Precursor Of Nitric Oxide Synthesis Research Articles

Role of superoxide and nitric oxide in platelet-activating factor-induced acute lung injury, hypotension, and mortality in rats.

To investigate the role of superoxide and nitric oxide in platelet-activating factor-induced acute lung injury, hypotension, and mortality. Prospective, randomized, controlled, experimental study. University research laboratory. Anesthetized male Wistar rats (180 to 220 g) were studied. In the first set of experiments, animals were divided into three groups. Group 1 received platelet-activating factor (2 microg/kg i.v.). Group 2 received recombinant human superoxide dismutase (50,000 U/kg i.v.) 30 mins before platelet-activating factor injection. Group 3 received vehicle agents. In the second set of experiments, animals were divided into six groups that received N(G)-nitro-L-arginine (L-NNA), a selective inhibitor of nitric oxide synthesis, or L-arginine, the physiologic precursor of nitric oxide synthesis: a) vehicles (i.v.); b) vehicle plus L-arginine (100 mg/kg i.v.); c) vehicle plus L-NNA (10 mg/kg i.v.); d) vehicle plus platelet-activating factor (2 microg/kg i.v.); e) L-arginine plus platelet-activating factor; and f) L-NNA plus platelet-activating factor. The first intravenous administration was given 5 mins before the second intravenous injection for each group. In the first set of experiments, vascular labeling with Monastral blue B demonstrated diffuse microvascular injury in the alveolar capillary beds 2 hrs after platelet-activating factor challenge. Thiobarbituric acid-reactive substances in the lung significantly increased at 2 hrs after platelet-activating factor injection. Platelet-activating factor treatment also resulted in an increased concentration of total protein, albumin, and Evans blue dye in bronchoalveolar lavage fluid at 2 hrs after administration, suggesting platelet-activating factor induction of increased alveolar permeability. The platelet-activating factor-induced alveolar microvascular injury, lipid peroxidation, and increased alveolar permeability were inhibited by pretreatment with recombinant human superoxide dismutase. Although L-NNA alone did not affect alveolar permeability in the second set of experiments, L-NNA treatment before platelet-activating factor challenge significantly aggravated platelet-activating factor-induced increased alveolar permeability 2 hrs after platelet-activating factor challenge. Platelet-activating factor also produced a rapid decrease in blood pressure that was not ameliorated by treatment with L-NNA. However, L-NNA pretreatment was associated with a significant increase in platelet-activating factor-caused mortality within 6 hrs. All rats survived with L-arginine treatment before platelet-activating factor challenge. L-NNA treatment decreased nitrate/nitrite concentration, an index of total nitric oxide production, in plasma. These results indicate that superoxide, the derived active oxygen species, and lipid peroxidation are implicated in the pathogenesis of platelet-activating factor-induced acute lung injury. Nitric oxide does not play a major role in platelet-activating factor-induced hypotension. Nitric oxide appears to play a protective role in the acute lung injury and mortality induced by platelet-activating factor.

Amylin and Food Intake in Mice: Effects on Motivation to Eat and Mechanism of Action

Amylin is a hormone produced by the pancreatic islets of Langerhans. Amylin decreased food pellet consumption. Amylin also decreased lever pressing for milk solution whether or not the mice were prefed. Amylin did not produce a conditioned taste aversion in a two bottle test, whereas lithium chloride did. In addition, l-arginine, a precursor for nitric oxide synthesis, was demonstrated to inhibit the ability of amylin to decrease food intake. Amylin did not alter nitric oxide synthase activity in the fundus of the stomach. These studies demonstrated that amylin inhibits food intake at a higher range of doses than is typical of anorectic agents such as cholecystokinin. Amylin does not appear to decrease food intake by reducing the release of nitric oxide but may affect appetite by modulating serum glucose levels when co-released with insulin.

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 ofL-NG-nitroarginine (L-NOARG, 10μM), an inhibitor of NO synthase, andL-arginine (L-ARG, 1mM), a precursor of NO synthesis, were evaluated. The cholinergic contractions induced by electrical field stimulation (EFS: 60V, 2ms, 60Hz) of pig lobar bronchial preparations increased (29%) in the presence ofL-NOARG (10μM). This effect may be released by nerves in pig large airways during EFS.

Altered endothelium-dependent responses in lambs with pulmonary hypertension and increased pulmonary blood flow.

To investigate early endothelial function associated with increased pulmonary blood flow, vascular shunts were placed between the ascending aorta and main pulmonary artery in 18 late-gestation fetal sheep. Four weeks after delivery, the lambs were instrumented to measure vascular pressures and blood flows, and blood was collected to measure plasma concentrations of guanosine 3',5'-cyclic monophosphate [cGMP, the second messenger to nitric oxide (NO)-mediated vasodilation] and L-arginine (the precursor for NO synthesis). The responses to the endothelium-dependent vasodilators acetylcholine (ACh, 1.0 microgram/kg) and ATP (0.1 mg.kg-1.min-1), the endothelium-independent vasodilators M & B-22948 (a cGMP-specific phosphodiesterase inhibitor, 2.5 mg/kg) and inhaled NO (40 ppm), and N omega-nitro-L-arginine (an inhibitor of NO synthase, 5 mg/kg) were then compared with responses in 12 age-matched controls. Vasodilator responses in control lambs were determined during pulmonary hypertension induced by U-46619 (a thromboxane A2 mimic). Shunted lambs displayed a selective impairment of endothelium-dependent pulmonary vasodilation, an augmented pulmonary vasoconstricting response to NO synthase inhibition, increased plasma cGMP concentrations, and decreased L-arginine concentrations. Taken together, these data suggest that lambs with pulmonary hypertension and increased pulmonary blood flow have early aberrations in endothelial function, as manifested by increased basal NO activity, that cannot be further increased by agonist-induced endothelium-dependent vasodilators.

Effects of in vitro 5-HT 1 receptor activation in guinea pig trachea

5-hydroxytryptamine (5-HT) has been reported to show some effects in respiratory tissues by activation of different subtype receptors. It has been demonstrated that 5-HT 2 receptor activation causes in vivo and in vitro airways contraction and enhances effects of cholinergic nerve-mediated responses, whereas 5-HT 1, receptor activation seems to be related to a relaxant effect. Moreover, in isolated guinea pig ascendens colon preparations 5-HT 1, activation causes relaxation by involvement of nitric oxide (NO). The aim of this study was to investigate the effects of 5-HT 1 receptor activation in guinea pig trachea as well as NO probable role in this activation. In tissues pretreated with both ketanserin (10 μM), an antagonist of 5-HT 2 receptors, and ondansetron (10 μM), an antagonist of 5-HT 3 receptors, 5-HT (from 10 nM to 10 mM) relaxed guinea pig trachea precontracted with acetylcholine (ACh, 100 μM). Carboxamidotryptamine (5-CT, from 10 nM to 10 mM), an agonist of 5-HT 1 receptors, as well relaxed guinea pig trachea precontracted with ACh (100 μM). A pretreatment with NAN-190 (from 10 nM to 100 μM), a 5-HT 1A selective antagonist, reduced the 5-HT and 5-CT relaxant effects but only at very high concentrations. Finally, a pretreatment with L-nitro-arginine-methyl-ester (L-NAME, 1 mM), an inhibitor of NO-synthase, and L-arginine (L-ARG, 1 mM), a precursor of NO synthesis, did not modify 5-HT and 5-CT responses in guinea pig trachea. In conclusion, this study suggests a 5-HT relaxant activity in guinea pig trachea via a 5-HT 1 receptor activation without any NO pathway involvement. However, further investigations are needed to clarify which 5-HT 1 receptor subtype is involved in 5-HT relaxant effect.

Relaxant effect of nitric oxide and prostacyclin on serotonin‐induced vasocontraction of human umbilical artery

We investigated the effect of nitric oxide and prostacyclin, which are endothelium-derived vasodilators, on serotonin (5-HT)-induced vasocontraction, and 5-HT1-serotoninergic receptor mediated relaxation in the human umbilical artery. Serotonin-stimulated vasocontraction was measured in umbilical artery segments treated with and without 1) L-arginine (10(-4) M approximately 10(-2) M), a precursor of nitric oxide synthesis; 2) methylene blue (10(-5) M) and L-NG-monomethyl arginine (LNMA, 2 X 1O(-4) M), which are specific inhibitors of nitric oxide action and nitric oxide synthesis, respectively; and 3) tranylcypromine (2 X 10(-6) M, 2 X 10(-5) M), an inhibitor of prostacyclin synthesis. 4) Vessels were pretreated with M1, an inhibitor of 5-HT2 serotoninergic receptors, and then prostaglandin F2 alpha (9.O X 1O(-7) M). Finally, we measured the 5-HT1, receptor-mediated relaxation induced by 5-HT. Treatment with L-arginine significantly inhibits 5-HT-induced contraction in a dose-dependent manner. Treatment with methylene blue and LNMA significantly potentiated 5-HT-induced contraction. Tranylcypromine caused no significant changes in 5-HT-induced vasocontraction. 5-HT1 serotoninetic receptor-mediated relaxation was found at higher concentrations of 5-HT (greater than 4.94 X 10(-5) M). Our results suggest that nitric oxide may exert a strong relaxant effect on 5-HT-induced vasocontraction compared with prostacyclin in human umbilical artery.

Nitric oxide modulates glomerular filtration and renal blood flow of the newborn rabbit.

The current study was performed in 17 anesthetized and mechanically ventilated newborn rabbits to investigate the role of nitric oxide (NO) in the regulation of basal renal function of the immature kidney. Renal blood flow and glomerular filtration rate were determined by the clearance of p-aminohippuric acid and inulin, respectively. In 9 newborn rabbits (group 1), L-NAME, a NO synthesis inhibitor, significantly increased the renal vascular resistance by 31 +/- 9% and decreased the renal blood flow by 20 +/- 6%. The fraction of filtration significantly increased by 8 +/- 5% despite a delayed decline in glomerular filtration rate by 13 +/- 5%. Mean arterial pressure and heart rate were not altered. In 8 additional newborn rabbits (group 2), L-arginine, the physiological precursor of NO synthesis, partially reversed the renal hemodynamic changes induced by L-NAME. The present results demonstrate that the decrease in NO production induced by L-NAME (1) significantly affects the renal microcirculation of the immature newborn rabbit kidney and (2) predominantly increases the postglomerular renal vascular resistance. Endogenous NO thus appears to play a major role in maintaining the basal perfusion of the immature kidney.

Mechanisms mediating NG-nitro- l-arginine methyl ester-induced hypophagia in mice

N G-Nitro- l-arginine methyl ester (50 mg/kg s.c.), an inhibitor of nitric oxide (NO) synthase, has been reported to increase brain serotonin (5-hydroxytryptamine, 5-HT) metabolism and induce hypophagia. Conversely, enhanced NO synthase activity is found to be accompanied by a decrease in 5-HT level. This negative correlation between NO and 5-HT in the regulation of food intake was further studied in mice. 5-HT depletion by p-chlorophenylalanine (250 mg/kg i.p., twice daily for 2 days) failed to antagonize the hypophagic effect of N G-nitro- l-arginine methyl ester. Similarly, treatment with the NO synthesis precursor, l-arginine (1000 mg/kg s.c.), did not reverse the anorexia induced by fenfluramine (10 mg/kg s.c.), a 5-HT releaser/uptake inhibitor. Pretreatment with (-)-pindolol, methysergide and ritanserin had no effect on the hypophagic action of N G-nitro- l-arginine methyl ester, suggesting the lack of involvement of 5-HT 1 and 5-HT 2 receptors. The selective neuronal NO synthase inhibitor, 7-nitroindazole (12.5–50.0 mg/kg i.p.), however, did not exhibit any hypophagic effect whilst N G-nitro- l-arginine methyl ester increased gastric retention, which may subsequently induce satiety. Moreover, the hypophagic effect of N G-nitro- l-arginine methyl ester, which was unassociated with changes in water intake and malaise induction, was also unattenuated by cholecystokinin (CCK) receptor antagonists, devazepide (10 mg/kg i.p.) and PD 135,158 ([1 S-[1α,2β[ S∗( S∗)],4α]]-4-[[2-[[3-(1 H-indol-3-myl)-2-methyl-1-oxo-2-[[[(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)oxy]carbonyl] acid N-methyl- d-glucamine salt; 1 mg/kg i.p.). Furthermore, a decrease in exploratory activity and diminished preference for favorable sensory cues were observed in N G-nitro- l-arginine methyl ester-treated animals. These results suggest that mechanisms other than 5-HT are possibly involved in mediating the hypophagic effect of N G-nitro- l-arginine methyl ester treatment in mice.

Possible modulation of auditory middle latency responses by nitric oxide in the inferior colliculus of anaesthetized rats

Nitric oxide (NO) is a short-lived radical species endowed with intercellular signalling functions in the mammalian brain. In the present study we have investigated the effects of focal injection into one inferior colliculus of N ω-nitro- L-arginine methyl ester ( L-NAME), a nitric oxide synthase inhibitor, on the acoustic middle latency responses (MLRs) evoked by click stimuli and recorded from the auditory cortex in anaesthetized rats. Microinfusion of L-NAME (1.0 mM) did not alter the latency of MLRs nor did it affect the evoked brain stem responses (ABRs). By contrast, L-NAME reduced P 1a—N 1 amplitude of MLRs by 51.7 ± 6.6% (mean ± SEM; n = 5) and almost complete recovery to background amplitude was obtained 15–25 min after treatment. The less active isomer, D-NAME (1.0 mM; n = 5), failed to produce consistent effects on the evoked MLRs. A higher concentration of L-NAME (5.0 mM; n = 5) yielded a 69.0 ± 13.3% inhibition whereas maximum inhibition produced by 0.5 mM ( n = 3) L-NAME was ≌10% of control value. The inhibitory effect typically evoked by 1.0 mM L-NAME was prevented by treating rats with L-arginine (5.0 mM; n = 5), the endogenous precursor of NO synthesis. Reduction of MLR amplitude was also obtained in rats receiving intracollicular injection of dizocilpine (MK801; 1.0 μM) and LY274614 (1.0 mM), two selective [ N-methyl- D-aspartate (NMDA) receptor antagonists. In conclusion, the present data support a role for intracollicular NO in the processing and transmission of the acoustic input to the auditory cortex in the rat.

Nitric oxide participates in the hypotensive effect induced by adenosine A2 subtype receptor stimulation.

In a previous study, we demonstrated that adenosine plays an important role in the central control of the cardiovascular system with involvement of adenosine A2 rather than A1 subtype receptors. In the present study, we investigated the putative relationship between nitric oxide (NO) and adenosine in the central and peripheral control of the cardiovascular system. Adult male normotensive anesthetized rats were treated with N6-cyclohexyladenosine (CHA), an A1-purinoceptor agonist, and 5'-N-cyclopropyl-carboxamidoadenosine (CPCA), an A2-purinoceptor agonist intracerebroventricularly (i.c.v. 3rd ventricle; 0.05-0.1-0.5 microgram/rat) and by intravenous injection (0.5-1-5 microgram kg-1 i.v.). CPCA and CHA induced a significant and dose-dependent decrease in arterial blood pressure (BP). CHA effects were less marked than CPA. Rats were pretreated with xanthine amine congener (XAC), and A1 adenosine antagonist, with 3,7-dimethyl-1-propargylxanthine (DMPX), an A2 adenosine antagonist (both administered at doses of 0.05 microgram/rat i.c.v. or 0.5 microgram kg-1 i.v.) and with N omega-nitro-L-arginine methyl ester, an NO synthase inhibitor, (L-NAME, 90 microgram/rat i.c.v. and 0.3 mg kg-1 i.v.). The intracerebroventricular and intravenous pretreatment with DMPX or L-NAME inhibited CPCA-induced hypotension; the effect of L-NAME was weaker than that of DMPX. The L-NAME inhibitory effect was reversed both in the central nervous system (CNS) and at the peripheral level by pretreatment with L-arginine (L-Arg; 90 mg kg-1 i.v.), a precursor of NO synthesis. Pretreatment with XAC, but not with L-NAME, reduced the hypotensive effect of CHA. Moreover, intracerebroventricular pretreatment with L-Arg (174 micrograms/rat) increased the hypotensive effect of CPCA.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide mediates C5a-induced vasodilation in the small intestine.

This study was designed to investigate the microvascular responses of the small intestine to complement C5a and to define the role of nitric oxide in the C5a-induced response. Male Sprague-Dawley rats were anesthetized with pentobarbital, and a loop of small intestine was exteriorized and suffused with Krebs solution. The diameters of large and small arterioles of the small intestinal wall were measured with in vivo videomicroscopy following the application of experimental mediators. Four 1-hr C5a dose-response trials were performed (10(-14) M, 10(-12) M, 10(-10) M, and 10(-8) M). Then, we completed acetylcholine dose-response curves with and without N omega-nitro-L-arginine (N-Arg) to document the adequacy of nitric oxide synthase inhibition. The microvascular response to the topical application of C5a (10(-12) M) was recorded in the presence of 2 x 10(-4) M N-Arg. Additionally, experiments of C5a-induced response with N-Arg were repeated in the presence of L-arginine (L-Arg; the precursor of nitric oxide synthesis) or with systemic administration of superoxide dismutase (SOD). (1) C5a induces a dose-dependent vasodilation in the small intestine, and the maximal vasodilation occurs in A3 arterioles at C5a concentration of 10(-12) M; (2) N-Arg inhibits the Ach-induced vasodilation in the rat small intestine; and (3) L-Arg or SOD partially reverses the inhibitory effect of N-Arg. Nitric oxide mediates the C5a-induced vasodilation in small intestinal microvessels. Superoxide is, at least partially, responsible for the vasoconstrictor response to C5a in the presence of N-Arg.

Functional role of nitric oxide in guinea pig tracheal epithelium.

Nitric oxide (NO) may play an important regulatory role in airway function. We have, thus, investigated in vitro whether epithelium derived NO may modulate cholinergic neurotransmission, via release of NO in guinea pig trachea, by using L-arginine (L-ARG), a precursor of NO synthesis, and L-N(G)-nitro-arginine-methyl-ester (L-NAME), an inhibitor of NO synthase. Results show that L-ARG and L-NAME modify acetylcholine sensitivity in epithelium-intact smooth muscle preparations, suggesting a probable NO synthesis by tracheal guinea pig epithelium.

Effects of E-series prostaglandins and their novel nitroderivatives on smooth muscle of small arteries

Nitric oxide (NO) is a unique interneuronal neurotransmitter and/or neuromodulator that is involved in a variety of physiological functions within the central nervous system (CNS). In neural tissue, NO is generated from an oxygen-dependent, constitutive NO synthase (NOS) by glutamatergic stimulation of N-methyl-d-aspartate (NMDA) receptors. Recent studies indicate that NO has excitatory effects on breathing within the CNS and mediates a central component of the hypoxic ventilatory reflex in mammals. Because NMDA receptors are important in central respiratory rhythmogenesis, we hypothesized that NO would have significant effects on the central pattern generator (CPG) for breathing in the brainstem. To test this hypothesis, the effects of NO on respiratory-related neural activity were investigated using an in vitro brainstem preparation from North American bullfrogs (Rana catesbeiana). Extracellular recordings of respiratory-related burst activity were made from cranial nerves V, X and XII before and during superfusion of the brainstem with NO-generating compounds, or inhibitors of NO synthesis. Addition of the NO donor, sodium nitroprusside (SNP; 0.1–1.0 mM), or the amino acid precursor for NO synthesis, l-arginine (L-Arg; 0.01–1.0 mM), caused significant increases in respiratory-related burst frequency. Inhibition of NOS with Nω-nitro-l-arginine (L-NA; 5–10 mM), a non-selective NOS inhibitor, caused a significant reduction in burst frequency or reversibly abolished neural activity. Brainstem perfusion with the specific neuronal NOS (nNOS) inhibitor, 7-nitro indazole (7-NI), produced significant, dose-dependent reversible reductions in burst frequency at concentrations of 0.1, 0.5 and 1.0 mM. These results suggest that production of NO, probably via nNOS, provides an excitatory input to the respiratory CPG in the amphibian brainstem. Our results suggest that NO may be a necessary inter- or intracellular messenger for neurotransmission and/or neuromodulation of central respiratory drive to motor effectors in the bullfrog.

Role of nitric oxide on the endothelium-dependent vasodilation in newborn piglet cerebral arteries

1. The present study was undertaken to determine whether endothelial nitric oxide (NO) is involved in the endothelium-dependent vasodilation elicited by bradykinin (BK) in rings of newborn (1–7-day-old) piglet cerebral arteries precontracted with KCl (25 mM). 2. In these rings, BK (10 −10−10 −6 M) induced concentration-dependent relaxation. The preincubation with the precursor of NO synthesis, l-arginine (10 −4 M), reduced KCl-induced contraction and increased the BK relaxation. However, preincubation with the NO synthase inhibitor, N G-nitro- l-arginine-methyl ester (L-NAME; 3 × 10 −5 M, increased KCl contraction and basal tone, and inhibited BK relaxation. 3. These results suggest that the endothelium of these arteries possesses the ability to produce NO, either basal or stimulated by agents like BK.

Is guanidino succinate a precursor for nitric oxide synthesis in rat vascular tissue?

Although L-Arginine (L-Arg) is considered the physiological precursor of nitric oxide (NO) synthesis in endothelial cells, recent experiments suggested that another guanidino derivative, guanidino succinate, may also serve as a major source of NO in this tissue. We tested this hypothesis in rat aortas, using two experimental situations in which L-Arg had previously shown significant activity, i.e., the ability to counteract contractile responses to the L-Arg analogue NG-nitro-L-arginine methyl ester (L-NAME) and the relaxation observed after prolonged incubation in physiologic buffer. Rat aortic rings, with or without endothelium, were suspended in organ chambers for recording of isometric tension and were contracted by phenylephrine (PE). After a brief incubation period (0.5 h), L-Arg, D-Arg, or guanidino succinate induced only minor relaxations in rings with or without endothelium. In the presence of L-NAME, L-Arg (but not the D-enantiomer), induced concentration-dependent relaxations of rings with endothelium (relaxation to L-Arg 10(-3) M 52 +/- 13%), reflecting a reversal by L-Arg of the L-NAME-induced potentiation of the contraction to PE. In contrast to L-Arg, guanidino succinate was less effective in the presence than in the absence of L-NAME (relaxation to guanidino succinate 10(-3) M before L-NAME 37 +/- 8% and after L-NAME 14 +/- 3%).(ABSTRACT TRUNCATED AT 250 WORDS)

Participation of nitric oxide in mouse anaphylactic hypotension

Mouse hen egg-white lysozyme-specific anaphylaxis was estimated by monitoring changes in blood pressure by using a tail-cuff method. Stimulation of histamine H 1 receptors of the vascular endothelium was suggested to be critical for mouse anaphylactic hypotension, because pretreatment with diphenhydramine but not with cimetidine completely inhibited the hypotension. Nitric oxide (NO) was indicated to play an important role in mouse anaphylaxis, because N G - nitro- L- argine methyl ester, a NO synthase inhibitor, significantly blocked the hypotension while a large amount of L-arginine, a precursor of NO synthesis, restored the hypotension.

Evidence for participation of nitric oxide in excitatory neurotransmission in rat vas deferens

A depression of the fast, non-adrenergic, and also of the slow, adrenergic, components of muscle contraction in response to intramural nerve stimulation was induced by the blocker of nitric oxide synthase N G-nitro-L-arginine methyl ester ( L-NAME), in rat vas deferens. Effects of exogenous noradrenaline or ATP were not reduced by L-NAME. However, L-arginine also caused an inhibition of electrically induced effects in most of the preparations, contrary to the expectations for a precursor of nitric oxide synthesis. In spite of these difficulties L-arginine antagonized the action of L-NAME. These results indicate that nitric oxide is involved in excitatory nerve-muscle transmission in vas deferens.

Nitric oxide activates cyclooxygenase enzymes.

We have evaluated the role of nitric oxide (NO) on the activity of the constitutive and induced forms of cyclooxygenase (COX; COX-1 and COX-2, respectively). Induction of NO synthase (NOS) and COX (COX-2) in the mouse macrophage cell line RAW264.7 by Escherichia coli lipopolysaccharide (1 microgram/ml, 18 h) caused an increase in the release of nitrite (NO2-) and prostaglandin E2 (PGE2), products of NOS and COX, respectively. Production of both NO2- and PGE2 was blocked by the NOS inhibitors NG-monomethyl-L-arginine or aminoguanidine. The effects of NG-monomethyl-L-arginine or aminoguanidine were reversed by coincubation with L-Arg, the precursor for NO synthesis, but not by D-Arg. RAW264.7 cells stimulated for 18 h with lipopolysaccharide in L-Arg-free medium (to reduce NO generation by the endogenous NOS pathway) failed to release NO2- and accumulated at least 4-fold less PGE2 when compared to cells in the presence of L-Arg. PGE2 production elicited by a 15-min arachidonic acid treatment of lipopolysaccharide-induced RAW264.7 cells in L-Arg-deficient medium was decreased 3-fold when compared to the release obtained with cells induced in medium containing L-Arg. To examine the NO activation of the induced form of COX in the absence of an endogenous L-Arg, human fetal fibroblasts were first stimulated for 18 h with interleukin 1 beta. These cells released PGE2 but not NO2-, consistent with the induction of COX but not NOS in the fibroblast. Exogenous NO either as a gaseous solution or released by a NO donor, sodium nitroprusside or glyceryl trinitrate, increased COX activity in the interleukin 1 beta-stimulated fibroblasts by 5-fold; these effects were abolished by coincubation with hemoglobin (10 microM), which binds and inactivates NO, but not by methylene blue, an inhibitor of the soluble guanylate cyclase. Furthermore, sodium nitroprusside (0.25-1 mM) increased arachidonic acid-stimulated PGE2 production by murine recombinant COX-1 and COX-2. These results demonstrate that NO enhances COX activity through a mechanism independent of cGMP and suggest that, in conditions in which both the NOS and COX systems are present, there is an NO-mediated increase in the production of proinflammatory prostaglandins that may result in an exacerbated inflammatory response. The data suggest that NO directly interacts with COX to cause an increase in the enzymatic activity.

Nitric oxide formation contributes to beta-adrenergic dilation of resistance coronary vessels in conscious dogs.

The contribution of the L-arginine/nitric oxide pathway to beta-adrenergic dilation of resistance coronary vessels was examined in conscious dogs instrumented for measuring coronary blood flow (CBF), left ventricular (LV) wall thickening, and LV and aortic pressures and for intracoronary injections of acetylcholine (0.003 micrograms/kg), nitroglycerin (0.175 micrograms/kg), and graded doses of isoproterenol (0.0005 to 0.004 micrograms/kg). Peak increases in CBF with intracoronary isoproterenol (0.001 micrograms/kg) averaged 105 +/- 10% from baseline. With acetylcholine, CBF increased by 158 +/- 11%, and with nitroglycerin, CBF increased by 139 +/- 10%. After the administration of intracoronary N omega-nitro-L-arginine methyl ester (L-NAME, 10 micrograms/kg per minute for 12 minutes) to block nitric oxide synthesis from L-arginine, baseline CBF was not altered, and CBF increased by 49 +/- 7% with isoproterenol and by 94 +/- 6% with acetylcholine; both values were smaller (P < .01) than those before the arginine analogue. With nitroglycerin, CBF was increased by 145 +/- 11%, not significantly different from the value before L-NAME. Intracoronary L-arginine (1.0 mg/kg per minute for 12 minutes), the precursor of nitric oxide synthesis, partially reversed the inhibition of L-NAME on CBF responses to acetylcholine and isoproterenol. After beta 1-adrenergic blockade, CBF responses to isoproterenol and acetylcholine were also reduced (P < .05) by the arginine analogue. When increases in CBF were prevented, peak changes in coronary vascular conductance with intracoronary bolus doses of acetylcholine and isoproterenol were attenuated (P < .01) by L-NAME. Thus, nitric oxide formation is an important intermediate in beta-adrenergic dilation of resistance coronary vessels in conscious dogs.

Endogenous nitric oxide protects against platelet aggregation and cyclic flow variations in stenosed and endothelium-injured arteries.

This study was designed to test the hypothesis that endogenously produced nitric oxide protects against platelet aggregation and cyclic flow variations in stenosed and endothelium-injured arteries of mongrel dogs. NG-Monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide formation, was administered at 5 mg/kg to 15 dogs after the left anterior descending coronary artery was mechanically injured and narrowed by external constrictors and to nine dogs before endothelial injury of the femoral artery and after injury and moderate arterial constriction. Treatment with L-NMMA resulted in cyclic flow variations (as detected by external Doppler flow probes) in the left anterior descending artery of seven of 15 dogs and in the femoral artery of four of nine dogs after endothelial injury. L-Arginine, the precursor for nitric oxide synthesis, was administered at 60 mg/kg and abolished cyclic flow variations in each of the 11 dogs. D-Arginine did not change the L-NMMA-induced cyclic flow variations. Saline infusion did not induce or change cyclic flow variations in any of the animals. Acetylcholine (1, 10, and 100 micrograms/min; n = 9) was administered in the femoral artery of nine additional dogs before and after endothelial injury in moderately stenosed femoral arteries. Acetylcholine did not induce cyclic flow variations in any animal; however, it did increase the severity of cyclic flow variations that developed in severely stenosed arteries. The diameter of the femoral artery was measured by intravascular ultrasound imaging. L-NMMA caused vasoconstriction of normal arteries, but no change was detected in endothelium-injured arteries. In contrast, L-arginine caused vasodilation of normal arteries, but, again, no change was noted in endothelium-injured arteries. Acetylcholine dilated normal femoral arteries but constricted arteries with endothelial injury. In both in vitro and ex vivo platelet studies, L-NMMA enhanced platelet aggregation, whereas L-arginine significantly reduced platelet aggregation. D-Arginine and acetylcholine showed no effect on platelet aggregation. Promotion of nitric oxide production decreases platelet aggregation and may eliminate cyclic flow variations, whereas a reduction in nitric oxide formation enhances platelet aggregation and may induce cyclic flow variations. Acetylcholine causes vasoconstriction at the femoral arterial site of endothelial injury and may increase the severity of cyclic flow variations.