Nitric Oxide Synthetic Pathway Research Articles

Predialysis and Dialysis Therapies Differently Affect Nitric Oxide Synthetic Pathway in Red Blood Cells from Uremic Patients: Focus on Peritoneal Dialysis.

Red blood cells (RBCs) have been found to synthesize and release both nitric oxide (NO) and cyclic guanosine monophosphate (cGMP), contributing to systemic NO bioavailability. These RBC functions resulted impaired in chronic kidney disease (CKD). This study aimed to evaluate whether predialysis (conservative therapy, CT) and dialysis (peritoneal dialysis, PD; hemodialysis, HD) therapies used during CKD progression may differently affect NO-synthetic pathway in RBCs. Our data demonstrated that compared to PD, although endothelial-NO-synthase activation was similarly increased, HD and CT were associated to cGMP RBCs accumulation, caused by reduced activity of cGMP membrane transporter (MRP4). In parallel, plasma cGMP levels were increased by both CT and HD and they significantly decreased after hemodialysis, suggesting that this might be caused by reduced cGMP renal clearance. As conceivable, compared to healthy subjects, plasma nitrite levels were significantly reduced by HD and CT but not in patients on PD. Additionally, the increased carotid intima-media thickness (IMT) values did not reach the significance exclusively in patients on PD. Therefore, our results show that PD might better preserve the synthetic NO-pathway in CKD-erythrocytes. Whether this translates into a reduced development of uremic vascular complications requires further investigation.

Nitric oxide synthetic pathway and cGMP levels are altered in red blood cells from end-stage renal disease patients

Red blood cells (RBCs) enzymatically produce nitric oxide (NO) by a functional RBC-nitric oxide synthase (RBC-NOS). NO is a vascular key regulatory molecule. In RBCs its generation is complex and influenced by several factors, including insulin, acetylcholine, and calcium. NO availability is reduced in end-stage renal disease (ESRD) and associated with endothelial dysfunction. We previously demonstrated that, through increased phosphatidylserine membrane exposure, ESRD-RBCs augmented their adhesion to human cultured endothelium, in which NO bioavailability decreased. Since RBC-NOS-dependent NO production in ESRD is unknown, this study aimed to investigate RBC-NOS levels/activation, NO production/bioavailability in RBCs from healthy control subjects (C, N=18) and ESRD patients (N=27). Although RBC-NOS expression was lower in ESRD-RBCs, NO, cyclic guanosine monophosphate (cGMP), RBC-NOS Serine1177 phosphorylation level and eNOS/Calmodulin (CaM)/Heat Shock Protein-90 (HSP90) interaction levels were higher in ESRD-RBCs, indicating increased enzyme activation. Conversely, following RBCs stimulation with insulin or ionomycin, NO and cGMP levels were significantly lower in ESRD- than in C-RBCs, suggesting that uremia might reduce the RBC-NOS response to further stimuli. Additionally, the activity of multidrug-resistance-associated protein-4 (MRP4; cGMP-membrane transporter) was significantly lower in ESRD-RBCs, suggesting a possible compromised efflux of cGMP across the ESRD-RBCs membrane. This study for the first time showed highest basal RBC-NOS activation in ESRD-RBCs, possibly to reduce the negative impact of decreased NOS expression. It is further conceivable that high NO production only partially affects cell function of ESRD-RBCs maybe because in vivo they are unable to respond to physiologic stimuli, such as calcium and/or insulin.

Nitric oxide synthetic pathway in patients with microvascular angina and its relations with oxidative stress.

A decreased nitric oxide (NO) bioavailability and an increased oxidative stress play a pivotal role in different cardiovascular pathologies. As red blood cells (RBCs) participate in NO formation in the bloodstream, the aim of this study was to outline the metabolic profile of L-arginine (Arg)/NO pathway and of oxidative stress status in RBCs and in plasma of patients with microvascular angina (MVA), investigating similarities and differences with respect to coronary artery disease (CAD) patients or healthy controls (Ctrl). Analytes involved in Arg/NO pathway and the ratio of oxidized and reduced forms of glutathione were measured by LC-MS/MS. The arginase and the NO synthase (NOS) expression were evaluated by immunofluorescence staining. RBCs from MVA patients show increased levels of NO synthesis inhibitors, parallel to that found in plasma, and a reduction of NO synthase expression. When summary scores were computed, both patient groups were associated with a positive oxidative score and a negative NO score, with the CAD group located in a more extreme position with respect to Ctrl. This finding points out to an impairment of the capacity of RBCs to produce NO in a pathological condition characterized mostly by alterations at the microvascular bed with no significant coronary stenosis.

Nitric Oxide Synthetic Pathway in Red Blood Cells Is Impaired in Coronary Artery Disease

BackgroundAll the enzymatic factors/cofactors involved in nitric oxide (NO) metabolism have been recently found in red blood cells. Increased oxidative stress impairs NO bioavailability and has been described in plasma of coronary artery disease (CAD) patients. The aim of the study was to highlight a potential dysfunction of the metabolic profile of NO in red blood cells and in plasma from CAD patients compared with healthy controls.MethodsWe determined L-arginine/NO pathway by liquid-chromatography tandem mass spectrometry and high performance liquid chromatography methods. The ratio of oxidized and reduced forms of glutathione, as index of oxidative stress, was measured by liquid-chromatography tandem mass spectrometry method. NO synthase expression and activity were evaluated by immunofluorescence staining and ex-vivo experiments of L-[15N2]arginine conversion to L-[15N]citrulline respectively.ResultsIncreased amounts of asymmetric and symmetric dimethylarginines were found both in red blood cells and in plasma of CAD patients in respect to controls. Interestingly NO synthase expression and activity were reduced in CAD red blood cells. In contrast, oxidized/reduced glutathione ratio was increased in CAD and was associated to arginase activity.ConclusionOur study analyzed for the first time the whole metabolic pathway of L-arginine/NO, both in red blood cells and in plasma, highlighting an impairment of NO pathway in erythrocytes from CAD patients, associated with decreased NO synthase expression/activity and increased oxidative stress.

Enhancement of the proline and nitric oxide synthetic pathway improves fermentation ability under multiple baking-associated stress conditions in industrial baker's yeast

BackgroundDuring the bread-making process, industrial baker's yeast, mostly Saccharomyces cerevisiae, is exposed to baking-associated stresses, such as air-drying and freeze-thaw stress. These baking-associated stresses exert severe injury to yeast cells, mainly due to the generation of reactive oxygen species (ROS), leading to cell death and reduced fermentation ability. Thus, there is a great need for a baker's yeast strain with higher tolerance to baking-associated stresses. Recently, we revealed a novel antioxidative mechanism in a laboratory yeast strain that is involved in stress-induced nitric oxide (NO) synthesis from proline via proline oxidase Put1 and N-acetyltransferase Mpr1. We also found that expression of the proline-feedback inhibition-less sensitive mutant γ-glutamyl kinase (Pro1-I150T) and the thermostable mutant Mpr1-F65L resulted in an enhanced fermentation ability of baker's yeast in bread dough after freeze-thaw stress and air-drying stress, respectively. However, baker's yeast strains with high fermentation ability under multiple baking-associated stresses have not yet been developed.ResultsWe constructed a self-cloned diploid baker's yeast strain with enhanced proline and NO synthesis by expressing Pro1-I150T and Mpr1-F65L in the presence of functional Put1. The engineered strain increased the intracellular NO level in response to air-drying stress, and the strain was tolerant not only to oxidative stress but also to both air-drying and freeze-thaw stresses probably due to the reduced intracellular ROS level. We also showed that the resultant strain retained higher leavening activity in bread dough after air-drying and freeze-thaw stress than that of the wild-type strain. On the other hand, enhanced stress tolerance and fermentation ability did not occur in the put1-deficient strain. This result suggests that NO is synthesized in baker's yeast from proline in response to oxidative stresses that induce ROS generation and that increased NO plays an important role in baking-associated stress tolerance.ConclusionsIn this work, we clarified the importance of Put1- and Mpr1-mediated NO generation from proline to the baking-associated stress tolerance in industrial baker's yeast. We also demonstrated that baker's yeast that enhances the proline and NO synthetic pathway by expressing the Pro1-I150T and Mpr1-F65L variants showed improved fermentation ability under multiple baking-associated stress conditions. From a biotechnological perspective, the enhancement of proline and NO synthesis could be promising for breeding novel baker's yeast strains.

EFFECTS OF HAEMODIALYSIS AND CONTINUOUS AMBULATORY PERITONEAL DIALYSIS ON NITRIC OXIDE SERUM CONCENTRATION IN PATIENTS WITH CHRONIC RENAL FAILURE

Introduction: Nitric oxide (NO) plays an important role in a wide range of physiologic and pathophysiological processes. A major mediator of endothelial function, NO regulates vasodilatory and antithrombotic actions in the vasculature and plays a role in reproductive functions, bronchodilation, bone formation, memory, insulin sensitivity, and gastrointestinal relaxation. Impaired NO bioactivity is strongly associated with endothelial dysfunction. NO, an L-arginine derivative, also exerts a variety of renal and extrarenal physiological and pathophysiological effects. It seems that NO synthetic pathway could have a key role in mediating the complex hemodynamic and hemostatic disorders associated to the progression of renal disease. It remains unclear whether endogenous NO production is increased or decreased in patients with chronic renal failure. The objective of this study was to present the effect of different dialysis treatment on NO serum concentration in patients with chronic renal failure.Patients and Methods: To evaluate endogenous NO production in these patients we studied plasma NO2 and NO3 levels (determined with the Griess method) in patients who underwent regular continuous ambulatory peritoneal dialysis or repeated haemodialysis and in healthy subjects. The study included 51 patients suffering from chronic renal failure and 30 healthy subjects.Results: Our results show that patients with chronic renal failure had a significantly higher NO serum concentration than controls. These values did not differ between patients on haemodialysis and those on continuous ambulatory peritoneal dialysis. NO serum concentration did not differ between female and male independently of the patient’s treatment.Discussion and Conclusion: From obtained results we can concluded that uremia is associated with excessive systemic NO release independently of the patient’s treatment. Alter (increase) NO synthesis may help to explain some pathological changes seen in uraemia such as bleeding tendency, a well-known complication of uremia and hemodialysis hypotension.

Docosahexaenoic Acid (DHA) Supplementation Benefits Pregnancy Complicated with Gestational Diabetes Mellitus (GDM)

GDM, characterized by an alteration of glucose regulation which is first detected during pregnancy, is associated with elevated blood glucose, insulin resistance, pro‐inflammatory agents, and increased nitric oxide (NO) synthesis. Our purpose was to assess supplementation of mothers with GDM with 600 mg/day DHA (randomized to receive DHA or placebo, corn oil) (week 24 of pregnancy‐delivery, GDM mothers, n=31). Blood samples were collected at weeks 24 and 36 in pregnancy. At delivery, blood from the mother and umbilical cord were collected to assess transfer of DHA from mother to fetus and placental tissue for NO synthesis. DHA supplementation significantly increased maternal red blood cell (RBC) DHA concentration but not fetal RBC DHA. Supplementation reduced fasting glucose in GDM compared to controls and insulin resistance tended to decrease substantially more in supplemented mothers than controls. Inflammatory markers were not different between the groups, likely explained on the basis of small sample size. Placental endothelial cells from controls had increased L‐arginine utilization and elevated NO synthesis compared to the DHA supplementation group. We hypothesize that DHA decreased expression of L‐arginine transport protein and/or diminished activity of endothelial NO synthase. In conclusion, DHA supplementation of women with GDM demonstrated favorable effects regarding glucose metabolism and the NO synthetic pathway compared to controls, underscoring the importance of this n‐3 long chain fatty acid in pregnancy complicated by diabetes. (Fondo Nacional de Innovacion en Salud de Chile and Nestle)

17beta-estradiol corrects hemostasis in uremic rats by limiting vascular expression of nitric oxide synthases.

Conjugated estrogens shorten the prolonged bleeding time in uremic patients and are similarly effective in a rat model of uremia. We have previously demonstrated that the shortening effect of a conjugated estrogen mixture or 17beta-estradiol on bleeding time was abolished by the nitric oxide (NO) precursor L-arginine, suggesting that the effect of these drugs on hemostasis in uremia might be mediated by changes in the NO synthetic pathway. The present study investigated the biochemical mechanism(s) by which conjugated estrogens limit the excessive formation of NO. 17beta-estradiol (0.6 mg/kg), given to rats made uremic by reduction of renal mass, significantly reduced bleeding time within 24 h and completely normalized plasma concentrations of the NO metabolites, nitrites and nitrates, and of NO synthase (NOS) catalytic activity, determined by NADPH-diaphorase staining in the thoracic aorta. Endothelial NOS (ecNOS) and inducible NOS (iNOS) immunoperoxidase staining in the endothelium of uremic aortas of untreated rats was significantly more intense than in control rats, while in uremic rats receiving 17beta-estradiol staining was comparable to controls. Thus 17beta-estradiol corrected the prolonged bleeding time of uremic rats and fully normalized the formation of NO by reducing the expression of ecNOS and iNOS in vascular endothelium. These results provide a possible biochemical explanation of the well-known effect of estrogens on primary hemostasis in uremia, in experimental animals and humans.

Endothelial cell calcium mobilization to acetylcholine is attenuated in copper-deficient rats.

Dietary copper deficiency significantly attenuates nitric oxide (NO)-mediated vascular smooth muscle relaxation and vasodilation. There is evidence for both increased inactivation of the NO radical by superoxide anion, and oxidative damage to the endothelium where NO is produced. The current study was designed to examine the NO synthetic pathway in the endothelium during copper deficiency. Male weanling rats were fed a copper-adequate (CuA, 6.4 mg Cu/kg diet) or copper-deficient (CuD, 0.4 mg Cu/kg diet) diet for four weeks. Cremasteric arterioles (approximately 100 microm diameter) were isolated and used for the experiments. Western blot analysis of the arteriole endothelial nitric oxide synthase (eNOS) concentration did not show a difference between dietary groups. Acetylcholine (Ach)-induced vasodilation was significantly reduced in the CuD group both before and after pretreatment with the eNOS substrate L-arginine. Endothelial intracellular calcium ([Ca2+]i) stimulated by 10(-6) M Ach was significantly inhibited in the arterioles from CuD rats. Coincident with the inhibition of [Ca2+]i and vasodilation was a depression of vascular Cu/Zn-SOD activity and an increase in plasma peroxynitrite activity. These data suggest that endothelial Ca2+ signaling and agonist-stimulated NO-mediated vascular dilation are likely reduced by increased oxidative damage in copper-deficient rats.

Physiology and pathophysiology of nitric oxide in chronic renal disease.

Nitric oxide (NO), an L-arginine derivative, exerts a variety of renal and extrarenal physiological and pathophysiological effects. NO is generated by three isoforms of nitric oxide synthases (NOS): two acutely responsive, constitutive isoforms, neuronal NOS (nNOS) and endothelial NOS (ecNOS), and the slower, more persistent, inducible NOS (iNOS). NO regulates glomerular ultrafiltration; tubular reabsorption, and intrarenal renin secretion. A number of recent studies, most of them in the experimental model of renal mass reduction (RMR) in rats, have raised the hypothesis that an impaired NO synthetic pathway could have a key role in mediating the complex renal hemodynamic and nonhemodynamic disorders associated with the progression of renal disease. Thus, kidneys from rats with RMR produce less NO than normal rats, and NO generation negatively correlates with markers of renal damage. The abnormality is due to a defect in iNOS in the kidney. Data are also available showing that drugs capable of enhancing renal NO activity may be renoprotective in a variety of experimental renal diseases, particularly those characterized by derangements of glomerular hemodynamics. Fewer studies are available in humans and these have shown less than conclusive results.

Effect of acetate, bicarbonate dialysis, and acetate-free biofiltration on nitric oxide synthesis: Implications for dialysis hypotension

The effect of acetate dialysis (AD), bicarbonate dialysis (BD), and acetate-free biofiltration (AFB) on nitric oxide (NO) synthesis and the implications for dialysis hypotension was studied. The finding that uremic plasma is a potent inducer of NO synthesis by endothelial cells in vitro suggested that the cardiovascular instability of dialysis patients might result from excessive NO formation. Cardiovascular instability is more frequent in patients undergoing AD than BD. To see whether these differences were attributable to NO, we studied the NO synthetic pathway ex vivo in patients undergoing different dialysis procedures. Five patients were treated, in a random order, with AD, BD, and AFB, a technique using a buffer-free dialysate and postdilution of a sterile bicarbonate solution. Each type of dialysis was used for 1 week, comprising three dialysis sessions. A polyacrylonitrile dialyzer was used for all three methods. Before and after the third dialysis, plasma was collected, added to [3H]L-arginine, and incubated with human umbilical vein endothelial cells (HUVECs) for 24 hours. NO synthesis was evaluated as [3H]L-citrulline formation. Plasma concentrations of interleukin-1beta (IL-1beta), a potent inducer of inducible NO synthase (iNOS) in endothelial cells, were also measured. Plasma collected from patients after AD stimulated endothelial NO synthesis more than plasma from the same patients before the dialysis session (pre-AD, 0.173+/- 0.028 nmol/10(5) cells v post-AD, 0.280+/-0.093 nmol/10(5) cells; P < 0.05). A slight, although not significant, increase was also observed when HUVECs were incubated with plasma drawn after BD (pre-BD, 0.151+/- 0.014 nmol/10(5) cells; post-BD, 0.230+/-0.055 nmol/10(5) cells). AFB did not aggravate the stimulatory effect of uremic plasma on endothelial NO synthesis (pre-AFB, 0.184+/-0.038 nmol/10(5) cells; post- AFB, 0.189+/-0.040 nmol/10(5) cells). Plasma IL-1beta was greater (P < 0.01) after AD than after BD and AFB (post-AD, 0.234+/-0.028 pg/mL; post-BD, 0.124+/-0.019 pg/mL; post-AFB, 0.120+/-0.013 pg/mL). With AD, there was a greater intradialytic decrease in systolic blood pressure than with BD or AFB. Weight and blood volume loss and sodium balance were similar in AD, BD, and AFB. These data were consistent with the possibility that NO and cytokines, released in excessive amounts during AD, may contribute to hemodynamic instability. (Am J Kidney Dis 1998 Jul;32(1):115-24)

Inhibition of Nitric Oxide Synthase byL-NAME Improves Ventricular Performance in Streptozotocin- diabetic Rats

The overall goal of this study was to determine if activation of the nitric oxide synthetic pathway suppressed basal ventricular performance and the responsiveness toβ-adrenergic stimulation characteristic of cardiac function in the 8-week streptozotocin (60 mg/kg, i.v.) diabetic (STZ-Db) rat. Left ventricular performance was measured in isolated working hearts, before and at the peak response to 0.8μm dobutamine, in the absence or presence of NG-nitro-l-arginine methyl ester (l-NAME, 1 mm), a non-selective inhibitor of nitric oxide synthase (NOS). Ventricular performance was suppressed in the STZ-Db heart under basal (decreased heart rate, cardiac output, aortic flow −dP/dt) and dobutamine-stimulated (diminished rise in +dP/dtand maximum systolic pressure) conditions.l-NAME had minimal effects on basal or dobutamine-stimulated ventricular performance in control hearts. In contrast,l-NAME infusion in hearts from STZ-Db returned the depressed heart rate to control values, which was correlated with an increase in aortic flow. In addition, the dobutamine-stimulated rise in maximum systolic pressure and +dP/dtwere similar in the control and STZ-Db rats in the presence ofl-NAME. Western blot analysis detected the presence of inducible nitric oxide synthase (NOS) and a significant (P<0.001) increase in the constitutive NOS in ventricular myocytes from STZ-Db rats. These data suggest that an increased production of nitric oxide by NOS in ventricular myocytes from STZ-Db animals suppressed basal ventricular performance and the responsiveness toβ-adrenergic stimulation in diabetic hearts.

Hyperdynamic circulation in patients with cirrhosis: direct measurement of nitric oxide levels in hepatic and portal veins

Background/Aims: Peripheral vasodilation represents the main vascular dysfunction associated with the hyperdynamic circulation of liver cirrhosis. This study was intended to measure directly regional and systemic levels of nitric oxide, a potent vasorelaxing mediator, in order to assess its role in the development of hemodynamic changes of cirrhosis. Methods: We compared nitric oxide levels in the splanchnic and systemic circulation of 25 patients with cirrhosis undergoing transjugular intrahepatic portosystemic stent shunt and in the hepatic vein and peripheral blood of 10 patients without cirrhosis submitted to venous catheterization. Nitric oxide levels were measured through electron paramagnetic resonance spectroscopy as nitrosylhemoglobin complexes. Results: Significantly higher nitric oxide levels were calculated in patients with cirrhosis with respect to controls, both in the peripheral and hepatic veins. In patients with cirrhosis, nitric oxide levels in the portal vein (3.44±2.17, expressed in arbitrary units) were higher than in the systemic circulation (1.89±1.15), but lower than in the hepatic vein (4.75±2.53; p<0.001 by variance analysis). Conclusions: These data suggest that nitric oxide synthetic pathway activity as well as nitric oxide release are enhanced at the level of splanchnic vasculature and, more important, in the hepatic tissue, confirming evidence of the predominant role of nitric oxide in the pathogenesis of hemodynamic changes in patients with cirrhosis with portal hypertension.

Increased production of nitric oxide by neutrophils and monocytes from cirrhotic patients with ascites and hyperdynamic circulation

An increased release of nitric oxide (NO), a powerful vasodilating agent, has been proposed to play a role in the pathogenesis of vasodilation and hyperdynamic circulation associated with advanced cirrhosis. We evaluated NO synthase (NOS) activity in peripheral leukocytes of 12 cirrhotic patients and 9 healthy subjects together with plasma endotoxin levels and systemic hemodynamic (by a noninvasive echocardiographic method). NOS activity was evaluated by (1) measuring the capacity of isolated polymorphonuclear cells (PMNs) and monocytes to convert [ 3[H]arginine to [ 3[H]citrulline; (2) measuring the ability of neutrophils and monocytes to inhibit thrombin-induced platelet aggregation and to increase guanosine 3′-5′-cyclic monophosphate content in coincubated platelets, an expression of NO release from these cells. Both neutrophils and monocytes from cirrhotic patients produced significantly higher amounts of [ 3h]citrulline than cells obtained from healthy subjects ( P < .001 and P < .02 for neutrophils and monocytes, respectively) and were more effective than control cells in inhibiting platelet aggregation ( P < .05 and P < .001, respectively for 2 ×10 6 cells) and in increasing guanosine 3′-5′-cyclic monophosphate content in coincubated platelets ( P < .05 and P < .001, respectively). The anti-aggregating activity expressed by leukocytes has a pharmacological profile similar to that described for NO, because it increased after addition of superoxide dismutase, a superoxide anion scavenger, and markedly decreased after inhibition of nitric oxide synthesis with N G-monomethyl- l-arginine (L-NMMA) and N G -nitro- l-arginine-methyl ester (LNAME). Cirrhotic patients had significantly higher plasma endotoxin levels ( P < .001) and cardiac index ( P < .01) when compared with controls. These data support the contention that the NO synthetic pathway is activated in cirrhotic patients with ascites, hyperdynamic circulation, and endotoxemia.

Rat Splenocytes Inhibit Antigen-Specific Lymphocyte Proliferation through a Reactive Nitrogen Intermediate (RNI)-Dependent Mechanism and Exhibit Increased RNI Production in Response to IFN-γ

Rat splenocytes inhibited antigen-specific proliferation of primed lymph node cells in vitro. This inhibition resided in the plastic-adherent splenocyte fraction and was radioresistant, suggesting that the effect was due to macrophages. While this suppression was more evident if spleen cells were derived from immunized rats, spleen cells from normal rats were just as suppressive when added to cocultures at higher numbers. Proliferative responses were greatly enhanced in the presence of NG -monomethyl-L-arginine, a specific inhibitor of the nitric oxide synthetic pathway, and significant levels of nitrite (NO-2), a product of this pathway, were detected in culture supernatants in association with suppressed responses, supporting the notion that suppression was mediated by the L-arginine-dependent production of reactive nitrogen intermediates (RNI). When the splenocytes were physically separated from the responding lymph node cell population, high levels of NO-2 were still detected but proliferative responses were no longer inhibited, suggesting that cell proximity or contact is necessary for delivery of the suppresslye signal. Adherent splenocytes cultured alone produced low levels of NO-2. Addition of 1 to 50 U/ml IFN-γ induced a dose-dependent increase in NO-2 production, with the maximal level approximating that found in suppressed cocultures; TNF-α, IL-2, or LPS did not synergize with IFN-γ to enhance NO-2 production. These findings suggest that by activating macrophages to upregulate RNI synthesis, IFN-γ-producing T cells may exert a negative influence over their own proliferation.

Inducible nitric oxide synthase activity of cloned murine microglial cells

Nitric oxide (NO) is a short-lived diffusable molecule now believed to participate in multiple physiologic functions in the CNS including neurotransmission and the maintenance of vascular tone. Previously, we reported that cell lines obtained by retroviral immortalization of tissue macrophages (M phi) could be induced to synthesize nitrite (NO2-), a stable end product of the NO synthetic pathway. We have further characterized the induction and activity of this pathway in a panel of seven microglial clones derived from primary embryonic mouse brain cultures. Like M phi, these clones were found to release high levels of NO2- in response to recombinant interferon-gamma (rIFN-gamma) as a priming signal together with either bacterial lipopolysaccharide (LPS) or exogenous recombinant tumor necrosis factor-alpha (rTNF-alpha). As previously demonstrated for M phi, phagocytosis of zymosan particles during induction of enzyme activity enhanced subsequent NO2- production, which is of interest in light of the postulated phagocytic role of microglia within the CNS. Biochemical characterization of enzyme activity in intact microglial clones and in isolated cytosolic fractions indicates that the microglial NO synthase present in these murine cell clones represents the M phi-like isotype. These findings suggest that microglial cells could represent a major source of NO within the CNS.

Involvement of nitric oxide synthetic pathway in inhibitory junction potentials in canine proximal colon

Experiments were performed to determine the involvement of nitric oxide synthase in the generation of nonadrenergic, noncholinergic (NANC) inhibitory junction potentials (IJPs) in the canine proximal colon. Smooth muscle cells were impaled near the myenteric border between the circular and longitudinal layers. Cells exhibited rhythmic myenteric potential oscillations. IJPs were evoked with electrical field stimulation in the presence of drugs to block adrenergic and cholinergic neurotransmission. N omega-nitro-L-arginine, methyl ester (L-NAME), a nitric oxide synthase inhibitor, progressively reduced the amplitude of IJPs. The effect of L-NAME was reversed by L-Arg but not by the stereoisomer D-Arg. IJPs disrupt the regular pattern of myenteric potential oscillations. This effect was also blocked by L-NAME and reversed by L-Arg. These experiments suggest that a product of the nitric oxide synthetic pathway is involved in NANC neurotransmission in the canine proximal colon.