Acute and chronic inhibition of nitric oxide synthase in conscious rabbits: role of nitric oxide in the control of vascular tone

Acute and chronic effects of Nw-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide synthase, were examined on the hindquarter hemodynamics of conscious rabbits. After pharmacological autonomic reflex blockade on four experimental days (days 0, 1, 2, and 7), responses to aortic occlusion (balloon cuff, 5-80 s inflation), intra-aortic infusion of acetylcholine, adenosine, and sodium nitroprusside (SNP) were measured before and after vehicle (day 0) or L-NNA (16 mg/kg/h i.v., days 1, 2, and 7). On day 1, L-NNA raised the mean arterial pressure (MAP), and lowered the heart rate (HR) and hindquarter vascular conductance (HVC = abdominal aortic Doppler blood flow/MAP). On days 2 and 7, L-NNA only slowly raised the MAP. The dilator response to acetylcholine was inhibited by L-NNA on day 1 and before and after L-NNA on days 2 and 7. The responses to aortic occlusion, adenosine, or SNP infusion were unaffected by L-NNA treatment on any day. Thus, if nitric oxide synthase inhibition by L-NNA abolishes NO release, then (i) reactive hyperaemia is independent of NO, (ii) basal NO release normalises the arterial pressure in the short term but other factors become important in the long term, and (iii) the blockade by L-NNA of receptor-stimulated NO release by acetylcholine is only very slowly reversible.     

Role of nitric oxide on cholinergic component of bronchial tone in pig

In vitro studies demonstrated that stimulation of intrinsic nerves of airway smooth muscle results in a predominantly contractile response, followed by a relaxant response which involves cholinergic, adrenergic and non-adrenergic non-cholinergic (NANC) nerve activation. Thus, in this paper it is determined whether endogenous nitric oxide (NO) modulates cholinergic neurotransmission in isolated pig airway smooth muscle. Bronchial rings were suspended in organ baths for isometric measurement of tension and the contractions were induced using electrical field stimulation (EFS) techniques. Then, the effects of L-NG-nitroarginine (L-NOARG, 10 microM), an inhibitor of NO synthase, and L-arginine (L-ARG, 1 mM), a precursor of NO synthesis, were evaluated. The cholinergic contractions induced by electrical field stimulation (EFS: 60 V, 2 ms, 60 Hz) of pig lobar bronchial preparations increased (29%) in the presence of L-NOARG (10 microM). This effect may be released by nerves in pig large airways during EFS.     

Nitric oxide and regulation of vascular tone: pharmacological and physiological considerations

The endothelial cells of the vascular system are responsible for many biological activities that maintain vascular homeostasis. Responding to a variety of chemical and physical stimuli, the endothelium elaborates a host of vasoactive agents. One of these agents, endothelium-derived relaxing factor, now accepted as nitric oxide, influences both cellular constituents of the blood and vascular smooth muscle. A principal intracellular target for nitric oxide is guanylate cyclase, which, when activated, increases the intracellular concentration of cyclic guanosine monophosphate, which in turn activates protein kinase G. Acting by this pathway, nitric oxide induces relaxation of vascular smooth muscle and inhibits platelet activation and aggregation. Derangements in endothelial production of nitric oxide are implicated as both cause and consequence of vascular diseases, including hypertension, atherosclerosis, and coronary artery disease.     

Role of vascular nitric oxide in physiological and pathological conditions

This review describes the ability of certain diseases, such as essential hypertension, atherosclerosis, angina, and vasospasm, to reduce vascular nitric oxide (NO) formation or to increase its metabolism. In contrast, others, such as hypotension, sepsis, stroke, myocardial depression, and inflammatory responses, increase NO synthesis. The mechanism implicated in the changes in the formation and metabolism of NO are described. To prevent or treat these pathological processes, in which a deficiency in vascular NO formation plays a causative role, NO may be provided through methods such as direct NO administration or indirect NO supply through either NO donors or L-arginine, which facilitates NO formation.     

Differential regulation of hepatic arterial and portal venous vascular resistance by nitric oxide and carbon monoxide in rats

Nitric oxide (NO), a gaseous mediator that accounts for the biological activity of endothelium-derived relaxing factor, has been shown to play an important role in the reduction of basal vascular tone in multiple vascular beds, including the hepatic circulation. On the other hand, recent studies have provided first evidence that endogenously generated carbon monoxide (CO) may exert vasodilatory effects in the hepatic portal vein and within sinusoids. Thus, we defined the differential role of NO and CO in the regulation of vascular resistance in the two inflows to the liver in the normal rat in vivo. Male Sprague-Dawley rats were anesthetized with pentobarbital sodium and surgically instrumented in order to study the change in hepatic arterial (Rha) and portal venous vascular resistance (Rpv) in response to intravenous bolus administration of either the NO-synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) (1 mg/kg; n = 7 animals) or of tin protoporphyrin-IX (SnPP-IX) (50 micromol/kg), a specific inhibitor of the CO-generating enzyme heme oxygenase (n = 8 animals). While L-NAME caused a substantial increase in Rha, Rpv increased only slightly under these conditions. In sharp contrast, SnPP-IX did not affect Rha, but caused a profound increase in Rpv. In conclusion, Rha and Rpv are differentially regulated by NO and CO in the normal rat liver in vivo, i.e., NO serves as a potent vasodilator in the hepatic arterial circulation, but exerts only a minor vasodilatory effect in the portal venous vascular bed. In contrast, while there is no intrinsic CO-mediated vasodilation in the hepatic artery, CO acts to maintain portal venous vascular tone in a relaxed state.     

Role of nitric oxide in the regulation of the hepatic microcirculation in vivo

BACKGROUND/AIMS: Nitric oxide (NO) is an important mediator in the regulation of vascular tone. However, no data exist on the physiological role of NO in the regulation of the hepatic microcirculation. This study was designed to evaluate the role of NO in the hepatic microcirculation in vivo under physiological conditions. METHODS: The hepatic microcirculation was investigated in anesthetized rats by intravital fluorescence microscopy after injection of fluorescein-isothiocyanate-labeled erythrocytes. Following assessment of baseline sinusoidal perfusion, animals were randomly treated with L-NMMA (n=6), L-arginine (n=6), nitroprusside sodium (NPS, n=5) or a comparable volume of NaCl (n=4). Drugs were given through a portal vein catheter at three doses (Dx), each followed by intravital microscopy. L-NMMA was given: 5 mg/kg (D1), 25 mg/kg (D2), 50 mg/kg (D3); L-arginine 30 mg/kg (D1), 150 mg/kg (D2), 300 mg/kg (D3); and NPS continuously 80 microg x kg(-1) x h(-1). RESULTS: L-NMMA induced a significant increase of mean arterial blood pressure (MAP) (114 vs. 129 mm Hg; p<0.05). In contrast, MAP of NPS-treated animals decreased (107 vs. 91 mm Hg; p<0.01) whereas MAP of animals receiving L-arginine did not significantly differ. Sinusoidal blood flow revealed dose-dependent changes: L-NMMA significantly decreased perfusion of sinusoids (D1: 65%, D2: 57%, D3: 50% of baseline, p<0.05). Injection of L-arginine increased the sinusoidal flow even with the lowest dose (D1: 137%, D2: 133%, D3: 123%, p<0.05). Continuous infusion of NPS had little effect on sinusoidal blood flow at the first and second times of microscopy but sinusoidal blood flow was significantly increased at the third time (D1: 103%, D2: 106%, D3: 122%). CONCLUSIONS: Inhibition of NOS results in a dose-dependent disturbance of the hepatic microcirculation despite significantly increased MAP, whereas L-arginine increases the sinusoidal blood flow. The results indicate an important role for NO in the regulatory mechanisms of hepatic sinusoidal perfusion under physiological conditions.     

Direct in vivo measurement of flow-dependent nitric oxide production in mesenteric resistance arteries

PURPOSE: To determine whether the concentration of nitric oxide (NO) at the arterial wall is increased subsequent to the abrupt elevation of blood flow in resistance arteries. METHODS: Eight dogs underwent laparotomy with anesthesia, and their small bowels were exteriorized. NO concentration was measured with NO-specific electrodes (200-micro-tip diameter) at the outer wall of the mesenteric arteries. Flow was increased by occlusion of the adjacent mesenteric arteries. In four animals, flow and NO concentration were measured after the administration of Nomega-nitro-L-arginine-methyl ester (L-NAME) to inhibit NO production. RESULTS: As arterial flow was increased from a baseline of 5.4 +/- 1.3 ml/min to 10.9 +/- 1.8 ml/min (p = 0.001), NO electrode current was elevated in every animal. With repetition of the flow stimulus, the response tended to be attenuated. In the first experimental trial, NO electrode current measured at the arterial wall increased from 2.86 +/- 0.56 to 3.00 +/- 0.60 nA (p = 0.02). L-NAME (10 mg/kg intravenous) effectively inhibited NO synthase as indicated by the elevation of mean arterial pressure (11 +/- 1.7 mm Hg; p = 0.04). After administration of L-NAME, NO electrode current measured at the outer arterial wall fell 0.23 +/- 0.05 nA (p = 0.02). CONCLUSIONS: The data indicate that a doubling of blood flow in the canine mesenteric resistance arteries is associated with an increase in NO concentration of at least 100 nm at the outer arterial wall. This association is probably a substantial underestimation of the actual concentration because of the geometry of the electrode tip. To our knowledge, ours is the first report of direct in vivo measurement of flow-dependent NO release in resistance arteries.     

Contribution of endogenous endothelin-1 to the maintenance of vascular tone: role of nitric oxide

Studies were designed to compare the effect of the nitric oxide inhibitor, N omega-nitro-L-arginine (L-NNA), and the novel ETB receptor antagonist, RES-701-1, on changes in blood pressure and renal blood flow induced by exogenous endothelin receptor agonists and to determine the effect of L-NNA on basal hemodynamics in conscious, chronically instrumented rats. Infusion of low (nonpressor) doses of L-NNA or RES-701-1 potentiated systemic and renal vasoconstriction induced by bolus injections of endothelin-1 or sarafotoxin 6c. Bolus intravenous injection or sustained infusion of L-NNA alone resulted in dose-dependent increases in blood pressure and decreases in renal blood flow, similar to our recently reported results with RES-701-1. Vasoconstriction induced by inhibition of nitric oxide was attenuated by SB 209670, a mixed ETA/B receptor antagonist, but not by BQ123, an ETA receptor antagonist; neither antagonist altered basal hemodynamics. Collectively, the results indicate that: (1) endothelin plays an important role in the control of basal vascular tone by mediating both vasodilation and vasoconstriction; (2) these effects are mediated by different ETB receptor subtypes in the rat, one located on the endothelium that mediates vasodilation via the nitric oxide pathway, the other located on the vascular smooth muscle that mediates contraction.     

Role of nitric oxide in the regulation of lymphocyte apoptosis and HIV-1 replication

Nitric oxide (NO) has been implicated in certain immunopathogenetic mechanisms during the course of infection with human immunodeficiency virus (HIV). We have evaluated the levels of NO release and lymphocyte apoptosis in peripheral blood mononuclear cell (PBMC) cultures from HIV-1 infected subjects and healthy controls. We have also examined these 2 parameters in parallel cultures maintained under conditions where either NO synthesis was inhibited or high level of NO was present. Nitrite contents in culture supernatants were measured as the stable end products of the released NO. Levels of spontaneous apoptosis and activation-induced cell death (AICD) by anti-CD3 or by phytohemagglutinin were evaluated using flow cytometry. Additional experiments were also aimed at addressing a potential link between NO synthesis and HIV-1 replication in human monocyte-derived macrophages (MDMs). Acutely infected MDMs with HIV-1Bal were maintained in culture, without any additional activation signal, for a period of 14 days. Nitrites in the supernatants and mRNA accumulation of the inducible NO synthase (iNOS) in infected cells were assessed over the whole culture period. In addition, the effect of blocking NO synthesis during and after infection of MDMs, using an inhibitor of NO, was evaluated on the level of viral replication as measured by the presence of P24 antigen in the supernatants. Similarly, the effect on HIV replication of high NO levels in MDM cultures, supplied by a donor of NO during the 24 h period of infection, was also studied. We conclude that no elevation in NO release could be detected in PBMC cultures from HIV-1 infected subjects and that modulation of NO content may slightly regulate the level of spontaneous lymphocyte apoptosis but not that of AICD. Infection of MDMs with HIV-1 does not seem to induce detectable NO release or iNOS mRNA accumulation. Similarly, neither inhibition of NO synthesis nor the presence of high NO levels during the infection period could modify the outcome of virus replication in macrophages.     

Role of nitric oxide in regulation of brain stem circulation during hypotension

We tested the hypothesis that nitric oxide (NO) plays a role in CBF autoregulation in the brain stem during hypotension. In anesthetized rats, local CBF to the brain stem was determined with laser-Doppler flowmetry, and diameters of the basilar artery and its branches were measured through an open cranial window during stepwise hemorrhagic hypotension. During topical application of 10(-5) mol/L and 10(-4) mol/L N(omega)-nitro-L-arginine (L-NNA), a nonselective inhibitor of nitric oxide synthase (NOS), CBF started to decrease at higher steps of mean arterial blood pressure in proportion to the concentration of L-NNA in stepwise hypotension (45 to 60 mm Hg in the 10(-5) mol/L and 60 to 75 mm Hg in the 10(-4) mol/L L-NNA group versus 30 to 45 mm Hg in the control group). Dilator response of the basilar artery to severe hypotension was significantly attenuated by topical application of L-NNA (maximum dilatation at 30 mm Hg: 16 +/- 8% in the 10(-5) mol/L and 12 +/- 5% in the 10(-4) mol/L L-NNA group versus 34 +/- 4% in the control group), but that of the branches was similar between the control and L-NNA groups. Topical application of 10(-5) mol/L 7-nitro indazole, a selective inhibitor of neuronal NOS, did not affect changes in CBF or vessel diameter through the entire pressure range. Thus, endothelial but not neuronal NO seems to take part in the regulation of CBF to the the brain stem during hypotension around the lower limits of CBF autoregulation. The role of NO in mediating dilatation in response to hypotension appears to be greater in large arteries than in small ones.     

Effect of dihydro-ouabain on vascular tone on the perfused cannine hindlimb

Digitalis compounds are known to cause contraction of vascular smooth muscle. However, it has been reported by Kahn and colleagues (J. Pharmacol. Exp. Ther. 142: 215-222, 1963) that dihydro-ouabain has no effect on vascular tone in dogs. The purpose of our study was to reassess the vasoconstrictor activity of dihydro-ouabain. This was done by monitoring perfusion pressure of the canine hindlimb preparation perfused at a constant rate of blood flow and administering intra-arterial injections of dihydro-ouabain. Increasing doses of this drug (i.e., 25, 50, 100, 200 and 300 mug) produced significant dose-dependent increases in perfusion pressure. Pretreatment with phentolamine prevented the effect of dihydro-ouabain on perfusion pressure. These results indicate that dihydro-ouabain causes contraction of vascular smooth muscle by an adrenergic mechanism.     

Role of nitric oxide in vascular hyper-responsiveness to norepinephrine in hypertensive Dahl rats

OBJECTIVE: To determine whether the abnormal vascular responses observed in salt-sensitive hypertension are caused by an impairment in vascular nitric oxide function. DESIGN: Isometric tension was measured in aortic rings isolated from Dahl salt-sensitive and salt-resistant rats fed a regular-salt (0.4% NaCl) or a high-salt (8% NaCl) diet, with and without inhibition of endogenous nitric oxide synthesis. METHODS AND RESULTS: Systolic arterial pressure, measured weekly by the tail-cuff method, increased markedly in DS rats with a high-salt diet but did not increase in the other groups. In aortic rings, norepinephrine evoked dose-dependent contractions which were significantly increased in rings from DS rats with a high-salt diet Pretreatment with Nomega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, increased the norepinephrine-induced contraction in all groups and abolished differences in contractile responses between high-salt DS rats and the other groups. Acetylcholine induced endothelium-dependent relaxation, which was significantly depressed in high-salt DS rats. L-NAME attenuated the acetylcholine-induced relaxation in all groups and abolished the difference in relaxation response between high-salt DS rats and the other groups. Sodium nitroprusside-induced relaxation was significantly depressed in high-salt DS rats. CONCLUSIONS: Vascular hypercontractile responses to norepinephrine in DS hypertensive rats can, in part, be explained by an impairment in endothelial nitric oxide production.     

Role for the Salmonella flavohemoglobin in protection from nitric oxide

Hemoglobin homologs are being identified in an expanding number of unicellular prokaryotic and eukaryotic organisms. Many of these hemoglobins are twodomain proteins that possess a flavin-containing reductase in their C terminus. Determination of a function for these flavohemoglobins has been elusive. A Salmonella typhimurium strain harboring a deletion in the flavohemoglobin gene shows no difference in growth under oxidative stress conditions but displays an increased sensitivity to acidified nitrite and S-nitrosothiols, both of which produce nitric oxide. The effect is seen aerobically or anaerobically, indicating that oxygen is not required for flavohemoglobin function. These results suggest a role for the bacterial flavohemoglobins that is independent of oxygen metabolism and provide evidence for a bacterial route of protection from nitric oxide that is distinct from oxidative stress responses.     

Intrauterine infection and the effects of inflammatory mediators on prostaglandin production by myometrial cells from pregnant women

OBJECTIVE: Our purpose was to evaluate the effects of known stimulants of prostaglandin production on cultured myometrial cells from women in labor with and without intrauterine infection. STUDY DESIGN: Myometrial segments were obtained from 16 patients between 33 and 40 weeks' gestation who had been in labor for > or = 8 hours at cesarean delivery; 8 patients had clinical chorioamnionitis and 8 did not. Myometrial cells were isolated and grown in culture. Incubations were conducted with interleukin-1 beta, tumor necrosis factor-alpha, or epidermal growth factor. Prostaglandin E2, prostaglandin F2 alpha, and 6-keto-prostaglandin F1 alpha (the stable metabolite of prostacyclin) were measured by radioimmunoassay, and cellular protein was determined. RESULTS: Cultured human myometrial cells from patients with and without prior intrauterine infection produced prostaglandins in response to interleukin-1 beta, tumor necrosis factor-alpha, and epidermal growth factor at a significantly increased rate (p<0.05 vs controls at and above 10 ng/ml of interleukin-1 beta, tumor necrosis factor-alpha, and epidermal growth factor). The major prostaglandin produced in response to each stimulant was 6-keto-prostaglandin F1 alpha; however, this response was attenuated in cells from patients with intrauterine infection. CONCLUSIONS: Cultured human myometrial cells from patients with and without prior intrauterine infection respond to known stimulants of prostaglandin production. Prior intrauterine infection has no effect on baseline prostaglandin production, but the amount of prostacyclin produced as a response to cellular stimulants is decreased with prior intrauterine infection. This effect may have a role in regulating myometrial function in intrauterine infection.     

Nitric oxide regulation of corticotropin-releasing hormone release from the human perfused placenta in vitro

We have investigated the regulatory role of nitric oxide (NO) in corticotropin-releasing hormone (CRH) release from the human perfused placental lobule in vitro. The effects of the NO donor sodium nitroprusside, the NO synthase inhibitor N omega-nitro-L-arginine, and the NO substrate L-arginine on human (h) placental CRH secretion have been studied. Single lobules of term placentae were bilaterally perfused with Krebs solution (5 mL/min; 95% O2-5% CO2; 37 C; pH 7.3). Fetal and maternal perfusates were collected at 4 C every 30 min for 3 h. CRH immunoreactivity (CRH-IR) in perfusates was measured by RIA using the 41-residue synthetic CRH as standard, 125I-labeled Tyr-hCRH as tracer, and a rabbit anti-CRH antibody Y2BO. The sensitivity of the assay was 0.13 pmol/L. Under basal conditions, human perfused placentae in vitro continuously secreted CRH-IR, which diluted in parallel to a synthetic hCRH-(1-41) standard curve. Size-exclusion chromatography of placental perfusates using a Sephadex G-50 column indicated that placental CRH-IR predominately coeluted with hCRH-(1-41) standard. Basal maternal perfusate CRH-IR levels (27 +/- 4 pmol/L) released from perfused placental lobules were nearly 10-fold greater than fetal perfusate CRH-IR levels (3.4 +/- 0.7 pmol/L; P < 0.05). Infusion of sodium nitroprusside (30-100 mumol/L) into the maternal and fetal placental circulations inhibited CRH-IR release into maternal perfusate in a concentration-dependent manner, but did not inhibit CRH-IR release into the fetal perfusate. N omega-nitro-L-arginine (100 mumol/L) increased placental CRH-IR secretion into fetal perfusate, and this effect was reversed by the infusion of L-arginine (100 mumol/L), which also reduced release below basal levels. In contrast, maternal perfusate CRH-IR levels were not affected by N omega-nitro-L-arginine or L-arginine. These results indicate that the human perfused placenta in vitro releases a substance of similar mol wt and hCRH-IR. Moreover, modulators of the NO signaling pathway differentially affect placental secretion of CRH-IR into the maternal and fetal perfusates. These data are consistent with the involvement of NO in the regulation of placental CRH release during pregnancy.     

Role of nitric oxide in the modulation of the vascular response to angiotensin II in hypertensive rats

Studies of phagocytic efficiency in cells of the macrophage lineage have assumed additional importance since the discovery that HIV infection of these cells impairs their immune function. A rapid method has been developed for measuring phagocytosis of the opportunistic pathogen Mycobacterium avium complex by human monocytes. Fluoresceinated M. avium complex (F-MAC) was incubated with whole blood at 37 degrees C and the fluorescence of extracellular F-MAC was quenched using a vital blue stain. Monocytes were then stained with a monoclonal antibody (mAb) to human CD14 conjugated to phycoerythrin (PE) red cells were lysed, and the percentage of monocytes which had phagocytosed F-MAC was measured by flow cytometry. The results were reproducible in samples of blood taken from individual donors over a period of 1 or 2 weeks, and optimum F-MAC concentrations and an optimum incubation time were determined by experiment. This method has the advantages of requiring only a small volume of blood, not necessitating manipulation of cells before testing, and using a phagocytic target relevant to the pathogenesis of HIV infection.     

Role of nitric oxide in the pancreatic vascular and metabolic responses associated with activation of capsaicin-sensitive neurons

A significant decrease in mean number of CD5+, CD8(+)-lymphocytes in persons, who worked in 30-km zone of Chernobyl nuclear power station was revealed. A significant increase in percent of CD5+, CD4(+)-cell percents was observed in workers, who worked for 1,2-2,5 years in zone, but absolute number, were decreased comparing a control and data received in people, who have just arrived to work in 30-km zone. The positive correlation exists between the percent of lymphocytes and years of service in 30-km zone. The lower level of alpha 1-thymosine was revealed in serum of the persons, who worked in zone for 4.5-5 years than data received in people, who worked for 0.5 year. Increase level of serum autoantibodies reacting with thymic epithelial cell was detected in men, who worked in zone for 3-3.5 years. In persons, who worked more 5 years and have just arrived in zone identical data were received.