Effects Of Nitric Oxide Synthase Inhibition Research Articles

Aminoguanidine, a selective inhibitor of the inducible nitric oxide synthase, has different effects on experimental allergic encephalomyelitis in the induction and progression phase

To elucidate the role of excessive nitric oxide (NO) via the inducible nitric oxide synthase (iNOS) in experimental allergic encephalomyelitis (EAE), the effect of a selective iNOS inhibitor, aminoguanidine, was investigated using mice with actively induced EAE. Administration of aminoguanidine by intraperitoneal or intracisternal injection from day 2 to day 12 after immunization produced a significant delay in the onset of EAE. On the other hand, administration of aminoguanidine by intraperitoneal or intracisternal injection for 10 days after the onset of EAE enhanced the clinical severity and mortality rate and hastened the onset of relapse significantly. The histological study at day 11 after the onset revealed that more inflammatory cells were present in the central nervous system of mice treated with aminoguanidine as compared with mice without aminoguanidine treatment. These results suggested that NO via iNOS was a pathogenetic factor in the induction phase of EAE, but had an inhibitory role in the progression phase of EAE. Although the effect of NO synthase inhibitors on EAE has been controversial, the present study suggested that the timing of administration might be an important consideration and might explain the previous contradictory reports.

Chronic inhibition of nitric oxide in central nervous system does not cause hypertension.

Acute inhibition of nitric oxide (NO) synthase in the brain causes elevation of blood pressure and sympathetic excitation under anesthetized conditions. To investigate chronic effects of NO synthase inhibition in the central nervous system on blood pressure regulation in conscious unrestrained animals, we administered NG-monomethyl-L-arginine (L-NMMA), a potent NO synthase inhibitor, at low (22.5 mumol/kg) and high (67.5 mumol/kg) doses for 1 wk into the cisterna magna with an osmotic pump and measured mean arterial pressure (MAP) and heart rate (HR) by a telemetry method. The same dose of NG-monomethyl-D-arginine (D-NMMA), an inactive isomer of L-NMMA, was administered to control rats. Chronic intracisternal administration of low-dose L-NMMA significantly decreased the brain nitrite/nitrate and NO metabolite contents as compared with D-NMMA (p < 0.05). However, MAP and its variability, HR and its variability, and plasma norepinephrine levels did not differ between the two groups of rats at either low- or high-dose treatment. Thus, chronic NO synthase inhibition in the central nervous system did not affect systemic hemodynamics or plasma norepinephrine concentrations despite the inhibition of brain NO. Our results suggest that endogenous NO in the central nervous system, at least as a whole, may not affect the systemic hemodynamics of chronic unanesthetized rats.

Hepatic O2 transport and energy balance in hyperdynamic porcine endotoxin shock: NG-monornethyl-L-arginine (L-NMMA) versus noradrenaline

Nitric oxide synthase (NOS) inhibition is currently investigated as a treatment of arterial hypotension associated with septic shock, but controversial data are available with respect to hepatic O2 balance. Therefore we studied the effect of NOS inhibition with L-NMMA on liver O2 transport as well as energy balance, in comparison to a standard treatment with noradrenaline (NOR) in a hyperdynamic endotoxin (ETX) shock model.

Role of NO in the O2 and CO2 responsiveness of cerebral and ocular circulation in humans.

It is well known that changes in PCO2 or PO2 strongly influence cerebral and ocular blood flow. However, the mediators of these changes have not yet been completely identified. There is evidence from animal studies that NO may play a role in hypercapnia-induced vasodilation and that NO synthase inhibition modulates the response to hyperoxia in the choroid. Hence we have studied the effect of NO synthase inhibition by NG-monomethyl-L-arginine (L-NMMA, 3 mg/kg over 5 min as a bolus followed by a continuous infusion of 30 micrograms.kg-1.min-1) on the changes of cerebral and ocular hemodynamic parameters elicited by hypercapnia and hyperoxia in healthy young subjects. Mean flow velocities in the middle cerebral artery and the ophthalmic artery were measured with Doppler ultrasound, and ocular fundus pulsation amplitude, which estimates pulsatile choroidal blood flow, was measured with laser interferometry Administration of L-NMMA reduced ocular fundus pulsation. (-19%, P < 0.005) but only slightly reduced mean flow velocities in the larger arteries. Hypercapnia (PCO2 = 48 mmHg) significantly increased mean flow velocities in the middle cerebral artery (+26%, P < 0.01) and fundus pulsation amplitude (+16%, P < 0.005) but did not change mean flow velocity in the ophthalmic artery. The response to hypercapnia in the middle cerebral artery (P < 0.05) and in the choroid (P < 0.05) was significantly blunted by L-NMMA. On the contrary, L-NMMA did not affect hyperoxia-induced (PO2 = 530 mmHg) hemodynamic changes. The hemodynamic effects of L-NMMA (at baseline and during hypercapnia) were reversed by coadministration of L-arginine. The present study supports the concept that NO has a role in hypercapnia induced vasodilation in humans.

Nonadrenergic–noncholinergic relaxations of isolated circular muscle from South American opossum esophagogastric junction: Is nitric oxide the inhibitory mediator?

Nonadrenergic–noncholinergic (NANC) inhibitory nerves are responsible for most of the nerve induced relaxations of gastrointestinal muscle. It has recently been proposed that NANC nerves may release nitric oxide (NO) or a related compound derived from l-arginine. We have recently shown that the South American (SA) opossum is another suitable model to elucidate the mechanism involved in these NANC relaxations. In the present study the effect of NO synthase inhibitors as well as NO inactivators on the NANC-nerve induced relaxations of the circular muscle of the esophagogastric junction (EGJ) of the SA opossum was investigated. It was observed that the NO synthase inhibitors, l-NOARG and l-NAME, caused a concentration-dependent reduction of NANC-nerve induced relaxations which was reversed by l- but not d-arginine. The NO-donors sodium nitroprusside and hydroxilamine as well as NO caused concentration-dependent relaxations of the EGJ circular muscle. In the myenteric plexus of this region, NADPH-diaphorase positive neurons and nerve fibers were observed while in the circular muscle layer only numerous positive fibers were found. The NO inactivators, hydroquinone, pyrogallol and carboxy-PTIO, reduced NO-induced relaxations but failed to affect NANC nerve- and sodium nitroprusside-induced relaxations. Taken together, these findings indicate that NANC nerve induced relaxation of the SA opossum EGJ circular muscle is dependent on neural NO synthase activity and suggest that the neurotransmitter being released is a superoxide resistant molecule, which is unlikely to be the NO radical, or that the activity of NO synthase is required for the release of the actual neurotransmitter rather than for synthesizing the neuromediator.

Nitric oxide enhances endothelin production in pancreas transplantation.

The role of endothelin and its relationship with nitric oxide (NO) production in ischemia-reperfusion associated with pancreas transplantation has been explored. For this purpose, pancreatic levels of endothelin were evaluated in an experimental model of pancreas transplantation after different periods of cold preservation. The effects of NO synthase inhibition were also evaluated. Results show posttransplantation increases in lipase and endothelin production. The release of lipase and endothelin was only prevented by NG-nitro-L-arginine methyl ester after a short ischemic period. Thus, endothelin synthesis could be a consequence of stimulation with NO in the ischemia-reperfusion associated with pancreas transplantation.

Nitric oxide(NO) and α-adrenergic reactivity of developing cerebral arteries: role of α1- and α2- adrenoceptor subtypes ♦ 339

Nitric oxide is an important endogenous inhibitor of norepinephrine(NE)-induced contraction accounting in part for the maturational decrease in NE sensitivity (i.e. ↑EC50) and in maximal force generated (Fmax) in ovine cerebral arteries. In fetal (F) and newborn (NB) sheep cerebral arteries, NE-induced contraction appears to be mediated byα1-adrenoceptors while the role of α2-adrenoceptors is not well characterized. However, α2-adrenoceptors have been demonstrated in cerebral arteries of some species. In order to evaluate the relative role of α-adrenoceptor subtypes and their susceptibility to attenuation by NO, we studied the effect of NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), on the contractile response to α1- and α2-adrenoceptor agonists, phenylephrine (PE) and UK 14304. Middle cerebral artery (MCA) rings from five F (125 d gest, 9 vessels) and three NB (3-7 d, 6 vessels) lambs were evaluated for isometric contraction using organ baths. Contractile reactivity to UK 14304 was poorly expressed in NB and F MCAs under control conditions; only 1/5 of NB and 3/8 of F MCAs responded. However, L-NAME (100 μM) dramatically enhanced the contractile response to this α2-agonist in both NB and F MCAs; 4/5 of NB and 8/8 of F responded (Fmax = 25±9 and 32±7% KCl(120 mM); EC50=45 and 50 nM, respectively). Theα1-agonist, PE, caused a dose-dependent contraction in both NB and F MCAs (Fmax=54±9 and 57±8% KCl, respectively). However, NB was three-fold less sensitive than F (EC50=1.30 μM vs 0.43 μM, respectively). L-NAME increased the sensitivity to PE in both NB and F MCA(EC50=0.55 μM and 0.23 μM, respectively) but not the Fmax. It is concluded that: 1) both α1- and α2-adrenoceptors are expressed in the developing ovine MCA, 2) NO attenuates bothα-adrenoceptor responses but α2-mediated contraction is most sensitive. The interplay between α2-stimulated endothelial NO production and smooth muscle contraction is pivotal to the ultimate adrenergic response. Accordingly, an immature or dysfunctional endothelium may significantly augment sympathetic reactivity via α2-dependent mechanisms.

EFFECT OF NITRIC OXIDE (NO) SYNTHASE INHIBITION ON PLASMA HYPOXANTHINE (Hx) IN NEWBORN PIGLETS WITH SURFACTANT DEFICIENCY: A POSSIBLE RELATIONSHIP BETWEEN PLASMA Hx AND PULMONARY HYPERTENSION † 1627

The Hx-xanthine oxidase system is an important generator of free radical which are reported to induce pulmonary hypertension. In vitro studies have shown that NO is an important free radical scavenger, and inhibition of NO synthase enhances production of superoxide anion in adult rats. The purpose of this study was to investigate the effect of NO synthase inhibition on plasma Hx in newborn piglets with surfactant deficiency, and to study a possible relationship between plasma Hx and pulmonary arterial pressure (Ppa). Methods: Nineteen anesthetized and instrumented newborn piglets were subjected to repeated lung lavages, and then randomly assigned to two groups: the L-NAME group (n = 12) received 3 mg/kg of L-NAME i.v; and the control group (n = 7) received same volume of saline i.v. Ppa was continuously recorded. Plasma Hx was analyzed with HPLC. Results: Plasma Hx was not modified by the repeated lung lavages, but increased significantly 45 minutes after L-NAME i.v. (p < 0.01)(fig). Saline injection, however, did not modify plasma Hx. Furthermore, the differences in Δ-plasma Hx between the two groups after L-NAME and saline i.v. were also significant (p < 0.05). Ppa was not changed after saline i.v., but increased significantly after L-NAME i.v. The change in Ppa after L-NAME i.v. was significantly correlated to plasma Hx (n = 24, r = 0.43, p < 0.05). Conclusion: These data show that inhibition of NO synthase may augments plasma Hx. We speculate that inhibition of NO synthase may potentiate production of free radicals during reperfusion which may contribute to pulmonary hypertension.

Further in vivo studies on attenuating morphine withdrawal: isoform-selective nitric oxide synthase inhibitors differ in efficacy

The N-methyl- d-aspartate (NMDA) receptor–nitric oxide (NO) pathway has been linked to opiate withdrawal. Pretreatments with four inhibitors of NO synthase, 7-nitro indazole, 3-bromo-7-nitro indazole, S-methyl- l-thiocitrulline and aminoguanidine, which exhibit different isoform selectivity in vitro, were evaluated for their ability to attenuate signs of naloxone-precipitated morphine withdrawal. In separate experiments, effects of NO synthase inhibitors on blood pressure were measured in naı̈ve and morphine-dependent rats. 7-Nitro indazole, 3-bromo-7-nitro indazole and S-methyl- l-thiocitrulline, which are specific inhibitors of the constitutive isoforms, produced dose-dependent reductions of several signs of withdrawal. Blood pressure was unaffected by the indazoles, whereas S-methyl- l-thiocitrulline produced a strong vasoconstrictor response. Aminoguanidine, which selectively inhibits inducible NO synthase, reduced fewer signs of opioid withdrawal, had a lower relative potency and exhibited no vasopressor activity. These data suggest that constitutive isoforms, but not the inducible isoform of NO synthase, have a primary role in NO-mediated processes that modulate the opioid withdrawal syndrome in the rat. © 1997 Elsevier Science B.V.

Obligatory role of NO in glutamate-dependent hyperemia evoked from cerebellar parallel fibers

Electrical stimulation of cerebellar parallel fibers (PF) increases cerebellar blood flow (BFcrb), a response that is attenuated by glutamate receptor antagonists and NO synthase (NOS) inhibitors. We investigated whether administration of NO donors could counteract attenuation by NOS inhibitors of vasodilation produced by PF stimulation. In halothane-anesthetized rats the cerebellar cortex was exposed and superfused with Ringer solution. PF were stimulated with microelectrodes (100 microA, 30 Hz), and BFcrb was recorded by a laser-Doppler probe. During Ringer superfusion, PF stimulation increased BFcrb by 56 +/- 7% and hypercapnia by 72 +/- 5% (n = 5). Superfusion with the nonselective NOS inhibitor N-nitro-L-arginine (L-NNA, 1 mM) reduced resting BFcrb and attenuated the response to PF stimulation (-47 +/- 5%) and hypercapnia (-46 +/- 7%; PCO2 = 50-60 mmHg). After L-NNA, superfusion with the NO donors 3-morpholinosydnonimine (100 microM, n = 5) or S-nitroso-N-acetyl-penicillamine (5 microM, n = 5) reestablished resting BFcrb (P > 0.05 vs. before L-NNA) and reversed L-NNA-induced attenuation of the response to hypercapnia (P > 0.05 vs. before L-NNA) but not PF stimulation (P > 0.05 vs. after L-NNA). Similar results were obtained when NOS activity was inhibited with the inhibitor of neuronal NOS 7-nitroindazole (50 mg/kg i.p.). Like NO donors, the guanosine 3',5'-cyclic monophosphate analog 8-bromoguanosine 3',5'-cyclic monophosphate (n = 5), administered after L-NNA, restored resting BFcrb and counteracted inhibition of the response to hypercapnia but not PF stimulation. In contrast to NO donors and 8-bromoguanosine 3',5'-cyclic monophosphate, the NO-independent vasodilator papaverine (100 microM, n = 5) had no effect on attenuation of responses to PF stimulation or hypercapnia. Thus NO donors are unable to reverse the effect of NOS inhibition on vasodilation produced by PF stimulation. The data support the hypothesis that the vascular response to PF stimulation, at variance with hypercapnia, requires NOS activation and NO production. Thus NO plays an obligatory role in vasodilation produced by increased functional activity in cerebellar cortex.

The potential role of peroxynitrite in the vascular contractile and cellular energetic failure in endotoxic shock.

1. Peroxynitrite is a toxic oxidant species produced from nitric oxide (NO) and superoxide. We have recently observed that the cell-permeable superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP) inhibits the suppression of mitochondrial respiration elicited by authentic peroxynitrite in vitro. Here we have investigated the relative potency of MnTBAP and a range of related compounds in terms of inhibition of peroxynitrite-induced oxidation and cytotoxicity. In addition, we tested the effects of MnTBAP on the vascular and the cellular energetic failure in rodent models of endotoxic shock. 2. We observed a dose-related inhibition of the peroxynitrite-induced oxidation of dihydrorhodamine 123 to rhodamine by MnTBAP, ZnTBAP and FeTBAP, but not by MnTMPyP [(5,10,15,20-tetrakis(N-methyl-4'-pirydyl)porphinato)-mangan ese (III)]. In addition, MnTBAP, ZnTBAP and FeTBAP, but not MnTMPyP prevented the suppression of mitochondrial respiration by authentic peroxynitrite in cultured J774 macrophages. 3. In rat cultured aortic smooth muscle cells, MnTBAP protected against the suppression of mitochondrial respiration in response to authentic peroxynitrite, immunostimulation and nitric oxide (NO) donor compounds. MnTBAP slightly reduced the amount of nitrite/nitrate produced in response to immunostimulation in these cells. 4. Administration of MnTBAP, 15 mg kg-1 i.v., before the administration of endotoxin (15 mg kg-1, i.v.) to rats ameliorated the development of vascular hyporeactivity and the development of endothelial dysfunction in the thoracic aorta ex vivo. 5. MnTBAP also prevented the endotoxin-induced decrease in mitochondrial respiration, the development of DNA single strand breaks, and the depletion of intracellular NAD+ in peritoneal macrophages ex vivo. 6. MnTBAP did not inhibit the expression by endotoxin of the inducible NO synthase in lung samples. 7. MnTBAP did not alter survival rate in mice challenged with high dose endotoxin. 8. Our findings, taken together with previous data demonstrating protective effects of NO synthase inhibitors against the endotoxin-induced contractile and energetic failure in the models of shock used in the current study, and with the known ability of peroxynitrite to cause cellular energy depletion, suggest a role for peroxynitrite in the pathogenesis of cellular energetic failure and contractile dysfunction in endotoxin shock.

Possible involvement of nitric oxide in NMDA-induced glutamate release in the rat striatum: an in vivo microdialysis study

The involvement of nitric oxide (NO) production in the release of striatal glutamate induced by local infusion of N-methyl- d-aspartate (NMDA) was investigated using microdialysis in freely moving rats. At concentrations of 0.1, 0.25, 0.5 or 1 mM NMDA induced concentration-dependent increases in striatal glutamate release. This effect of NMDA (0.5 mM) was significantly inhibited by tetrodotoxin (10 μM), by striatal perfusion with Ca 2+-free medium containing EGTA (5 mM), or by the putative antagonist of intracellular Ca 2+, 8-( N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) (1, 10 or 100 μM). Local infusion of the competitive inhibitors of NO synthase (NOS), N G-nitro- l-arginine methyl ester ( l-NAME) or N G-monomethyl- l-arginine ( l-NMMA) (both at concentrations 0.1, 0.25, 0.5 or 1 mM) caused the concentration-dependent inhibition of the glutamate response to 0.5 mM NMDA. This effect of NOS inhibition was stereospecific, inasmuch as N G-nitro- d-arginine methyl ester ( d-NAME) (0.5 or 1 mM) failed to affect NMDA-induced glutamate release. These findings suggest that increased NO production following NMDA receptor activation mediates the increase in release of neurotransmitter glutamate triggered by activation of striatal NMDA receptors.

A Role for Nitric Oxide in the Development of the Ferret Retinogeniculate Projection

The ferret retinogeniculate projection segregates into eye-specific layers during the first postnatal week and into ON/OFF sublaminae, which receive inputs from either on-center or off-center retinal ganglion cells, during the third and fourth postnatal weeks. The restriction of retinogeniculate axon arbors into eye-specific layers appears to depend on action potential activity () but does not require activation of NMDA receptors (). The formation of ON/OFF sublaminae is also activity-dependent and is disrupted by in vivo blockade of NMDA receptors (). To investigate a possible mechanism whereby blockade of postsynaptic NMDA receptors in the lateral geniculate nucleus (LGN) results in changes in the size and position of presynaptic axon arbors, we tested the role of the diffusible messenger nitric oxide (NO) in the development of the retinogeniculate pathway. We found previously that NO synthase (NOS) is transiently expressed in LGN cells during the refinement of retinogeniculate projections (). In this study, treatment with NG-nitro-L-arginine (L-NoArg), an arginine analog that inhibits NOS, during the third and fourth postnatal weeks resulted in an overall pattern of sublamination that was significantly reduced compared with normal and control animals. Single retinogeniculate axon arbors were located in the middle of eye-specific layers rather than toward the inner or outer half as in normal or control animals. The effect of NOS inhibition was not a consequence of the hypertensive effect of L-NoArg. In contrast to the effect of L-NoArg on the formation of ON/OFF sublaminae, treatment with L-NoArg during the first postnatal week did not disrupt the formation of eye-specific layers. Biochemical assays indicated significant inhibition of NOS during both treatment periods. These data suggest that NO acts together with NMDA receptors in activity-dependent refinement of connections during a specific phase of retinogeniculate development.

Nitric oxide synthase inhibition does not affect somatosensory evoked potentials in the rat

Nitric oxide synthase (NOS) inhibition reduces the regional cerebral blood floww (rCBF) responses to somatosensory stimulations. It is controversial whether this is caused by a signalling role of nitric oxide (NO) between neurons and vascular smooth muscle, or by effects of NOS inhibition on neuronal activity. We here report that more than 85% inhibition of NOS activity by topical application of the NOS inhibitor N ω-nitro- l-arginine ( l-NNA) for 2 h does not affect somatosensory evoked potentials (SEPs) elicited by vibrissal deflection or electrical forepaw stimulation in chloralose anaesthetised rats equipped with a closed cranial window, whereas cerebral blood flow (CBF) responses due to these stimulation paradigms are reduced by approximately 60%. We conclude that the decrease of the regional vascular response to increased neuronal activity during NOS inhibition is not caused by a suppression of neuronal activity.

Acetylcholine relaxation of renal artery and nitric oxide synthase activity of renal cortex increase with fetal and postnatal age.

Endothelium-derived nitric oxide (NO) regulates hemodynamics in the fetal kidney and modulates renal perfusion during postnatal maturation. We hypothesize that NO release by renal arteries increases with fetal maturation and contributes to the increased renal perfusion before and after birth. We tested the effect of maturation on relaxation to acetylcholine (ACh; 10(-9) M to 10(-5) M), the prototypic endothelium-dependent relaxing agent, and sodium nitroprusside (10(-9) M to 10(-5) M), an NO donor, on isolated main renal arteries obtained from anesthetized fetal guinea pigs of varying gestational age (0.5-0.8, 0.8-0.9, and 0.9-0.97 gestation), and neonatal (1-50 d) and reproductively mature adult guinea pigs. The effect of NO synthase inhibition by nitro-L-arginine (LNA; 10(-4) M) and cyclooxygenase inhibition by indomethacin (10(-5) M) on ACh relaxation was also measured. Ca(2+)-dependent NO synthase activity was measured in fetal (0.5-0.87 gestation), neonatal (1-10 d), and adult (mature) renal cortex by the conversion of [L-14C]arginine to [L-14C]citrulline and the time course compared with the relaxation responses. Sensitivity and maximal relaxation to ACh increased with fetal age. In neonatal renal arteries, maximal relaxation but not sensitivity to ACh increased relative to the fetal arteries. In adult renal arteries, both sensitivity and maximal relaxation increased compared with fetal arteries. Sensitivity but not maximal responses to sodium nitroprusside increased with age but exhibited a different maturational pattern than ACh relaxation. LNA inhibited ACh relaxation in arteries of all ages. Indomethacin reduced the sensitivity to ACh only in the fetal arteries. Ca(2+)-dependent NO synthase activity of the renal cortex increased during fetal development reaching levels at near term similar to those found in both the newborn and adult kidneys. These results suggest that endothelium-derived NO release by the renal artery and constitutive NO synthase activity in the renal microvasculature increases with fetal and postnatal maturation. Further, the sensitivity of vascular smooth muscle to NO also increases after birth. Thus, functional adaptations in both the endothelium and the vascular smooth muscle contribute to the maturational changes in mechanisms regulating renal hemodynamics before and after birth.

Effects of nitric oxide donor SIN-1 on oxygen availability and regional blood flow during endotoxic shock.

An excessive release of nitric oxide (NO) has been incriminated in the circulatory disturbances of septic shock. To study the effects of an NO donor, 3-morpholinosydnonimine (SIN-1), an oxygen availability and regional blood flow during endotoxic shock to see if a beneficial effect of NO synthase inhibitors in septic shock could be conclusively demonstrated. In 14 anesthetized and mechanically ventilated dogs, global invasive hemodynamic monitoring was completed and ultrasonic flow probes were placed around the superior mesenteric, left renal, and left femoral arteries for simultaneous measurements of regional blood flow. All dogs received Escherichia coli endotoxin, 2 mg/kg. A control group (n = 7) was administered saline at 20 mL/kg per hour, and a SIN-1 group (n = 7) was given a combination of saline with SIN-1 at successive doses of 1, 2, and 4 micrograms/kg per minute. Neither systemic nor pulmonary arterial pressures were influenced by SIN-1. Cardiac index, stroke index, and left ventricular stroke work index did increase at low to moderate doses of SIN-1 but tended to decrease at the highest dose. Systemic and pulmonary vascular resistances decreased. Fractional blood flow increased in the mesenteric bed at all doses used, was not influenced in the renal bed, but decreased in the femoral bed at the highest dose. Oxygen-derived variables were similar in the 2 groups. Blood lactate and plasma concentrations of tumor necrosis factor were not significantly influenced. At the end of the SIN-1 infusion, the administration of 5 mg/kg of methylene blue increased arterial pressure, pulmonary arterial pressure, and systemic and pulmonary vascular resistances but decreased cardiac index and regional blood flow. The administration of low to moderate doses of the NO donor SIN-1 can significantly increase cardiac index and superior mesenteric blood flow without deleterious effects on arterial pressure in this model of endotoxic shock. These findings support the hypothesis that NO is essential to maintain organ blood flow even during endotoxic shock.

Toxic dilatation of colon in a rat model of colitis is linked to an inducible form of nitric oxide synthase.

The contribution of nitric oxide (NO) to the altered colonic contractility of acute colitis was investigated in the 2,4,6-trinitroben-zenesulfonic acid model. NO synthase was measured in colonic tissue; the effects of NO synthase inhibition on colonic contractility were studied in vitro and in vivo. Inducible NO synthase was not detected in normal colons, whereas inflamed colons showed high activity. Acute inflammation was associated with enlarged colonic perimeter. NO synthase inhibitors or selective inhibitors of the inducible enzyme prevented colonic dilatation. In vitro, contractile responses to KCl were lower in muscle from colitic than control rats. After NO synthase inhibition, however, no difference was observed between colitic and control muscle contractility. In vivo, intracolonic pressure was lower in colitic than in control rats. Selective inhibition of inducible NO synthase increased intracolonic pressure in colitic but not in control rats. In conclusion, NO generation by inducible enzymes impairs smooth muscle contractility in colitis and may be involved in the pathogenesis of toxic dilatation of the colon.

Chapter 21 Modulation of acetylcholine release by nitric oxide

This chapter reviews the studies in which the effects of endogenous and exogenous nitric oxide (NO) on the release of acetylcholine were investigated. As tools for the involvement of endogenous NO, the effects of NO synthase inhibitors on release of acetylcholine were studied. The cholinergic neurons are tonically stimulated by endogenous NO. On the other hand, NO synthase inhibitors fail to change either basal or electrically evoked acetylcholine release from slices of rat hippocampus and striatum. Endogenous and exogenous NO increase basal acetylcholine release from central and peripheral cholinergic neurons. The effect is tetrodotoxin sensitive and calcium-dependent, thus reflecting stimulation of exocytotic release. The facilitation of acetylcholine release is likely to be mediated via the cGMP system. In addition, NO has been shown to decrease the action potential-evoked acetylcholine release from peripheral neurons. The mechanism of inhibition is not known. Contraction experiments on various intestinal preparations suggest that the presynaptic nitrergic inhibition of acetylcholine release has functional consequences.

The influence of skin flap ischemia on serum nitric oxide concentrations.

Nitric oxide (NO), identified as a mediator of endothelium-dependent relaxation of vascular smooth muscle, is known to cause a number of inflammatory diseases, especially ischemia-reperfusion injury. This experimental study, using a rabbit epigastric island flap, was designed to investigate whether skin flap ischemia followed by reperfusion influences serum NO concentrations. In addition, the author investigated the effects of NO synthase inhibitors and heparin on skin flap ischemia. Serum NO concentrations after 15, 30, 45, and 60 minutes of ischemia followed by reperfusion were significantly increased compared with non-ischemic controls and elevated flaps. On the other hand, serum NO concentrations were suppressed in nitro-amino-methyl-L-arginine- and aminoguanidine-treated animals. Furthermore, administration of heparin increased serum NO concentrations in controls and animals with elevated flaps, but decreased serum NO concentrations in ischemic flaps with subsequent reperfusion. These results suggest that NO is one of the factors responsible for ischemia-reperfusion injury and that NO synthase inhibitors and heparin may protect against such injury.

Nitric oxide synthase inhibitors decrease human polymorphonuclear leukocyte luminol-dependent chemiluminescence

Nitric oxide synthase (NOS) inhibitors have been reported to modulate luminol-dependent chemiluminescence (CL) in rat macrophages, whereas the potent oxidant peroxynitrite (ONOO -) was shown to react with luminol to yield CL in a cellfree system. We evaluated the role of the l-arginine/NOS pathway in luminol CL by phorbol ester-activated human polymorpho-nuclear (PMN) leukocytes using the NOS inhibitors N G-monomethyl- l-arginine ( l-NMMA) and N-iminoethyl- l-omithine ( l-NIO). Nitric oxide (·NO) release was determined by oxidation of oxymyoglobin. In addition, the effect of NOS inhibitors on superoxide anion O 2 -) production was measured. Luminol CL was notably diminished by l-NMMA in a dose-dependent manner. Superoxide dismutase (SOD) also decreased luminol CL and l-NMMA potentiated light emission decrease produced by SOD. Nitric oxide and O 2 ·- production was significantly decreased by l-NMMA; moreover, luminol-dependent CL but not O 2 ·- production was attenuated by l-NIO. These data suggest that products of catalytic activity of both ·NO synthase and NADPH oxidase are required to elicit maximal luminol CL in this system. These studies demonstrate that the NOS synthase pathway is involved in luminol CL by human PMN, and they suggest that ONOO would be an unrecognized mediator in this phenomenon.