar rats (150-200 g, 10-12 weeks old) rats. Isoproterenol (5 mg·(kg body weight)(-1)) was injected subcutaneously once daily for 14 days to induced cardiac hypertrophy. Genistein (0.1 and 0.2 mg·kg(-1), subcutaneous injection once daily) was administered along with isoproterenol. Heart tissue was studied for myocyte size and fibrosis. Myocardial thiobarbituric acid reactive substances (TBARS), glutathione (GSH), superoxide dismutase (SOD), catalase levels, and 1-OH proline (collagen content) were also estimated. Genistein significantly prevented any isoproterenol-induced increase in heart weight to body weight ratio, left ventricular mass (echocardiographic), myocardial 1-OH proline, fibrosis, myocyte size and myocardial oxidative stress. These beneficial effects of genistein were blocked by a nonselective NOS inhibitor (L-NAME), but not by a selective iNOS inhibitor (aminoguanidine). Thus, the present study suggests that the salutary effects of genistein on isoproterenol-induced cardiac hypertrophy may be mediated through inhibition of iNOS and potentiation of eNOS activities.     

Disruption of COX-2 and eNOS does not confer protection from cardiovascular failure in lipopolysaccharide-treated conscious mice and isolated vascular rings

It was hypothesized that a serial stimulation of vascular cyclooxygenase-2 (COX-2) with subsequent activation of endothelial nitric oxide synthase (eNOS) is responsible for decrease in blood pressure, cardiac performance, and vascular reactivity in endotoxemia caused by LPS. The hypothesis was tested in catheterized, conscious, freely moving, wild-type mice and mice (C57BL/6J background) with targeted deletion of COX-2 and eNOS that were given an intravenous LPS bolus (2 mg/kg, 055:B5). In vitro studies were performed on murine aorta rings. LPS caused a concomitant decrease in mean arterial blood pressure (MAP) and heart rate (HR) that was significant after 3 h and was sustained throughout the experiment (8 h). The LPS-induced changes in MAP and HR were not different from control in COX-2(-/-) and eNOS(-/-) mice. A prostacyclin receptor antagonist (BR5064) blocked the hypotensive effect of a prostacyclin agonist (beraprost), but did not attenuate the LPS-induced decrease in MAP and HR. LPS decreased eNOS and neuronal NOS mRNA abundances in several organs, while inducible NOS mRNA was enhanced. In aortic rings, LPS suppressed α(1)-adrenoceptor-mediated vascular tone. Inhibition of COX-2 activity (NS 398), disruption of COX-2, endothelium removal, or eNOS deletion (eNOS(-/-)) did not improve vascular reactivity after LPS, while the NO synthase blockers 1400W and N(G)-nitro-l-arginine methyl ester prevented loss of tone. COX-2 and eNOS activities are not necessary for LPS-induced decreases in blood pressure, heart rate, and vascular reactivity. Inducible NOS activity appears crucial. COX-2 and eNOS are not obvious therapeutic targets for cardiovascular rescue during gram-negative endotoxemic shock.     

Increased expression of iNOS is associated with endothelial dysfunction and impaired pressor responsiveness in streptozotocin-induced diabetes

Studies in streptozotocin (STZ)-induced diabetic rats have demonstrated cardiovascular abnormalities such as depressed mean arterial blood pressure (MABP) and heart rate (HR), endothelial dysfunction, and attenuated pressor responses to vasoactive agents. We investigated whether these abnormalities are due to diabetes-associated activation of inducible nitric oxide synthase (iNOS). In addition, the effect of the duration of diabetes on these abnormalities was also evaluated. Diabetes was induced by administration of 60 mg/kg STZ via the tail vein. One, 3, 9, or 12 wk after STZ injection, MABP, HR, and endothelial function were measured in conscious unrestrained rats. Pressor response curves to bolus doses of methoxamine (MTX) and angiotensin II (ANG II) were constructed in the presence of N-[3(aminomethyl)benzyl]-acetamidine, dihydrochloride (1400W), a specific inhibitor of iNOS. Depressed MABP and HR and impairment of endothelial function were observed as early as 3 wk after induction of diabetes. Acute inhibition of iNOS with 1400W (3 mg/kg i.v.) restored the attenuated pressor responses to both MTX and ANG II without affecting the basal MABP and HR. Immunohistochemical and Western analysis blot studies in cardiovascular tissues revealed decreased expression of endothelial nitric oxide synthase (eNOS) concomitant with increased expression of iNOS and nitrotyrosine with the progression of diabetes. Our findings suggest that induction of iNOS in cardiovascular tissues is dependent on the duration of diabetes and contributes significantly to the depressed pressor responses to vasoactive agents and potentially to endothelial dysfunction.     

Effect of calcitonin gene-related peptide on nitric oxide production in osteoblasts: an experimental study

The aim of this study was to investigate the in vitro effects and regulatory mechanism of CGRP (calcitonin gene-related peptide) on NO (nitric oxide) production in osteoblasts. MOB (primary human mandibular osteoblasts) and osteoblast-like cells (MG-63) were either cultured with CGRP or co-incubated with inhibitors targeting eNOS (endothelial nitric oxide synthase), iNOS (inducible nitric oxide synthase), nNOS (neuronal nitric oxide synthase) and [Ca2+]i (intracellular Ca2+). The NO concentration in cell culture supernatants was measured during the first 24 h using the Griess test; cellular NO was marked with the fluorescent marker DAF-FM, DA (3-amino, 4-aminomethyl-2',7'-difluorescein; diacetate) and measured by fluorescence microscopy from 1 to 4 h after treatment. eNOS and iNOS mRNA expression levels were measured by quantitative RT-PCR during the first 24 h after treatment. CGRP-induced NO production in the supernatants was high between 1 to 12 h, while cellular NO was highest between 1 to 2 h after treatment and returned to basal levels by 3 h. Both in MG-63 cells and MOBs, the most effective CGRP concentration was 10 nM with a peak time of 1 h. CGRP-induced NO production decreased when eNOS activity was inhibited or when voltage-dependent L-type Ca2+ channels were blocked at 4 h. CGRP was not able to induce changes in iNOS or eNOS mRNA levels and had no effect on the cytokine-induced increase of iNOS expression. Our results suggest that CGRP transiently induces NO production in osteoblasts by elevating intracellular Ca2+ to stimulate the activity of eNOS in vitro.     

Mechanisms involved in the cardiovascular alterations immediately after spinal cord injury

The early cardiovascular effects resulting from an acute spinal cord injury (SCI) produced by a contusion procedure at T5-T6 were evaluated in anaesthetized rats. The mean arterial pressure (MAP) and heart rate (HR) were measured during one hour after the injury. A marked decrease in MAP and HR was observed immediately after injury, followed by an abrupt increase in MAP. These changes were observed between 3 and 9 min and the basal values were recovered after 20 min. Fall in the MAP and HR and increase in MAP induced by SCI were abolished by atropine. The interruption of the parasympathetic outflow by vagotomy also significantly diminished the fall and increase in MAP and the fall in HR. Likewise, pre-treatment with nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) completely abolished the effects produced by SCI. These data suggest that after SCI the decrement in MAP and HR was probably due to acetylcholine release from parasympathetic fibers and NO from endothelial source probably by a cholinergic stimulation. Additionally, the MAP increase observed was probably due to a reflex compensatory vasoconstriction.     

Resveratrol protects myocardial ischemia-reperfusion injury through both NO-dependent and NO-independent mechanisms

We previously showed that resveratrol (3,4',5-trihydroxystilbene) stimulates NO production and is cardioprotective in rat heart subjected to ischemia-reperfusion (I/R rat heart). We now show that in I/R rat heart, inducible nitric oxide synthase (iNOS) expression is markedly induced, while expression of endothelial nitric oxide synthase (eNOS) and nueronal nitric oxide synthase (nNOS) is unchanged. In animals preconditioned with resveratrol (0.5 to 1 mg/kg body wt), I/R-induced iNOS induction is abrogated; however, expression of eNOS and nNOS is greatly upregulated. The protective effects of resveratrol on I/R rat heart include reduced rhythm disturbances, reduced cardiac infarct size, and decreased plasma levels of lactate dehydrogenase (LDH) and creatine kinase (CK). Among these, the reductions in LDH/CK levels and infarct size are NO-dependent as the coadministration of N(omega)-nitro-L-arginine methyl ester (L-NAME, 1 mg/kg body wt) with resveratrol abolishes the resveratrol effect. In contrast, the reductions in the severity of ventricular arrhythmia and mortality rate are not affected by L-NAME coadministration, suggesting that a NO-independent mechanism is involved.     

红花黄色素对新生鼠缺氧后一氧化氮合酶表达的影响

目的:观察红花黄色素对缺氧后脑内诱生型一氧化氮合酶(iNOS)、神经原型一氧化氮合酶(nNOS)及内皮型一氧化氮合酶(eNOS)基因表达的影响,探讨红花黄色素抗缺氧脑损伤的作用.方法:采用SD新生鼠缺氧模型,于缺氧前30 min腹腔注射红花黄色素生药7g/kg,缺氧40 min后复氧48 h,提取脑组织总RNA,应用RT-PCR技术检测三种NOS mRNA的表达量.结果:新生鼠缺氧再复氧48 h,脑内iNOS、nNOS基因表达上升(P＜0.05),预先给予红花黄色素能抑制iNOS、nNOS基因的表达(P＜0.05),但eNOS基因表达不受影响.结论:红花黄色素对缺氧脑损伤的保护作用与NOS基因表达有关.     

白细胞介素—10对血管一氧化氮／一氧化氮合酶系统的影响

为探讨抗炎因子－－白细胞介素－10（IL－10）对大鼠主动脉一氧化氮（NO）／一氧化氮合酶（NOS）系统的影响,应用Griess试剂、^3H－瓜氨酸生成及蛋白免疫印迹杂交等方法,测定IL－10孵育对血管NO释放、NOS活性及表达的影响。结果发现细菌脂多糖（LPS）呈浓度领带性地激活诱导型NOS（iNOS）,促进NO生成。IL－10（10^-10-10^-8g/ml）呈浓度依赖性地上调内皮型NOS（eNOS）蛋白表达及其活性,但对iNOS活性及表达无明显影响,IL－10（10^-9-10^-8g/ml）显著抑制10μg/ml LPS诱导的NO生成和iNOS激活;而高浓度IL－10（10^-7g/ml）则上调iNOS的活性,对eNOS蛋白的表达知活性无明显影响。因此IL－10对NO／NOS系统具有双重影响,一方面可抑制炎症介质诱发的作为炎性物质的iNOS的表达及激活,另一方面可上调内皮源扩血管物质NO的释放。     

Prevention of concentric hypertrophy and diastolic impairment in aortic-banded rats treated with resveratrol

This study was designed to examine the effects of the antioxidant resveratrol on cardiac structure and function in pressure overload (PO)-induced cardiac hypertrophy. Male Sprague-Dawley rats were subjected to sham operation and the aortic banding procedure. A subgroup of sham control and aortic-banded rats were treated with resveratrol for 2 wk after surgery. Echocardiographic analysis of cardiac structure and function along with Western blot analysis of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and redox factor-1 (ref-1) were performed in all groups after 4 wk of surgery. Banded rats showed significantly increased left ventricle-to-body weight ratio. Echocardiographic analysis showed that the interventricular septal wall thickness and left ventricular posterior wall thickness at systole and diastole were significantly increased in banded rats. Also, a significant increase in isovolumic relaxation time was observed in banded rats. Measured eNOS, iNOS, and ref-1 protein levels were significantly reduced in banded rats. Resveratrol treatment prevented the above changes in cardiac structure, function, and protein expression in banded rats. Aortic banding after 4 wk resulted in concentric remodeling and impaired contractile function due to PO on the heart. The 2-wk treatment with resveratrol was found to abolish PO-induced cardiac hypertrophy. Resveratrol may therefore be beneficial against PO-induced cardiac hypertrophy found in clinical settings of hypertension and aortic valve stenosis.     

Is there a role for neuronal nitric oxide synthase (nNOS) in cytokine toxicity to pancreatic beta cells?

Nitric oxide (NO), produced by the action of the inducible NO synthase, plays a crucial role in cytokine toxicity to pancreatic beta cells during type 1 diabetes development. It was the aim of this study to analyze the role of the neuronal NOS (nNOS) in proinflammatory cytokine-mediated beta cell toxicity. Expression of different isoforms of nitric oxide synthase in insulin-secreting INS1E cells and rat islets was analyzed by quantitative real-time PCR and Western blotting. The expression of nNOS in insulin-secreting INS1E cells was similar to that found in rat brain, while two other isoforms, namely the endothelial eNOS and inducible iNOS were not expressed in untreated cells. IL-1β alone or in combination with TNF-α and/or IFNγ induced iNOS but not eNOS expression. In contrast, nNOS expression was strongly decreased by the mixture of the three proinflammatory cytokines (IL-1β, TNF-α and IFNγ) both on the gene and protein level in INS1E cells and rat islet cells. The effects of cytokines on glucose-induced insulin-secretion followed the pattern of nNOS expression reduction and, on the other hand, of the iNOS induction. The data indicate that a low level of nitric oxide originating from the constitutive expression of nNOS in pancreatic beta cells is not deleterious. In particular since proinflammatory cytokines reduce this expression. This nNOS suppression can compensate for NO generation by low concentrations of IL-1β through iNOS induction. Thus, this basal nNOS expression level in pancreatic beta cells represents a protective element against cytokine toxicity.     

Indapamide-induced prevention of myocardial fibrosis in spontaneous hypertension rats is not nitric oxide-related

We studied the effect of thiazide-like diuretic--indapamide on fibrosis development in the left ventricle of young spontaneously hypertensive rats (SHR) and assessed the involvement of nitric oxide in this process. Six-week-old male SHR were treated with indapamide (1 mg/kg/day) for six weeks. Age-matched SHR were used as hypertensive and Wistar-Kyoto rats (WKY) as normotensive control. Systolic blood pressure was measured by tail-cuff plethysmography. Nitric oxide synthase (NOS) activity, protein expressions of endothelial (eNOS) and inducible NOS (iNOS), myocardial fibrosis and collagen type I and III were determined in the left ventricle. Indapamide treatment partially prevented SBP increase in SHR (SHR+Indapamide: 157+/-4, SHR: 171+/-3, WKY: 119+/-3 mmHg). Indapamide prevented myocardial fibrosis development in SHR, but without affecting collagen type I to type III ratio. Indapamide did not affect NOS activity as well as eNOS and iNOS protein expressions in the left ventricles evaluated by both Western blot and immunohistochemically. In conclusion, our results indicate that indapamide-induced prevention of myocardial fibrosis is not mediated by nitric oxide-related mechanism.     

Naringin protects viscera from ischemia/reperfusion injury by regulating the nitric oxide level in a rat model

We investigated the effects of naringin on small intestine, liver, kidney and lung recovery after ischemia/reperfusion (I/R) injury of the gut. Rats were divided randomly into four groups of eight. Group A was the sham control; group B was ischemic for 2 h; group C was ischemic for 2 h and re-perfused for 2 h (I/R); group D was treated with 50 mg/kg naringin after ischemia, then re-perfused for 2 h. Endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) expressions were detected by immunolabeling. We also measured arginase activity, amounts of nitric oxide (NO) and total protein. iNOS was increased significantly in the small intestine, liver and kidney in group C. iNOS was decreased significantly only in small intestine and lung in group D. eNOS was increased significantly in the small intestine, liver and lung in group C. eNOS was decreased in small intestine, liver and lung in group D; however, eNOS was decreased in the kidney in group C and increased in the kidney in group D. The amount of NO was decreased significantly in all tissues in group D, but arginase activity was decreased in the small intestine and lung, increased in the kidney and remained unchanged in the liver in group D. The total protein increased in the small intestine and liver in group D, but decreased significantly in the kidney and lung in group D. Naringin had significant, salutary effects on the biochemical parameters of I/R by decreasing the NO level, equilibrating iNOS and eNOS expressions, and decreasing arginase activity.     

Nitric oxide synthases in infants and children with pulmonary hypertension and congenital heart disease

Rationale

Nitric oxide is an important regulator of vascular tone in the pulmonary circulation. Surgical correction of congenital heart disease limits pulmonary hypertension to a brief period.

Objectives

The study has measured expression of endothelial (eNOS), inducible (iNOS), and neuronal nitric oxide synthase (nNOS) in the lungs from biopsies of infants with pulmonary hypertension secondary to cardiac abnormalities (n = 26), compared to a control group who did not have pulmonary or cardiac disease (n = 8).

Methods

eNOS, iNOS and nNOS were identified by immunohistochemistry and quantified in specific cell types.

Measurements and main results

Significant increases of eNOS and iNOS staining were found in pulmonary vascular endothelial cells of patients with congenital heart disease compared to control infants. These changes were confined to endothelial cells and not present in other cell types. Patients who strongly expressed eNOS also had strong expression of iNOS.

Conclusion

Upregulation of eNOS and iNOS occurs at an early stage of pulmonary hypertension, and may be a compensatory mechanism limiting the rise in pulmonary artery pressure.     

Hypoxia-induced regulation of nitric oxide synthase in cardiac endothelial cells and myocytes and the role of the PI3-K/PKB pathway

The roles of endothelial nitric oxide synthase (eNOS), and its putative association with protein kinase B (PKB), and inducible nitric oxide synthase (iNOS) are not well characterized in hypoxic cardiac cells and there is a lack of studies that measure nitric oxide (NO) directly. Objective To measure NO production in cardiomyocytes and cardiac microvascular endothelial cells (CMECs) under baseline and hypoxic conditions and to evaluate the expression, regulation and activation of eNOS, iNOS and PKB. The effect of PI3-K/PKB inhibition on NO production and eNOS expression/activation was also investigated. Methods Adult rat cardiomyocytes and rat CMECs were made hypoxic by cell pelleting and low PO₂ incubation. Intracellular NO was measured by FACS analysis of DAF-2/DA fluorescence, and eNOS, iNOS and PKB were evaluated by Western blotting or flow cytometry. Upstream PKB inhibition was achieved with wortmannin. Results (1) NO levels increased in both cell types after exposure to hypoxia. (2) In hypoxic CMECs, eNOS was upregulated and activated, no iNOS expression was observed and PKB was activated. (3) In myocytes, hypoxia did not affect eNOS expression, but increased its activation. Activated PKB also increased during hypoxia. FACS analysis showed increased iNOS in hypoxic myocytes. (4) Wortmannin resulted in decreased hypoxia-induced NO production and reduced activated eNOS levels. Conclusions Cardiomyocytes and CMECs show increased NO production during hypoxia. eNOS seems to be the main NOS isoform involved as source of the increased NO generation, although there may be a role for iNOS and other non-eNOS sources of NO in the hypoxic myocytes. Hypoxia-induced PKB and eNOS activation occurred simultaneously in both cell types, and the PI3-K/PKB pathway was associated with hypoxia-induced NO production via eNOS activation.