nitroarginine and pimagedine

Resistance exercise (RE) is characterized to increase strength, tone, mass, and/or muscular endurance and also for produces many beneficial effects, such as blood pressure and osteoporosis reduction, diabetes mellitus control, and analgesia. However, few studies have investigated endogenous mechanisms involved in the RE-induced analgesia. Thus, the aim of this study was evaluate the role of the NO/CGMP/KATP pathway in the antinociception induced by RE. Wistar rats were submitted to acute RE in a weight-lifting model. The nociceptive threshold was measured by mechanical nociceptive test (paw-withdrawal). To investigate the involvement of the NO/CGMP/KATP pathway the following nitric oxide synthase (NOS) non-specific and specific inhibitors were used: N-nitro-l-arginine (NOArg), Aminoguanidine, N5-(1-Iminoethyl)-l-ornithine dihydrocloride (l-NIO), Nω-Propyl-l-arginine (l-NPA); guanylyl cyclase inhibitor, 1H-[1,2,4]oxidiazolo[4,3-a]quinoxalin-1-one (ODQ); and KATP channel blocker, Glybenclamide; all administered subcutaneously, intrathecally and intracerebroventricularly. Plasma and cerebrospinal fluid (CSF) nitrite levels were determined by spectrophotometry. The RE protocol produced antinociception, which was significantly reversed by NOS specific and unspecific inhibitors, guanylyl cyclase inhibitor (ODQ) and KATP channel blocker (Glybenclamide). RE was also responsible for increasing nitrite levels in both plasma and CSF. These finding suggest that the NO/CGMP/KATP pathway participates in antinociception induced by RE.

Topics: Animals; Arginine; Cyclic GMP; Enzyme Inhibitors; Glyburide; Guanidines; Guanylate Cyclase; KATP Channels; Male; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Nitroarginine; Nociception; Ornithine; Oxadiazoles; Pain Measurement; Physical Conditioning, Animal; Quinoxalines; Rats; Signal Transduction

Nitric oxide synthase (NOS) isoforms are hemoenzymes that are only active as homodimers. We have examined the effect of the substrate-analogue inhibitors, N(G)-monomethyl-L-arginine (L-NMA), N(G)-nitro-L-arginine (L-NNA), N(G)-nitro-L-arginine methyl ester (L-NAME), N(5)-(1-iminoethyl)-L-ornithine (L-NIO), and N(6)-(1-iminoethyl)-L-lysine (L-NIL), the guanidine-containing inhibitor aminoguanidine (AG), and the amidine moiety-containing iNOS-specific inhibitor 1400W, on the formation of NOS dimer. Of these inhibitors, L-NMA effectively not only inhibited iNOS dimerization, but also destabilized its dimeric form in RAW264.7 cells stimulated with lipopolysaccharide plus interferon-γ, but not eNOS dimerization in endothelial cells. Importantly, this inhibition was highly correlated with NO production. These inhibitory effects were significantly reversed by addition of L-arginine. However, L-NNA, L-NAME, and AG in part or significantly increased dimerization of iNOS and eNOS in intact cells, and the other inhibitors assessed did not alter dimerization of iNOS and eNOS. These data taken together suggest that substituted groups of an arginine guanidino moiety play an important role in NOS dimerization as well as its catalytic activity. Our results indicate that l-NMA can inhibit iNOS-dependent NO production by preventing iNOS dimerization and destabilizing its dimeric form.

Topics: Animals; Cell Line; Enzyme Inhibitors; Guanidines; Lysine; Mice; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitroarginine; omega-N-Methylarginine; Ornithine; Protein Multimerization; Substrate Specificity

In liver cirrhosis/portal hypertension, collaterals as varices may bleed and are influenced by vasoresponsiveness. An angiotensin blockade ameliorates portal hypertension but the influence on collaterals is unknown.. Portal hypertension and cirrhosis were induced by portal vein (PVL) and common bile duct ligation (BDL). Hemodynamics, real-time PCR of angiotensin II receptors (AT(1)R, AT(2)R) in the left adrenal vein (LAV, sham) and splenorenal shunt derived from LAV (PVL, BDL) were performed. With an in situcollateral perfusion model, angiotensin II vasoresponsiveness with different preincubations was evaluated: (1) vehicle; (2) AT(1)R blocker losartan; (3) losartan plus nonselective nitric oxide synthase (NOS) inhibitor (N(ω)-nitro-L-arginine); (4) AT(2)R blocker PD123319; (5) PD123319 plus N(ω)-nitro-L-arginine; (6) N(ω)-nitro-L-arginine, and (7) losartan plus inducible NOS inhibitor aminoguanidine.. LAV AT(1)R and AT(2)R expression decreased in PVL and BDL rats. Losartan attenuated angiotensin II-elicited vasoconstriction but PD123319 had no effect. N(ω)-nitro-L-arginine but not aminoguanidine reversed the losartan effect.. Angiotensin receptors are downregulated in the collateral vessel of portal hypertensive and cirrhotic rats. The AT(1)R blockade attenuates the angiotensin II vasoconstrictive effect, suggesting AT(1)R mediates collateral vasoconstriction and the influence of AT(2)R is negligible. The lack of aminoguanidine influence indicates that endothelial NOS participates in the losartan effect.

Topics: Animals; Collateral Circulation; Down-Regulation; Guanidines; Hypertension, Portal; Imidazoles; Ligation; Liver Cirrhosis; Losartan; Male; Nitric Oxide Synthase; Nitroarginine; Portal Vein; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Vasoconstriction

Zinc is an important trace element in biological systems; however, excessive extracellular zinc could lead to neuronal cell death following ischemia, seizures, and brain trauma. In this study, we investigated whether the intracortical injection of zinc sulphate (200 μg/kg, i.c.) changes total number of Purkinje cells in the cerebellum and whether different types nitric oxide synthase inhibitors, N-(G)-nitro-L-arginine methyl ester (L-NAME), N(omega)-nitro-L-arginine (L-NNA), aminoguanidine and 7-nitroindazole (7-NI), have protective effects against zinc neurotoxicity in Wistar albino rats. Animals were divided into 6 groups: control, zinc, zinc+L-NAME (100 mg/kg, i.p.), zinc+L-NNA (100 mg/kg, i.p.), zinc+7-NI (100 mg/kg, i.p.) and zinc+aminoguanidine (100 mg/kg, i.p.) groups. Total number of Purkinje cells in the cerebellum was estimated using unbiased stereological technique as 318,947 ± 20,549, 123,483 ± 23,762, 206,537 ± 43,128, 178,135 ± 26,635, 193,148 ± 46,104 and 212,910 ± 26,399 in the control, zinc, zinc+L-NAME, zinc+L-NNA, zinc+7-NI and zinc+aminoguanidine groups, respectively (mean ± SD). The number of Purkinje cells in zinc group was significantly lower than that of the other groups (P<0.001). It was found that the nitric oxide synthase inhibitors have neuroprotective effect against zinc neurotoxicity on Purkinje cells. These data show that the inhibition of the nitric oxide synthase could prevent some of the deleterious effects of zinc on Purkinje cells.

Topics: Animals; Cell Count; Cell Death; Cerebellum; Enzyme Inhibitors; Guanidines; Indazoles; Neuroprotective Agents; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Purkinje Cells; Rats; Rats, Wistar; Zinc Sulfate

Supplementation of L-arginine, a nitric oxide precursor, during the late phase of myocardial ischemia/reperfusion increases myocyte apoptosis and exacerbates myocardial injury, but the underlying mechanism is unclear. During myocardial ischemia/reperfusion, apoptosis of endothelial cells precedes that of cardiomyocyte. Tumor necrosis factor-alpha (TNF) production is increased during myocardial ischemia/reperfusion, which may exacerbate myocardial injury by inducing endothelial cell apoptosis. We postulated that L-arginine may exacerbate TNF-induced endothelial cell apoptosis by enhancing peroxynitrite-mediated nitrative stress. Cultured human umbilical vein endothelial cells were either not treated (control) or treated with TNF alone or with TNF in the presence of L-arginine, the nonselective nitric oxide synthase inhibitor N (omega)-nitro-L-arginine (L-NNA), propofol (an anesthetic that scavenges peroxynitrite), or L-arginine plus propofol, respectively, for 24 hours. TNF increased intracellular superoxide and hydrogen peroxide production accompanied by increases of inducible nitric oxide synthase (iNOS) protein expression and nitric oxide production. This was accompanied by increased protein expression of nitrotyrosine, a fingerprint of peroxynitrite and an index of nitrative stress, and increased endothelial cell apoptosis. L-arginine did not enhance TNF-induced increases of superoxide and peroxynitrite production but further increased TNF-induced increase of nitrotyrosine production and exacerbated TNF-mediated cell apoptosis. L-NNA and propofol, respectively, reduced TNF-induced nitrative stress and attenuated TNF cellular toxicity. The L-arginine-mediated enhancement of nitrative stress and TNF toxicity was attenuated by propofol. Thus, under pathological conditions associated with increased TNF production, L-arginine supplementation may further exacerbate TNF cellular toxicity by enhancing nitrative stress.

Topics: Apoptosis; Arginine; Cell Line; Cell Survival; Endothelial Cells; Enzyme Inhibitors; Glutathione Peroxidase; Guanidines; Humans; Hydrogen Peroxide; L-Lactate Dehydrogenase; Malondialdehyde; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitrites; Nitroarginine; Oxidative Stress; Propofol; Superoxide Dismutase; Superoxides; Tumor Necrosis Factor-alpha; Tyrosine

The aim of this study was to investigate the effects of peritonitis on spontaneous contractions of ileum and jejunum smooth muscles isolated from rats. Peritonitis was induced by cecal ligation and puncture in 8 rats. Another group of 8 rats underwent a sham operation and acted as controls. Twenty-four hours after the operation, the rats were killed, and their ileum and jejunum smooth muscles were excised and placed in circular muscle direction in a 10 ml organ bath. Changes in the amplitude and frequency of spontaneous contractions were analyzed before and after the addition of different antagonists. Peritonitis induced the decrease in the amplitude and frequency of spontaneous contractions in ileum and jejunum smooth muscles. In control groups, both ileum and jejunum, the amplitude and frequency of spontaneous contractions were significantly elevated in the presence of N(G)-nitro-L-arginine (L-NNA) and indomethacin. In peritonitis groups, both ileum and jejunum, the amplitude and frequency of spontaneous contractions were significantly enhanced in the presence of L-NNA, aminoguanidine, indomethacin and celecoxib compared to control values. In conclusion, peritonitis induces the decrease in the amplitude and frequency of spontaneous contractions of ileum and jejunum that can be attributed to the rise of nitric oxide synthase and cyclooxygenase isoforms levels.

Topics: Animals; Celecoxib; Guanidines; Ileum; In Vitro Techniques; Indomethacin; Jejunum; Male; Muscle Contraction; Muscle, Smooth; Nitric Oxide Synthase; Nitroarginine; Peritonitis; Prostaglandin-Endoperoxide Synthases; Pyrazoles; Rats; Rats, Wistar; Sulfonamides

Ca2+-dependent and Ca2+-independent nitric oxide synthase (NOS) activity, and neuronal and inducible NOS immunoreactivity (nNOS-IR and iNOS-IR), were investigated in the rabbit lower lumbar spinal cord after i) sciatic nerve transection and survival of experimental animals for 2 weeks, ii) treatment of animals with N-nitro-L-arginine (NNLA), an inhibitor of nNOS dosed at 20 mg/b.w. for 12 days, and iii) after treatment of animals with the inducible NOS (iNOS) inhibitor, aminoguanidine, dosed at 100 mg/b.w. for 4 and 12 days. Our attention was focused on the dorsal part of L4-L6 segments receiving sensory inputs from the sciatic nerve, and on the ventral part consisting of sciatic nerve motor neurons. Sciatic nerve transection increased Ca2+-dependent NOS activity and the density of nNOS in the dorsal part of the spinal cord on the ipsilateral side. NNLA treatment effectively reduced nNOS-IR in both the dorsal horn and the dorsal column, and decreased Ca2+-dependent NOS activity in the lower lumbar segments. Immunocytochemical analysis disclosed the up-regulation of iNOS immunoreactive staining after peripheral axotomy in alpha-motoneurons. The changes in iNOS expression and Ca2+-independent NOS activity were not significantly corrected by aminoguanidine treatment for 4 days. Long-lasting iNOS inhibition decreased Ca2+-independent NOS activity, but caused motor neuron degeneration and mediated small necrotic foci in the ventrolateral portion of the ventral horn. The results of the present study provide evidence that constitutive NOS inhibition by NNLA is more effective than specific long-lasting inhibition of iNOS by aminoguanidine treatment.

Topics: Animals; Axotomy; Calcium; Enzyme Inhibitors; Female; Guanidines; Immunohistochemistry; Male; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitroarginine; Rabbits; Sciatic Nerve; Spinal Cord; Tissue Distribution

The involvement of matrix metalloproteinases (MMPs) in ischemic tissue damage and remodeling has been reported by many investigators. Our study was designed to investigate the involvement of MMPs and of tissue inhibitors of metalloproteinases (TIMPs) in rat retinal ischemic injury, the effect of nitric oxide synthase (NOS) inhibitors on MMPs' activity in this model and whether minocycline (an MMP inhibitor) is protective in retinal ischemia.. Ninety-four rats were used in the study. Ischemia was induced by 90 min elevation of intraocular pressure. MMPs' activities and the effect of NOS inhibitors [aminoguanidine (AG) or N-nitro-L-arginine (NNA)] and minocycline on MMPs' activities were assessed by zymography and TIMPs expression by Western analysis. Morphological damage was quantified by morphometry of hematoxylin and eosin-stained retinal sections.. Retinal extracts exhibited activities of proMMP-9 and proMMP-2. The activity of proMMP-9 increased immediately post ischemia (PI) and peaked to 4.6 times that of normal untreated controls in ischemic retinas and to 2.6 times that of controls in retinas of fellow sham-treated eyes at 24 h PI. The relative amount of TIMP-1 increased to 1.9-fold following ischemia and 2.5-fold in fellow sham-treated eyes at 24 h PI. ProMMP-2 activity increased more than two-fold immediately, at 24 h and at 48 h PI in ischemic retinas, and insignificantly in fellow sham-treated eyes. Treatment with 25 mg/kg AG or NNA caused a non-significant increase in proMMP-9 activity at 24 h PI (3.7- and 2.9-fold, respectively, p>0.6). There was no effect of AG or NNA on the activity of proMMP-2. Minocycline significantly attenuated the retinal ischemic damage, primarily by partially preserving ganglion cells and the inner plexiform layer. Minocyline (0.5 mg/ml or 5 mg/ml) inhibited MMPs' activities in ischemic retinal extracts in vitro.. MMPs participated in morphological ischemic damage to rat retina. Treatment with minocycline dramatically attenuated damage to the retina.

Topics: Animals; Blotting, Western; Disease Models, Animal; Enzyme Inhibitors; Guanidines; Ischemia; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Minocycline; Nitric Oxide Synthase; Nitroarginine; Rats; Rats, Sprague-Dawley; Retinal Diseases; Retinal Vessels; Tissue Inhibitor of Metalloproteinase-1

Brief ischemia was reported to protect retinal cells against injury induced by subsequent ischemia-reperfusion with de novo protein synthesis, and this phenomenon is known as late ischemic preconditioning. The aims of the present study were to determine whether nitric oxide synthase (NOS) was involved in the mechanism of late ischemic preconditioning in rat retina using pharmacological tools. Under anesthesia with pentobarbital sodium, male Sprague-Dawley rats were subjected to 60 min of retinal ischemia by raising intraocular pressure to 130 mm Hg. Ischemic preconditioning was achieved by applying 5 min of ischemia 24 hrs before 60 min of ischemia. Retinal sections sliced into 5 microm thick were examined 7 days after ischemia. Additional groups of rats received NG-nitro-L-arginine and NG-monomethyl-L-arginin, non-selective NO synthase inhibitors, 7-nitroindazole, a neuronal NOS inhibitor, and aminoguanidine and L-N6-(1-iminoethyl) lysine, inducible NO synthase (iNOS) inhibitors before preconditioning, and were subjected to 60 min of ischemia. In the non-preconditioned group, cell loss in the ganglion cell layer and thinning of the inner plexiform and inner nuclear layer were observed 7 days after 60 min of ischemia. Ischemic preconditioning for 5 min completely protected against the histological damage induced by 60 min of ischemia applied 24 hrs thereafter. Treatment of rats with aminoguanidine and L-N6-(1-iminoethyl) lysine, but not NG-nitro-L-arginine, NG-monomethyl-L-arginine or 7-nitroindazole, wiped off the protective effect of ischemic preconditioning. The inhibitory effect of aminoguanidine was abolished by L-arginine, but not D-arginine. The results in the present study suggest that NO synthesized by iNOS is involved in the histological protection by late ischemic preconditioning in rat retina.

Topics: Animals; Enzyme Inhibitors; Guanidines; Indazoles; Ischemia; Ischemic Preconditioning; Lysine; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitroarginine; Rats; Rats, Sprague-Dawley; Retina; Retinal Vessels

Previous reports from our laboratory have shown that ethanol elicits hypotension in female but not in male rats and that this effect of ethanol is estrogen dependent (El-Mass MM and Abdel-Rahman AA. Alcohol Clin Exp Res 23: 624-632, 1999; El-Mass MM and Abdel-Rahman AA. Clin Exp Hypertens 21: 1429-1445, 1999). In the present study, we tested the hypothesis that ethanol lowers blood pressure in female rats via upregulation of the inducible nitric oxide synthase (iNOS) in vascular tissues. The effects of pretreatment with NG-nitro-L-arginine (NOARG; nonselective nitric oxide synthase inhibitor) or aminoguanidine (selective iNOS inhibitor) on hemodynamic responses elicited by intragastric (ig) ethanol were determined in conscious female rats. Changes in vascular (aortic) iNOS protein expression evoked by ethanol in the presence and absence of aminoguanidine were also measured by immunohistochemistry. Compared with control (water treated) female rats, ethanol (1 g/kg ig) elicited hypotension that was associated with a significant increase in the aortic iNOS activity. The hypotensive effect of ethanol was virtually abolished in rats infused with the nitric oxide synthase inhibitor NOARG, suggesting a role for nitric oxide in ethanol hypotension. The inability of ethanol to lower blood pressure in NOARG-treated rats cannot be attributed to the presence of elevated blood pressure in these rats because ethanol produced hypotension when blood pressure was raised to comparable levels with phenylephrine infusion. Selective inhibition of iNOS by aminoguanidine (45 mg/kg ip), which had no effect on baseline blood pressure, abolished both the hypotensive action of subsequently administered ethanol and the associated increases in aortic iNOS content. These findings implicate vascular iNOS, at least partly, in the acute hypotensive action of ethanol in female rats.

Topics: Animals; Aorta; Blood Pressure; Consciousness; Enzyme Inhibitors; Ethanol; Female; Guanidines; Heart Rate; Hemodynamics; Immunohistochemistry; Lipopolysaccharides; Nitric Oxide Synthase Type II; Nitroarginine; Rats; Rats, Sprague-Dawley; Up-Regulation

Melatonin, the major hormone produced by the pineal gland, is shown to have anticonvulsant effects. Nitric oxide (NO) is a known mediator in seizure susceptibility modulation. In the present study, the involvement of NO pathway in the anticonvulsant effect of melatonin in pentylenetetrazole (PTZ)-induced clonic seizures was investigated in mice. Acute intraperitoneal administration of melatonin (40 and 80 mg/kg) significantly increased the clonic seizure threshold induced by intravenous administration of PTZ. This effect was observed as soon as 1 min after injection and lasted for 30 min with a peak effect at 3 min after melatonin administration. Combination of per se non-effective doses of melatonin (10 and 20 mg/kg) and nitric oxide synthase (NOS) substrate L-arginine (30, 60 mg/kg) showed a significant anticonvulsant activity. This effect was reversed by NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg), implying an NO-dependent mechanism for melatonin effect. Pretreatment with L-NAME (30 mg/kg) and N(G)-nitro-L-arginine (L-NNA, 10 mg/kg) inhibited the anticonvulsant property of melatonin (40 and 80 mg/kg) and melatonin 40 mg/kg, respectively. Specific inducible NOS (iNOS) inhibitor aminoguanidine (100 and 300 mg/kg) did not affect the anticonvulsant effect of melatonin, excluding the role of iNOS in this phenomenon, while pretreatment of with 7-NI (50 mg/kg), a preferential neuronal NOS inhibitor, reversed this effect. The present data show an anticonvulsant effect for melatonin in i.v. PTZ seizure paradigm, which may be mediated via NO/L-arginine pathway by constitutively expressed NOS.

Topics: Animals; Anticonvulsants; Arginine; Convulsants; Dose-Response Relationship, Drug; Drug Therapy, Combination; Enzyme Inhibitors; Guanidines; Indazoles; Injections, Intraperitoneal; Kindling, Neurologic; Male; Melatonin; Mice; Mice, Inbred Strains; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Pentylenetetrazole; Seizures

Oxygen free radicals have been implicated in the pathogenesis of acoustic injury of the cochlea. The purpose of this study was to evaluate the effects of tempol (a superoxide anion scavenger), 3-aminobenzamide (a poly (ADP-ribose) synthetase (PARS) inhibitor), N-nitro-l-arginine (a non-selective nitric oxide synthase (NOS) inhibitor), 7-nitroindazole (a selective neuronal NOS inhibitor) and aminoguanidine (a selective inducible NOS inhibitor) on acoustic injury. Mice were exposed to a 4 kHz pure tone of 110-128 dB SPL for 4h. Tempol, 3-aminobenzamide or N-nitro-l-arginine was intraperitoneally administered immediately before the onset of acoustic overexposure, while 7-nitroindazole or aminoguanidine was intraperitoneally administered every 12h starting immediately before the onset of acoustic overexposure. The threshold shift of the auditory brainstem response (ABR) and hair cell loss were then evaluated one and two weeks after acoustic overexposure. Tempol and 3-aminobenzamide significantly protected the cochlea against acoustic injury, whereas the NOS inhibitors did not exert any protective effect. These findings suggest that reactive oxygen species and PARS are involved in acoustic injury of the cochlea. However, further study is necessary to elucidate the roles of nitric oxide and nitric oxide synthase in acoustic injury.

Topics: Analysis of Variance; Animals; Antioxidants; Auditory Threshold; Benzamides; Cochlea; Cyclic N-Oxides; Enzyme Inhibitors; Evoked Potentials, Auditory, Brain Stem; Female; Free Radical Scavengers; Guanidines; Hearing Loss, Noise-Induced; Indazoles; Mice; Neuroprotective Agents; Nitric Oxide Synthase; Nitroarginine; Noise; Poly(ADP-ribose) Polymerase Inhibitors; Spin Labels

As an important biological messenger, nitric oxide (NO) exhibits a wide range of effects during physiological and pathophysiological processes, including mammalian oocyte meiotic maturation. The present study investigated whether NO derived from two nitric oxide synthase (NOS) isoforms, inducible NOS (iNOS) or endothelial NOS (eNOS), is involved in the meiotic maturation of porcine oocytes. Meanwhile, the cumulus cells' function in meiotic maturation and their interaction with oocyte development and degeneration were also investigated using cumulus-enclosed oocytes (CEOs) and denuded oocytes (DOs). Different inhibitors for NOS were supplemented to the medium. Cumulus expansion, cumulus cell DNA fragmentation and oocyte meiotic resumption were evaluated 48 h after incubation. Aminoguanidine (AG), a selective inhibitor for iNOS, suppressed cumulus expansion and inhibited CEOs to resume meiosis (p < 0.05), but did not inhibit cumulus cell DNA fragmentation. Both Nomega-nitro-L-arginine (L-NNA) and Nomega-nitro-L-arginine methyl ester (L-NAME), inhibitors for both iNOS and eNOS, delayed cumulus expansion, inhibited cumulus cell DNA fragmentation and inhibited CEOs to resume meiosis. Such effects were not seen in DOs. These results indicate that iNOS-derived NO is necessary for cumulus expansion and meiotic maturation by mediating the function of the surrounding cumulus cells, and eNOS-derived NO is also involved in porcine meiotic maturation.

Topics: Animals; Cells, Cultured; DNA Fragmentation; Enzyme Inhibitors; Female; Guanidines; Meiosis; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitroarginine; Oocytes; Ovarian Follicle; Swine

The role of prostaglandins and nitric oxide (NO), generated after peripheral lipopolysaccharide (LPS) administration, in the adaptation of hypothalamic-pituitary-adrenal (HPA) axis under stressful circumstances remains to be elucidated. The aim of the present study was to assess the effect of chronic repetitive restraint or social crowding stress on the involvement of nitric oxide and prostaglandins in the LPS-induced pituitary-adrenocortical response. Male Wistar rats were restrained in metal tubes 2 x 10 min/day or crowded in cages for 7 days prior to treatment. All compounds were injected i.p., cyclooxygenase (COX) and nitric oxide synthase (NOS) inhibitors 15 min before LPS. Two hrs after injection LPS induced a significant increase in ACTH and corticosterone secretion. Repeated restraint impaired more potently than crowding stress the LPS-induced HPA-response. Indomethacin, a non-selective COX inhibitor, considerably reduced the LPS-induced HPA response in non-stressed rats and to a lesser extent diminished this response in repeatedly restrained or crowded rats. Neuronal NOS inhibitor, Nomega-nitro-L-arginine decreased the LPS-induced HPA response, more potently in control than crowded rats. Aminoguanidine, an iNOS inhibitor, diminished the LPS-elicited ACTH response in crowded rats. These results indicate that prostaglandins and NO generated by neuronal and inducible NOS are involved in the LPS-induced HPA axis response under basal conditions and during its adaptation to chronic social stress circumstances.

Topics: Adrenocorticotropic Hormone; Animals; Corticosterone; Guanidines; Hypothalamo-Hypophyseal System; Indomethacin; Interleukin-1; Lipopolysaccharides; Male; Nitric Oxide; Nitroarginine; Pituitary-Adrenal System; Prostaglandins; Rats; Rats, Wistar; Stress, Psychological

We investigated the role of nitric oxide (NO) in the interleukin 1beta (IL-1beta) and nicotine induced hypothalamic-pituitary-adrenal axis (HPA) responses, and a possible significance of CRH and vasopressin in these responses under basal and social stress conditions. Male Wistar rats were crowded in cages for 7 days prior to treatment. All compounds were injected i.p., nitric oxide synthase (NOS) inhibitors, alpha-helical CRH antagonist and vasopressin receptor antagonist 15 min before IL-1beta or nicotine. Identical treatment received control non-stressed rats. Plasma ACTH and serum corticosterone levels were measured 1 h after IL-1beta or nicotine injection. L-NAME (2 mg/kg), a general nitric oxide synthase (NOS) inhibitor, considerably reduced the ACTH and corticosterone response to IL-1beta (0.5 microg/rat) the same extent in control and crowded rats. CRH antagonist almost abolished the nicotine-induced hormone responses and vasopressin antagonist reduced ACTH secretion. Constitutive endothelial eNOS and neuronal nNOS inhibitors substantially enhanced the nicotine-elicited ACTH and corticosterone response and inducible iNOS inhibitor, aminoguanidine, did not affect these responses in non-stressed rats. Social stress significantly attenuated the nicotine-induced ACTH and corticosterone response. In crowded rats L-NAME significantly deepened the stress-induced decrease in the nicotine-evoked ACTH and corticosterone response. In stressed rats neuronal NOS antagonist did not alter the nicotine-evoked hormone responses and inducible NOS inhibitor partly reversed the stress-induced decrease in ACTH response to nicotine. These results indicate that NO plays crucial role in the IL-1beta-induced HPA axis stimulation under basal and social stress conditions. CRH and vasopressin of the hypothalamic paraventricular nucleus may be involved in the nicotine induced alterations of HPA axis activity. NO generated by eNOS, but not nNOS, is involved in the stress-induced alterations of HPA axis activity by nicotine.

Topics: Adrenocorticotropic Hormone; Animals; Arginine Vasopressin; Corticosterone; Corticotropin-Releasing Hormone; Crowding; Enzyme Inhibitors; Guanidines; Hypothalamo-Hypophyseal System; Interleukin-1; NG-Nitroarginine Methyl Ester; Nicotine; Nicotinic Agonists; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Pituitary-Adrenal System; Rats; Social Environment; Stress, Psychological

We examined the contribution of nitric oxide (NO) on the contractile impairment in diaphragm muscles of endotoxemic rats. Force-frequency relationship was depressed 24 h after lipopolysaccharide administration. 7-Nitroindazole, aminoguanidine and 1H-[1,2,4]Oxadiazole (4,3-a)quinoxalin-1-one (ODQ) partially restored the contractile impairment, Nomega-Nitro-L-Arginine (L-NNA) was ineffective. K+ contractions were reduced by 50% in endotoxemic muscles, 7-nitroindazole partially recovered, while aminoguanidine and L-NNA were ineffective. Verapamil reduced contractility to a greater extent in endotoxemic muscles. Caffeine and ryanodine contractions were augmented during endotoxemia without NOS contribution. L-NNA, 7-nitroindazole, ODQ and hemoglobin did not affect, but aminoguanidine completely restored partially inhibited neurotransmission by d-tubocurarine. Endotoxemia did not change membrane potentials and neurotransmitter release but slightly increased excitability. At this stage of endotoxemia, (1) constitutive NOS appears to be the dominant isoform, (2) NO does not have a major role on contractile dysfunction and (3) impairment could be explained by altered sensitivity of the voltage sensor. (4) NO does not substantially modulate neuromuscular transmission in normal and endotoxemic rats.

Topics: Animals; Caffeine; Diaphragm; Endotoxemia; Enzyme Inhibitors; Guanidines; In Vitro Techniques; Indazoles; Lipopolysaccharides; Male; Muscle Contraction; Neuromuscular Nondepolarizing Agents; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Oxadiazoles; Quinoxalines; Rats; Rats, Wistar; Ryanodine; Tubocurarine; Vasodilator Agents; Verapamil

Possible involvement of nitric oxide (NO) in the protective effect of ischemic preconditioning against the ischemia/reperfusion-induced acute renal failure was investigated. Ischemic preconditioning, which consists of three cycles of 2-minute ischemia followed by 5-minute reperfusion, was performed prior to 45-minute ischemia. Ischemic preconditioning significantly improved the renal dysfunction induced by 45-minute ischemia followed by 24-hour reperfusion. Histopathological examination of the kidney of ischemia/reperfusion rats revealed severe renal damage, and suppression of the damage was seen with the ischemic preconditioning treatment. NO metabolites (NOx) production in the kidney after 45-minute ischemia and reperfusion was markedly increased in ischemia/reperfusion rats with ischemic preconditioning, compared with animals not subjected to ischemic preconditioning, and these increases correlated with changes in endothelial NO synthase (eNOS) protein expression in renal tissues. The improvement of renal dysfunction in ischemic preconditioning rats was abolished by the pretreatment with NG-nitro-L-arginine, a nonselective NOS inhibitor, but not with aminoguanidine, an inducible NOS inhibitor. In addition, increment of endothelin-1 (ET-1) content in the kidney after the reperfusion was markedly suppressed by ischemic preconditioning treatment. These findings suggest that the protective effect of ischemic preconditioning on ischemia/reperfusion -induced acute renal failure is closely related to the renal nitric oxide production following the increase in eNOS expression after the reperfusion and that the suppressive effect of ischemic preconditioning on the ischemia/reperfusion -induced renal ET-1 overproduction may be partly involved in the ameliorating effect of ischemic preconditioning.

Topics: Acute Kidney Injury; Animals; Blood Urea Nitrogen; Endothelin-1; Enzyme Inhibitors; Guanidines; Ischemic Preconditioning; Kidney; Male; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Rats; Rats, Sprague-Dawley

To assess the systemic and nociceptive effects of nitric oxide synthase (NOS) inhibitors in the modulation of acute pain in rats subjected to the formalin test.. Formalin 5% was injected in the hind paw in the presence and absence of NOS inhibitors (e.g., 7-nitro indazole, N-nitro-L-arginine and aminoguanidine). Catheters were chronically implanted to continuously record mean arterial blood pressure (MAP) and heart rate (HR). MAP, HR and paw lifting time were recorded at control and every five minutes for 35 min following formalin and NOS inhibitors.. Formalin injected into the rat hind paw induced a biphasic nociceptive behaviour: an initial acute phase (phase 1: during zero to five minutes after the formalin injection) followed by a prolonged tonic response (phase 2: beginning about ten minutes after the formalin injection). Aminoguanidine, an inhibitor of the inducible NOS and 7-nitro indazole, an inhibitor of the neuronal NOS, did not affect phase 1, whereas N-nitro-L-arginine, a non-selective NOS inhibitor decreased it (49%). All three NOS inhibitors diminished nociceptive behaviours during phase 2. L-arginine reversed antinociceptive effects of N-nitro-L-arginine in phase 1 and in phase 2. Pressor effects induced by formalin in phase 1 were abolished following all three NOS inhibitors. During phase 2, formalin-induced pressor effects remained unaffected by N-nitro-L-arginine and aminoguanidine but were inhibited by 7-nitro indazole.. Our data demonstrate that NO is predominantly generated by vascular endothelial NOS in phase 1 and phase 2, whereas the neuronal NOS and the inducible NOS exhibit antinociceptive effects through a non-NO related pathway in phases 1 and 2 in rats subjected to the formalin test.

Topics: Analgesics; Animals; Arginine; Blood Pressure; Enzyme Inhibitors; Formaldehyde; Guanidines; Heart Rate; Indazoles; Nitric Oxide Synthase; Nitroarginine; Pain; Pain Measurement; Rats; Rats, Sprague-Dawley; Sodium Chloride; Time Factors

There is controversy in the literature over whether nitric oxide (NO) released during the inflammatory process has a pro- or inhibitory effect on neutrophil migration. The aim of the present investigation was to clarify this situation. Treatment of rats with non-selective, NG-nitro-L-arginine (nitro), or selective inducible NO synthase (iNOS), aminoguanidine (amino) inhibitors enhanced neutrophil migration 6h after the administration of low, but not high, doses of carrageenan (Cg) or Escherichia coli endotoxin (LPS). The neutrophil migration induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was also enhanced by nitro or amino treatments. The enhancement of Cg-induced neutrophil migration by NOS inhibitor treatments was reversed by co-treatment with L-arginine, suggesting an involvement of the L-arginine/NOS pathway in the process. The administration of Cg in iNOS deficient (iNOS(-/-)) mice also enhanced the neutrophil migration compared with wild type mice. This enhancement was markedly potentiated by treatment of iNOS(-/-) mice with nitro. Investigating the mechanisms by which NOS inhibitors enhanced the neutrophil migration, it was observed that they promoted an increase in Cg-induced rolling and adhesion of leukocytes to endothelium and blocked the apoptosis of emigrated neutrophils. Similar results were observed in iNOS(-/-) mice, in which these mechanisms were potentiated and reverted by nitro and L-arginine treatments, respectively. In conclusion, these results suggest that during inflammation, NO released by either constitutive NOS (cNOS) or iNOS down-modulates the neutrophil migration. This NO effect seems to be a consequence of decreased rolling and adhesion of the neutrophils on endothelium and also the induction of apoptosis in migrated neutrophils.

Topics: Animals; Apoptosis; Carrageenan; Cell Adhesion; Chemotaxis, Leukocyte; Enzyme Inhibitors; Guanidines; Inflammation; Leukocyte Rolling; Lipopolysaccharides; Male; Mice; Mice, Knockout; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Rats; Rats, Wistar

Blood vessels express estrogen receptors, but their role in cardiovascular physiology is not well understood. We show that vascular smooth muscle cells and blood vessels from estrogen receptor beta (ERbeta)-deficient mice exhibit multiple functional abnormalities. In wild-type mouse blood vessels, estrogen attenuates vasoconstriction by an ERbeta-mediated increase in inducible nitric oxide synthase expression. In contrast, estrogen augments vasoconstriction in blood vessels from ERbeta-deficient mice. Vascular smooth muscle cells isolated from ERbeta-deficient mice show multiple abnormalities of ion channel function. Furthermore, ERbeta-deficient mice develop sustained systolic and diastolic hypertension as they age. These data support an essential role for ERbeta in the regulation of vascular function and blood pressure.

Topics: Adrenergic alpha-Agonists; Animals; Aorta; Blood Pressure; Cells, Cultured; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Estrogen Receptor beta; Fulvestrant; Guanidines; Humans; Hypertension; In Vitro Techniques; Male; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Patch-Clamp Techniques; Phenylephrine; Potassium Channels; Receptors, Estrogen; Vasoconstriction

Some of the effects of bacterial toxins are mediated through the local production of nitric oxide (NO) or its products. This study examined if NO inhibition in the intestinal mucosa had effects on the responses to intravenous lipopolysaccharide (LPS) in anesthetized rats. Aminoguanidine (AMGU, 500 microM), a relatively selective inducible NO synthase (iNOS) inhibitor, or N(G)-nitro-L-arginine (NOLARG, 50 or 500 microM), a nonselective inhibitor of iNOS and constitutive NOS (cNOS), were perfused through the ileal lumen during intravenous LPS (17 mg/kg) or saline administration. Intestinal H20 transport, NO3- + NO2- (NOx) secretion, absorptive site mucosal blood flow (ASBF), blood pressure, plasma [NOx], tissue damage, and blood leukocytes were measured for 4 hr. LPS increased luminal NOx secretion. At 50 microM, luminal NOLARG attenuated the LPS-induced NOx secretion and increased blood pressure. There were no significant changes in lethality, plasma [NOx] or other parameters. At 500 microM, luminal NOLARG converted a nonlethal dose of LPS into a lethal dose, but AMGU did not increase lethality. The LPS-induced luminal NOx secretion was blocked by 500 microM intraluminal AMGU and NOLARG. Luminal NOx secretion also increased in control animals. This increase was blocked by 500 microM NOLARG but not AMGU. Luminal 500 microM NOLARG increased blood pressure, but AMGU did not. Luminal 500 microM NOLARG prevented the LPS-induced increase in plasma [NOx] and the decrease in leukocytes, but AMGU did not. Tissue damage occurred with intravenous LPS plus intraluminal 500 microM NOLARG. It was concluded that luminal AMGU inhibited mucosal iNOS. Luminal NOLARG inhibited mucosal cNOS and iNOS, and cNOS inhibition primed a lethal LPS effect. NOLARG, but not AMGU, was absorbed from the intestine and had systemic effects.

Topics: Animals; Blood Pressure; Escherichia coli; Female; Guanidines; Ileum; Lipopolysaccharides; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Rats; Rats, Sprague-Dawley; Water

The nitric oxide (NO) synthase inhibitor N(omega)-nitro-L-arginine (L-NNA) inhibits heat stress (HS)-induced NO production and the inducible 70-kDa heat shock protein (HSP-70i) in many rodent organs. We used human intestinal epithelial T84 cells to characterize the inhibitory effect of L-NNA on HS-induced HSP-70i expression. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured using fura-2, and protein kinase C (PKC), and PKA activities were determined. HS increased HSP-70i mRNA and protein in T84 cells exposed to 45 degrees C for 10 min and allowed to recover for 6 h. L-NNA treatment for 1 h before HS inhibited the induction of HSP-70i mRNA and protein, with an IC(50) of 0.0471 +/- 0.0007 microM. Because the HS-induced increase in HSP-70i mRNA and protein is Ca(2+) dependent, we measured [Ca(2+)](i) after treating cells with L-NNA. L-NNA at 100 microM significantly decreased resting [Ca(2+)](i). Likewise, treatment with 1 microM GF-109203X or H-89 (inhibitors of PKC and PKA, respectively) for 30 min also significantly decreased [Ca(2+)](i) and inhibited HS-induced increase in HSP-70i. GF-109203X- or H-89-treated cells failed to respond to L-NNA by further decreasing [Ca(2+)](i) and HSP-70i. L-NNA effectively blocked heat shock factor-1 (HSF1) translocation from the cytosol to the nucleus, a process requiring PKC phosphorylation. These results suggest that L-NNA inhibits HSP-70i by reducing [Ca(2+)](i) and decreasing PKC and PKA activity, thereby blocking HSF1 translocation from the cytosol to the nucleus.

Topics: Calcium; Cell Nucleus; Chelating Agents; Colonic Neoplasms; Cyclic AMP-Dependent Protein Kinases; Cycloheximide; Cytosol; Dactinomycin; DNA-Binding Proteins; Egtazic Acid; Enzyme Inhibitors; Guanidines; Heat Shock Transcription Factors; Hot Temperature; HSP70 Heat-Shock Proteins; Humans; Indoles; Intestinal Mucosa; Intestines; Isoquinolines; Maleimides; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Protein Kinase C; Protein Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulfonamides; Transcription Factors; Tumor Cells, Cultured

Left ventricular (LV) dysfunction caused by myocardial infarction (MI) is accompanied by endothelial dysfunction, most notably a loss of nitric oxide (NO) availability. We tested the hypothesis that endothelial dysfunction contributes to impaired tissue perfusion during increased metabolic demands as produced by exercise, and we determined the contribution of NO to regulation of regional systemic, pulmonary, and coronary vasomotor tone in exercising swine with LV dysfunction produced by a 2- to 3-wk-old MI. LV dysfunction resulted in blunted systemic and coronary vasodilator responses to ATP, whereas the responses to nitroprusside were maintained. Exercise resulted in blunted systemic and pulmonary vasodilator responses in MI that resembled the vasodilator responses in normal (N) swine following blockade of NO synthase with N(omega)-nitro-L-arginine (L-NNA, 20 mg/kg iv). However, L-NNA resulted in similar decreases in systemic (43 +/- 3% in N swine and 49 +/- 4% in MI swine), pulmonary (45 +/- 5% in N swine and 49 +/- 4% in MI swine), and coronary (28 +/- 4% in N and 35 +/- 3% in MI) vascular conductances in N and MI swine under resting conditions; similar effects were observed during treadmill exercise. Selective inhibition of inducible NO synthase with aminoguanidine (20 mg/kg iv) had no effect on vascular tone in MI. These findings indicate that while agonist-induced vasodilation is already blunted early after myocardial infarction, the contribution of endothelial NO synthase-derived NO to regulation of vascular tone under basal conditions and during exercise is maintained.

Topics: Adenosine Triphosphate; Animals; Coronary Circulation; Coronary Vessels; Endothelium, Vascular; Enzyme Inhibitors; Guanidines; Lung; Myocardial Infarction; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Nitroprusside; Physical Exertion; Reproducibility of Results; Swine; Vasodilation; Ventricular Dysfunction, Left

To determine whether type II nitric oxide synthase (NOS II) contributes to the NO-mediated fall in pulmonary vascular resistance (PVR) at birth, we studied the effects of selective NOS II antagonists N-(3-aminomethyl) benzylacetamidine dihydrochloride (1400W) and aminoguanidine (AG) and a nonselective NOS antagonist, nitro-L-arginine (L-NA), during mechanical ventilation with low FIO(2) (<10%), high FIO(2) (100%), and inhaled NO (20 ppm) in 23 near-term fetal lambs. Intrapulmonary infusions of AG, 1400W, and L-NA increased basal PVR before delivery (P<0.05). In control animals, ventilation with low and high FIO(2) decreased PVR by 62% and 85%, respectively. Treatment with AG and 1400W attenuated the fall in PVR by 50% during ventilation with low and high FIO(2) (control versus treatment, P<0.05 for each intervention). L-NA treatment attenuated the fall in PVR during ventilation with low and high FIO(2) to a similar degree as the NOS II antagonists. To test the selectivity of the NOS II antagonists, we studied the effects of acetylcholine and inhaled NO in each study group. Acetylcholine-induced pulmonary vasodilation remained intact after treatment with selective NOS II antagonists but not after treatment with nonselective NOS blockade with L-NA. In contrast, the response to inhaled NO was similar between treatment groups. We conclude that selective NOS II inhibition is as effective as nonselective NOS blockade in attenuating pulmonary vasodilation at birth and speculate that NOS II activity contributes to NO-mediated pulmonary vasodilation at birth. We additionally speculate that stimulation of the airway epithelium by rhythmic distension and increased FIO(2) may activate NOS II release at birth.

Topics: Acetylcholine; Administration, Inhalation; Amidines; Animals; Benzylamines; Enzyme Inhibitors; Female; Fetus; Guanidines; Hemodynamics; Labor, Obstetric; Lung; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Oxygen; Pregnancy; Pulmonary Circulation; Respiration, Artificial; Sheep; Vascular Resistance; Vasodilation; Vasodilator Agents

We have investigated the role of nitric oxide (NO) as mediator of the secondary growth of a traumatic cortical necrosis. For this purpose, a highly standardized focal lesion of the brain was induced in 46 Sprague-Dawley rats by cold injury. Twenty-four hours later--the timepoint of maximal lesion spread--the animals were sacrificed and brains were removed for histomorphometry of the maximal necrosis area and volume. The animals were divided into five experimental groups. Group I received the NO donor L-arginine as i.v. bolus 10 min prior to trauma (300 mg/kg body weight; n = 10) and a second bolus of the same dosage intraperitoneally 1 h after trauma. Group II (n = 10)--serving as control of group I--was infused with an i.v. bolus of 1 mL/kg isotonic saline 10 min prior to and a subsequent bolus i.p. 1 h after trauma. Group III (n = 8) received 100 mg/kg b.w. of the inducible NOS (iNOS) inhibitor aminoguanidine (AG) 1 h before and 8 h after trauma by intraperitoneal route. Group IV was administered with the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine (L-NNA; 100 mg/kg b.w., i.p.; n = 8); group V--the controls of group III and IV--was administered with isotonic saline (1 mL/kg b.w. i.p.; n = 10) 1 h before and 8 h after trauma. In the control group with i.v./i.p. sham treatment (II), the focal lesion led to a cortical necrosis with a maximum area of 3.1 +/- 0.3 mm2 and a lesion volume of 5.7 +/- 0.5 mm3 at 24 h after trauma. In animals with administration of L-arginine, the focal lesion had a maximum area of 3.1 +/- 0.3 mm2 and a volume of 5.3 +/- 0.5 mm3. Hence, the NO donor did not affect the secondary growth of necrosis. Animals with i.p. sham treatment (group V) had a maximal lesion area of 3.6 +/- 0.2 mm2 and lesion volume of 6.2 +/- 0.4 mm3. Administration of aminoguanidine afforded significant attenuation of the lesion growth. Accordingly, the maximal area of necrosis spread only to 2.8 +/- 0.2 mm2 with a volume of 4.5 +/- 0.5 mm3, respectively, at 24 h after trauma (p < 0.01 vs group V). On the other hand, administration of L-NNA did not influence the maximal lesion area (3.7 +/- 0.2 mm2) or lesion volume (6.5 +/- 0.5 mm3) evolving at 24 h after trauma. Thus, neither the enhancement of the formation of NO by L-arginine nor gross inhibition of the synthesis of NO by L-NNA did affect the secondary spread of the necrosis from a focal trauma. The marked attenuation of the posttraumatic necrosis growth by the iNOS inhibitor aminoguanidin

Topics: Animals; Arginine; Brain; Brain Injuries; Cold Temperature; Enzyme Inhibitors; Guanidines; Male; Necrosis; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Rats; Rats, Sprague-Dawley; Time Factors

Arterial vasodilation with concomitant hyperdynamic circulation are common findings in liver cirrhosis. Nitric oxide acting at a local level has been suggested to be pathophysiologically relevant in this context. Several systemic factors in conjunction with nitric oxide might interfere with the observed phenomena.. The study has been designed to demonstrate the influence of cirrhotic serum on the nitric oxide system and vascular contractility.. The contractile response of aortic segments from healthy rats was studied in vitro after incubation with serum of healthy and cirrhosis-induced rats (1 week, 2 weeks, 3 weeks and 4 weeks after bile duct ligation). A cumulative dose response curve to phenylephrine (10--10-4 mol) was established before and after incubation with nitric oxide synthesis blocker N(omega)-nitro-L-arginine, the more selective aminoguanidine (nitric oxide synthase [NOS]-2 inhibitor) and W7 (NOS-3 inhibitor). NOS-2 expression in incubated aortic rings was evaluated by Western blot analysis.. A 4-hour incubation with serum of cirrhosis-induced rats reduced the maximum contractile response to phenylephrine to 66.8 +/- 9.1% after 1 week, 50.4 +/- 7.8% after 2 weeks, 43.2 +/- 2.8% after 3 weeks and 35 +/- 5.2% after 4 weeks of bile duct ligation. This reduction in the contractility response to phenylephrine was completely reversed by blocking nitric oxide synthesis with N(omega)-nitro-L-arginine and aminoguanidine, but not after W7. Incubation with cirrhotic serum induced NOS-2 expression in aortic rings. In Western blot analysis, the most intensive signal for NOS-2 protein was obtained in rings incubated with serum from rats 3 weeks and 4 weeks after induction of cirrhosis.. Cirrhotic serum decreases the contractile response to phenylephrine even in an early stage of secondary cirrhosis. Reversibility of this effect after nitric oxide synthesis blockade suggests an induction of nitric oxide synthesis by systemic factors as a major point in vascular hyporeactivity to vasoconstrictors in cirrhosis.

Topics: Animals; Aorta; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanidines; In Vitro Techniques; Liver Cirrhosis, Experimental; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Phenylephrine; Rats; Rats, Sprague-Dawley; Sulfonamides; Vasoconstriction; Vasoconstrictor Agents; Vasodilator Agents

The effects of a range of nitric oxide (NO)-related compounds on histamine release from human basophils and rat peritoneal mast cells were studied. Basal and immunologic histamine releases from human basophils were not affected by N(omega)-nitro-L-arginine, N(omega)-nitro-L-arginine methyl ester, aminoguanidine or methylene blue (all inhibitors of NO production), sodium nitroprusside (an NO donor), L-arginine (a substrate for NO synthase) or D-arginine (the inactive enantiomer of L-arginine). In rat peritoneal mast cells, NO donors such as sodium nitroprusside, sodium nitrite and sodium nitrate, and lipopolysaccharide (an inducer of NO synthase) had little effect on basal histamine release, while 3-morpholino-sydnonimine (SIN-1, an NO donor), L-arginine and D-arginine increased this release by up to threefold. None of the inhibitors of NO production had any striking effect on histamine release induced by anti-rat immunoglobulin E (IgE), compound 48/80, sodium fluoride, phospholipase C, 1,2-dioctanoyl-sn-glycerol or ionophore A23187. However, haemoglobin was found to inhibit histamine release by anti-rat IgE or A23187 by ca. 40%. Alone of the NO donors, low concentrations of L-arginine produced a mild inhibition of histamine release induced by anti-IgE, compound 48/80 and A23187, but not other ligands, while sodium nitroprusside dose-dependently inhibited (by a maximum of ca. 30%) histamine release by anti-rat IgE, sodium fluoride or A23187. Stimulation with a variety of secretagogues or treatment with L-arginine, D-arginine, lipopolysaccharide, SIN-1 or sodium nitroprusside had no effect on NO production. Similarly, L-arginine, D-arginine or sodium nitroprusside did not change intracellular cGMP levels. On the basis of these results, it is suggested that NO does not play a significant role in the modulation of histamine release from human basophils or rat peritoneal mast cells. The effects of L-arginine, D-arginine and sodium nitroprusside may involve mechanisms unrelated to NO.

Topics: Animals; Antibodies; Arginine; Basophils; Calcimycin; Dose-Response Relationship, Drug; Guanidines; Hemoglobins; Histamine; Humans; Immunoglobulin E; Male; Mast Cells; Methylene Blue; Molsidomine; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitroarginine; Nitroprusside; p-Methoxy-N-methylphenethylamine; Peritoneal Cavity; Rats; Rats, Sprague-Dawley; Sodium Fluoride; Sodium Nitrite

To study the effects of aminoguanidine (AG) and two L-arginine analogues N(omega)-nitro-L-arginine methyl ester (L-NAME) and N(omega)-nitro-L-arginine (L-NNA) on nitric oxide (NO) production induced by cytokines (TNF-alpha, IL-1 beta, and IFN-gamma) and bacterial lipopolysaccharide (LPS) mixture (CM) in the cultured rat hepatocytes, and examine their mechanisms action.. Rat hepatocytes were incubated with AG, L-NAME, L-NNA, Actinomycin D (ActD) and dexamethasone in a medium containing CM (LPS plus TNF-alpha, IL-1 beta, and IFN-gamma) for 24h. NO production in the cultured supernatant was measured with the Griess reaction. Intracellular cGMP level was detected with radioimmunoassy.. NO production was markedly blocked by AG and L-NAME in a dose-dependent manner under inflammatory stimuli condition triggered by CM in vitro. The rate of the maximum inhibitory effects of L-NAME (38.9%) was less potent than that obtained with AG(53.7%, P < 0.05). There was no significant difference between the inhibitory effects of AG and two L-arginine analogues on intracellular cGMP accumulation in rat cultured hepatocytes. Non-specific NOS expression inhibitor dexamethasone (DEX)and iNOS mRNA transcriptional inhibitor ActD also significantly inhibited CM-induced NO production. AG(0.1 mmol x L(-1)) and ActD (0.2 ng x L(-1)) were equipotent in decreasing NO production induced by inflammatory stimuli in vitro, and both effects were more potent than that induced by non-selectivity NOS activity inhibitor L-NAME (0.1 mmol x L(-1)) under similar stimuli conditions (P<0.01).. AG is a potent selective inhibitor of inducible isoform of NOS,and the mechanism of action may be not only competitive inhibition in the substrate level, but also the gene expression level in rat hepatocytes.

Topics: Animals; Antineoplastic Agents; Cells, Cultured; Cyclic GMP; Cytokines; Dactinomycin; Dexamethasone; Enzyme Inhibitors; Glucocorticoids; Guanidines; Hepatocytes; Interferon-gamma; Interleukin-1; Lipopolysaccharides; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Protein Synthesis Inhibitors; Rats; Rats, Wistar; Tumor Necrosis Factor-alpha

This study investigated the effect of nitric oxide on lipid peroxide formation during endotoxaemia. Nitric oxide synthase inhibitors N(G)-monomethyl-L-arginine acetate (L-NMMA, 20 mg/kg, intravenously), N(G)-nitro-L-arginine-methyl ester (L-NAME, 10 mg/kg, intravenously), and N(G)-nitro-L-arginine (L-NA, 10 mg/kg, intravenously), and a relatively selective inducible nitric oxide synthase inhibitor aminoguanidine (10 mg/kg, intravenously), did not protect against endotoxin-induced death of mice. Superoxide dismutase activity in liver 18 hr after administration of endotoxin (6 mg/kg, intraperitoneally) to L-arginine analogues (L-NMMA, L-NAME, L-NA)-treated mice was lower than in mice treated with endotoxin alone, whereas the administration of L-arginine analogues increased xanthine oxidase activity in the livers of endotoxin-injected mice compared with mice treated with endotoxin alone. In mice treated with L-arginine analogues and aminoguanidine, the levels of non-protein sulfhydryl and lipid peroxide in liver 18 hr after endotoxin injection did not show significant differences from mice treated with endotoxin alone. L-Arginine analogues and aminoguanidine had little effect on lipid peroxide formation in liver caused by endotoxin. Treatment with aminoguanidine (300 microM) significantly inhibited endotoxin-induced intracellular peroxide in J774A.1 cells, however, aminoguanidine did not affect endotoxin-induced cytotoxicity in J774A.1 cells. Our results clearly demonstrate that treatment with catalase (10 microg/ml), D-mannitol (10 mM), or superoxide dismutase (100 U/ml), has little or no effect on nitric oxide production by endotoxin (1 microg/ml)-activated J774A.1 cells. These findings suggest that nitric oxide is not crucial for lipid peroxide formation during endotoxaemia. Therefore, it is unlikely that nitric oxide plays a significant role in liver injury caused by free radical generation in endotoxaemia.

Topics: Animals; Antioxidants; Cell Count; Cell Line; Cell Survival; Endotoxins; Enzyme Inhibitors; Free Radical Scavengers; Guanidines; Lipid Peroxides; Lipopolysaccharides; Liver; Male; Mice; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; omega-N-Methylarginine; Sulfhydryl Compounds; Superoxides; Survival Rate

The purpose of this study was to analyse the influence of experimental diabetes on vascular response of rabbit carotid artery to acetylcholine (Ach). We compared the Ach-induced relaxant response of isolated arterial segments obtained from both control and diabetic animals. To assess the influence of the endothelium, this cell layer was mechanically removed in some of the arterial segments ("rubbed arteries") from each experimental group. Ach induced a concentration-related endothelium-mediated relaxation of carotid artery from control rabbits that was significantly higher with respect to that obtained in diabetic animals. Pre-treatment with N(G)-nitro-L-arginine (L-NA) induced a concentration-dependent inhibition of relaxant response to Ach, which was significantly higher in carotid arteries isolated from diabetic rabbits. Incubation of rubbed arteries with L-NA almost abolished the relaxant response to Ach in arterial segments from both control and diabetic animals. Indomethacin potentiated Ach-induced response of carotid arteries from control rabbits, without modifying that obtained in those from diabetic animals. Aminoguanidine did not significantly inhibit the relaxant action of Ach in arterial segments from either control or diabetic rabbits. These results suggest that diabetes impairs endothelial modulatory mechanisms of vascular response of rabbit carotid artery to Ach. This endothelial dysfunction is neither related with a lower release of nitric oxide (NO) or prostacyclin. Diabetes impairs the production of some arachidonic acid vasoconstrictor derivative. There has been observed an increased modulatory activity of NO, but this is not related with the expression of an inducible isoform of NO synthase.

Topics: Acetylcholine; Alloxan; Animals; Anti-Inflammatory Agents, Non-Steroidal; Carotid Arteries; Diabetes Mellitus, Experimental; Endothelium, Vascular; Enzyme Inhibitors; Guanidines; Indomethacin; Male; Nitroarginine; Rabbits; Vasodilator Agents

The cecal ligation and puncture (CLP) model was used to investigate whether failure of neutrophil migration occurs in sepsis and whether it correlates with disease outcome. It was observed that the severity of sepsis correlates with the number of punctures in the cecum: mice with 2 punctures (sublethal [SL]-CLP) developed mild peritonitis (100% survived), whereas mice with 12 punctures (lethal [L]-CLP) developed severe peritonitis and bacteremia that evolved to sepsis (none survived). The production of tumor necrosis factor-alpha, interleukin-1beta, and interleukin-10 was higher in L-CLP than in SL-CLP mice. The impairment of neutrophil migration to the peritoneum and to the cecum wall was observed only in L-CLP mice. This phenomenon was shown to be mediated by nitric oxide, because aminoguanidine prevented the failure of neutrophil migration and improved the survival of L-CLP animals. In conclusion, impairment of neutrophil migration is a crucial event in the worsening of sepsis, and nitric oxide seems to be responsible for the phenomenon.

Topics: Animals; Ascitic Fluid; Bacteremia; Cell Movement; Colony Count, Microbial; Cytokines; Disease Models, Animal; Enzyme Inhibitors; Guanidines; Heart; Liver; Lung; Male; Mice; Mice, Inbred C57BL; Neutrophils; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Peritoneal Cavity; Peritoneal Diseases; Sepsis; Wounds, Penetrating

The influence of diabetes on endothelial mechanisms implicated in the response of isolated rabbit carotid arteries to 5-hydroxytryptamine (5-HT) was studied. 5-HT induced a concentration-dependent contraction that was potentiated in arteries from diabetic rabbits with respect to that in arteries from control rabbits. Endothelium removal potentiated 5-HT contractions in arteries from both control and diabetic rabbits but increased the maximum effect only in arteries from diabetic rabbits. Incubation of arterial segments with N(G)-nitro-L-arginine (L-NA) enhanced the contractile response to 5-HT. This L-NA enhancement was greater in arteries from diabetic rabbits than in arteries from control rabbits. Aminoguanidine did not modify the 5-HT contraction in arteries from control and diabetic rabbits. Indomethacin inhibited the 5-HT-induced response, and this inhibition was higher in arteries from control rabbits than in arteries from diabetic rabbits. In summary, diabetes enhances the sensitivity of the rabbit carotid artery to 5-HT. In control animals, the endothelium modulated the arterial response to 5-HT by the release of both nitric oxide (NO) and a vasoconstrictor prostanoid. Diabetes enhances endothelial constitutive NO activity and impairs the production of the endothelial vasoconstrictor.

Topics: Animals; Carotid Arteries; Diabetes Mellitus, Experimental; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Inhibitors; Guanidines; In Vitro Techniques; Indomethacin; Ketamine; Male; Nitric Oxide Synthase; Nitroarginine; Rabbits; Serotonin; Vasoconstriction

Gram-negative sepsis and subsequent endotoxic shock after surgery remain problematic in the United States and throughout the world. While morphine is widely prescribed for postoperative trauma pain management, there are reports that morphine may compromise the immune system and contribute to postoperative sepsis. The current study tested the hypothesis that morphine attenuates leukocyte rolling and sticking in both arterioles and venules via nitric oxide production. Nude mice implanted with slow-release morphine pellets were used in this study. The dorsal skinfold chamber model for intravital fluorescence microscopy on awake mice was used. Leukocyte/endothelial interactions were evaluated after bolus injection of oxidized low density lipoprotein. Morphine was found to significantly attenuate leukocyte rolling and sticking in both the arterial and venular side of the microcirculation. This attenuation was reversed by simultaneous implantation of naloxone pellets. The mechanisms of this attenuation were further investigated by administration of the nitric oxide synthase inhibitors NG-nitro-l-arginine (NOLA) and aminoguanidine (AG) in drinking water. NOLA was found to significantly reverse this morphine-induced attenuation of leukocyte rolling and sticking in both arterioles and venules. However, AG did not have the same effect. The results indicate that morphine interferes with leukocyte/endothelial cell interactions via stimulation of nitric oxide production.

Topics: Analgesics, Opioid; Animals; Cell Adhesion; Cell Communication; Endothelium, Vascular; Enzyme Inhibitors; Guanidines; Leukocytes; Mice; Mice, Hairless; Morphine; Nitroarginine

The aim of this work was to examine whether the non-insulin-dependent diabetic Goto-Kakizaki (GK) rats develop retinal changes with similar characteristics to those observed in insulin-dependent diabetic rats in what concerns blood-retinal barrier (BRB) permeability, nitric oxide (NO) production, and retinal IL-1beta level. BRB permeability was evaluated by vitreous fluorophotometry. NO synthase (NOS) activity was assessed by the production of l-[(3)H]-citrulline and retinal IL-1beta level was determined by ELISA. The expression of the inducible isoform of NOS (iNOS) protein was evaluated by Western blot analysis and immunohistochemistry. The in vivo studies indicated that in GK rats the BRB permeability to fluorescein was increased (787.81 +/- 68 min(-1)) in comparison to that in normal Wistar rats (646.6 +/- 55 min(-1)). The ex vivo studies showed that in retinas from GK rats the NOS activity was higher (207 +/- 28.9 pmol l-[(3)H]-citrulline/mg protein/30 min) than that in normal Wistar rats (125 +/- 32.3 pmol l-[(3)H]-citrulline/mg protein/30 min). These results were correlated with an increase in the protein level of iNOS in the retinas of GK rats, which was confirmed not only by the study of the iNOS protein expression but also by the use of NOS activity inhibitors. Indeed, the data about the effect of specific inhibitors on the NOS activity revealed that in retinas from GK rats the most effective inhibitor was aminoguanidine, which predominantly inhibits the iNOS isoform whereas in retinas from normal Wistar rats it was N(G) nitro l-arginine that predominantly inhibits the constitutive isoforms of NOS. In summary, in retinas from GK rats there is an increased production of NO which may contribute to the BRB breakdown.

Topics: Animals; Arginine; Blood Glucose; Blood-Retinal Barrier; Blotting, Western; Densitometry; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Diabetic Retinopathy; Disease Models, Animal; Fluorescein Angiography; Guanidines; Immunohistochemistry; Interleukin-1; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Rats; Rats, Inbred Strains; Rats, Wistar; Retina; Retinal Vessels

Endothelial nitric oxide (NO) synthase (eNOS) mRNA and protein and NO production are increased in hypoxia-induced hypertensive rat lungs, but it is uncertain whether eNOS gene expression and activity are increased in other forms of rat pulmonary hypertension. To investigate these questions, we measured eNOS mRNA and protein, eNOS immunohistochemical localization, perfusate NO product levels, and NO-mediated suppression of resting vascular tone in chronically hypoxic (3-4 wk at barometric pressure of 410 mmHg), monocrotaline-treated (4 wk after 60 mg/kg), and fawn-hooded (6-9 mo old) rats. eNOS mRNA levels (Northern blot) were greater in hypoxic and monocrotaline-treated lungs (130 and 125% of control lungs, respectively; P < 0.05) but not in fawn-hooded lungs. Western blotting indicated that eNOS protein levels increased to 300 +/- 46% of control levels in hypoxic lungs (P < 0.05) but were decreased by 50 +/- 5 and 60 +/- 11%, respectively, in monocrotaline-treated and fawn-hooded lungs (P < 0.05). Immunostaining showed prominent eNOS expression in small neomuscularized arterioles in all groups, whereas perfusate NO product levels increased in chronically hypoxic lungs (3.4 +/- 1.4 microM; P < 0.05) but not in either monocrotaline-treated (0.7 +/- 0.3 microM) or fawn-hooded (0.45 +/- 0.1 microM) lungs vs. normotensive lungs (0.12 +/- 0.07 microM). All hypertensive lungs had increased baseline perfusion pressure in response to nitro-L-arginine but not to the inducible NOS inhibitor aminoguanidine. These results indicate that even though NO activity suppresses resting vascular tone in pulmonary hypertension, there are differences among the groups regarding eNOS gene expression and NO production. A better understanding of eNOS gene expression and activity in these models may provide insights into the regulation of this vasodilator system in various forms of human pulmonary hypertension.

Topics: Animals; Enzyme Inhibitors; Guanidines; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; In Vitro Techniques; Male; Monocrotaline; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Nitroarginine; Pulmonary Circulation; Rats; Rats, Mutant Strains; Rats, Sprague-Dawley; RNA, Messenger; Tissue Distribution; Vasomotor System

Neuroprotection against cerebral ischemia can be realized if the brain is preconditioned by previous exposure to a brief period of sublethal ischemia. The present study was undertaken to test the hypothesis that nitric oxide (NO) produced from the neuronal isoform of NO synthase (NOS) serves as a necessary signal for establishing an ischemia-tolerant state in brain. A newborn rat model of hypoxic preconditioning was used, wherein exposure to sublethal hypoxia (8% oxygen) for 3 hours renders postnatal day (PND) 6 animals completely resistant to a cerebral hypoxic-ischemic insult imposed 24 hours later. Postnatal day 6 animals were treated 0.5 hour before preconditioning hypoxia with the nonselective NOS inhibitor L-nitroarginine (2 mg/kg intraperitoneally). This treatment, which resulted in a 67 to 81% inhibition of calcium-dependent constitutive NOS activity 0.5 to 3.5 hours after its administration, completely blocked preconditioning-induced protection. However, administration of the neuronal NOS inhibitor 7-nitroindazole (40 mg/kg intraperitoneally) before preconditioning hypoxia, which decreased constitutive brain NOS activity by 58 to 81%, was without effect on preconditioning-induced cerebroprotection, as was pretreatment with the inducible NOS inhibitor aminoguanidine (400 mg/kg intraperitoneally). The protective effects of preconditioning were also not blocked by treating animals with competitive [3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate; 5 mg/kg intraperitoneally] or noncompetitive (MK-801; 1 mg/kg intraperitoneally) N-methyl-D-aspartate receptor antagonists prior to preconditioning hypoxia. These findings indicate that NO production and activity are critical to the induction of ischemic tolerance in this model. However, the results argue against the involvement of the neuronal NOS isoform, activated secondary to a hypoxia-induced stimulation of N-methyl-D-aspartate receptors, and against the involvement of the inducible NOS isoform, but rather suggest that NO produced by the endothelial NOS isoform is required to mediate this profound protective effect.

Topics: Animals; Animals, Newborn; Brain Ischemia; Calcium; Dizocilpine Maleate; Enzyme Inhibitors; Guanidines; Hypoxia; Indazoles; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitroarginine; Oxygen; Piperazines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate

Little is known about the changes in hepatic blood flow associated with acute hepatic injury. The aim of this study was to investigate the effect of nitric oxide (NO) on hepatic blood flow and the severity of hepatic injury in rats with carbon tetrachloride (CCl4)-induced acute hepatic injury.. Rats were pretreated with CCl4 to induce acute hepatic injury. Hepatic blood flow was measured using a radioactive microsphere method. The role of NO in the regulation of hepatic blood flow and the severity of hepatic injury was investigated by administering NG-nitro-L-arginine (L-NNA) and aminoguanidine (AG). Plasma nitrite/nitrate levels, hepatic NO synthase (NOS) activity, and expression of hepatic NOS messenger RNA (mRNA) were measured, and histological examinations were performed.. Hepatic arterial and portal venous blood flow was increased significantly by CCl4, without any change in mean arterial pressure or cardiac output. L-NNA and AG dose-dependently decreased hepatic arterial blood flow, with the highest dose resulting in complete blockade of hepatic arterial dilation, but failed to change portal venous blood flow. Histologically, administration of AG aggravated the hepatic injury in CCl4-treated rats. Plasma nitrite/nitrate levels and hepatic NOS activity were increased significantly by CCl4 treatment. Inducible NOS mRNA was detected in CCl4-treated rats but not in the controls.. The results of this study suggest that the increased hepatic arterial blood flow in CCl4-induced acute hepatic injury is mediated by excessive NO production and up-regulated by inducible NOS, which plays a role in reducing hepatic injury.

Topics: Acute Disease; Animals; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Enzyme Inhibitors; Guanidines; Hepatic Artery; Liver; Liver Circulation; Liver Diseases; Male; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitrites; Nitroarginine; Rats; Rats, Sprague-Dawley; Regional Blood Flow; RNA, Messenger

Glyoxal, methylglyoxal (MG), and 3-deoxyglucosone (3-DG) are physiological alpha-oxoaldehydes formed by lipid peroxidation, glycation, and degradation of glycolytic intermediates. They are enzymatically detoxified in cells by the cytosolic glutathione-dependent glyoxalase system (glyoxal and MG only) and by NADPH-dependent reductase and NAD(P)+-dependent dehydrogenase. In this study, the changes in the cellular and extracellular concentrations of these alpha-oxoaldehydes were investigated in murine P388D1 macrophages during necrotic cell death induced by median toxic concentrations of hydrogen peroxide and 1-chloro-2,4-dinitrobenzene (CDNB). Alpha-oxoaldehyde concentrations were determined by derivatization with 1,2-diamino-4,5-dimethoxybenzene. There were relatively small increases in cellular and extracellular glyoxal concentration, except that extracellular glyoxal was decreased with hydrogen peroxide. The cytosolic concentration of 3-DG and the cytosolic and extracellular concentrations of MG, however, increased markedly. Aminoguanidine inhibited alpha-oxoaldehyde accumulation and prevented cytotoxicity induced by hydrogen peroxide and CDNB. The accumulation of glyoxal and MG in toxicant-treated cells was a likely consequence of decreased in situ activity of glyoxalase 1. The effect was marked for MG but not for glyoxal, suggestive of a greater metabolic flux of MG formation than of glyoxal. The accumulation of 3-DG in toxicant-treated cells was probably due to the decreased availability of pyridine nucleotide cofactors for the detoxification of 3-DG. Impairment of alpha-oxoaldehyde detoxification is cytotoxic, and this may contribute to toxicity associated with GSH oxidation and S conjugation in oxidative stress and chemical toxicity, and to chronic pathogenesis associated with diabetes mellitus where there is oxidative stress and the formation of glyoxal, MG, and 3-DG is increased.

Topics: Animals; Cell Line; Cell Survival; Deoxyglucose; Dinitrochlorobenzene; Glutathione; Glyoxal; Guanidines; Hydrogen Peroxide; Macrophages; Mice; Necrosis; Nitroarginine; Oxidative Stress; Pyruvaldehyde

TNF-alpha, inducible NO synthase (iNOS), and ICAM-1 are considered to be key proteins in the inflammatory response of most tissues. We tested the hypothesis that cell walls of Streptococcus pneumoniae (PCW), the most common cause of adult bacterial meningitis, induce TNF-alpha, iNOS, and ICAM-1 expression in rat primary brain microvascular endothelial cell cultures. We detected TNF-alpha mRNA by RT-PCR already 1 h after stimulation with PCW, while TNF-alpha protein peaked at 4 h (9.4 +/- 3.6 vs 0.1 +/- 0.1 pg/microgram protein). PCW induced iNOS mRNA 2 h after stimulation, followed by an increase of the NO degradation product nitrite (18.1 +/- 4 vs 5.8 +/- 1.8 at 12 h; 18.1 +/- 4 vs 5.8 +/- 1.8 pmol/microgram protein at 72 h). The addition of TNF-alpha Ab significantly reduced nitrite production to 62.2 +/- 14.4% compared with PCW-stimulated brain microvascular endothelial cells (100%). PCW induced the expression of ICAM-1 (measured by FACS), which was completely blocked by TNF-alpha Ab (142 +/- 18.6 vs 97.5 +/- 12.4%; 100% unstimulated brain microvascular endothelial cells). Cerebral endothelial cells express TNF-alpha mRNA as well as iNOS mRNA and release the bioactive proteins in response to PCW. PCW-induced NO production is mediated in part by an autocrine pathway involving TNF-alpha, whereas ICAM-1 expression is completely mediated by this autocrine loop. By these mechanisms, cerebral endothelial cells may regulate critical steps in inflammatory blood-brain-barrier disruption of bacterial meningitis.

Topics: Animals; Autocrine Communication; Brain; Cell Wall; Cells, Cultured; Dexamethasone; Dose-Response Relationship, Immunologic; Endothelium, Vascular; Guanidines; Immune Sera; Intercellular Adhesion Molecule-1; Kinetics; Microcirculation; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Rats; Rats, Wistar; RNA, Messenger; Streptococcus pneumoniae; Tumor Necrosis Factor-alpha; Up-Regulation

Tetrahydrobiopterin (BH4) is one of the cofactors of nitric oxide synthase (NOS), and the synthesis of BH4 is induced as well as inducible NOS (iNOS) by lipopolysaccharide (LPS) and/or cytokines. BH4 has a protective effect against the cytotoxicity induced by nitric oxide (NO) and/or reactive oxygen species in various types of cells. The purpose of this study was to examine whether or not an excess of BH4 is present during the production of NO by iNOS in LPS-treated de-endothelialized rat aorta. Addition of LPS (10 microg/ml) to the aorta bath solution caused L-arginine (L-Arg)-induced relaxation from 1.5 hr after the addition of LPS in de-endothelialized rat aorta pre-contracted with 30 mM KCl. The L-Arg-induced relaxation was prevented by NOS inhibitors. BH4 content also increased from 3 hr after the addition of LPS. mRNAs of iNOS and GTP cyclohydrolase I (GTPCH), a rate-limiting enzyme of BH4 synthesis, were increased from 1.5 hr after addition of LPS. Although the expression of iNOS and GTPCH mRNAs was observed in the media, the expression levels in the media were much lower than those in the adventitia. Ten millimolar 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTPCH, strongly reduced L-Arg-induced relaxation, and decreased BH4 content to below the basal level in LPS-treated aorta, whereas 0.5 mM DAHP reduced the LPS-induced increase in BH4 content to the basal level but did not affect L-Arg-induced relaxation. The inhibition of L-Arg-induced relaxation by 10 mM DAHP was overcome by the addition of BH4 (10 microM). These results suggest that although BH4 is essential for NO production from iNOS, the increase in BH4 content above the basal level is not needed for eliciting L-Arg-induced relaxation by the treatment with LPS. Thus, an excess amount of BH4 may be synthesized during NO production by iNOS in LPS-treated rat aorta.

Topics: Animals; Aorta, Thoracic; Arginine; Biopterins; Endothelium, Vascular; Glyceraldehyde-3-Phosphate Dehydrogenases; GTP Cyclohydrolase; Guanidines; Hypoxanthines; In Vitro Techniques; Lipopolysaccharides; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Rats; RNA, Messenger

Nitric oxide (NO) produced by NO synthase (NOS) modulates fetal pulmonary vascular tone and contributes to the fall in pulmonary vascular resistance (PVR) at birth. Although the inducible (type II) NOS isoform is present in human and rat fetal lungs, it is uncertain whether type II NOS activity contributes to vascular NO production in the fetal lung. To determine whether type II NOS is present in the ovine fetal lung and to study the potential contribution of type II NOS on the regulation of basal PVR in the fetus, we measured the hemodynamic effects of three selective type II NOS antagonists: aminoguanidine (AG), 2-amino-5,6-dihydro-6-methyl-4H-1,3 thiazine (AMT), and S-ethylisothiourea (EIT). Studies were performed after at least 72 h of recovery from surgery in 19 chronically prepared fetal lambs (133+/-3 d; 147 d, term). Brief intrapulmonary infusions of AG (140 mg), AMT (0.12 mg), and EIT (0.12 mg) increased basal PVR by 82, 69, and 77%, respectively (P < 0.05). The maximum increase in PVR occurred within 20 min, but often persisted up to 80 min. These agents also increased mean aortic pressure but did not alter the pressure gradient between the pulmonary artery and aorta, suggesting little effect on tone of the ductus arteriosus. Acetylcholine-induced pulmonary vasodilation remained intact after treatment with selective type II NOS antagonists, but not after treatment with the nonselective NOS blocker, nitro-L-arginine. Using Northern blot analysis with poly(A)+ RNA, we demonstrated the presence of two mRNA transcripts for type II NOS (4.1 and 2.6 kb) in the fetal lung. We conclude that the type II NOS isoform is present in the ovine fetal lung, and that selective type II NOS antagonists increase PVR and systemic arterial pressure in the late-gestation fetus. We speculate that type II NOS may play a physiological role in the modulation of vascular tone in the developing fetal lung.

Topics: Acetylcholine; Animals; Aorta; Blotting, Northern; Enzyme Inhibitors; Female; Gestational Age; Guanidines; Hemodynamics; Isothiuronium; Lung; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Pulmonary Artery; Rats; Sheep; Thiazines; Vascular Resistance

Nitric oxide (NO) is a free radical involved in the regulation of many cell functions and in the expression of several diseases. We have found that the antimalarial and antiinflammatory drug, chloroquine, is able to stimulate NO synthase (NOS) activity in murine, porcine, and human endothelial cells in vitro: the increase of enzyme activity is dependent on a de novo synthesis of some regulatory protein, as it is inhibited by cycloheximide but is not accompanied by an increased expression of inducible or constitutive NOS isoforms. Increased NO synthesis is, at least partly, responsible for chloroquine-induced inhibition of cell proliferation: indeed, NOS inhibitors revert the drug-evoked blockage of mitogenesis and ornithine decarboxylase activity in murine and porcine endothelial cells. The NOS-activating effect of chloroquine is dependent on its weak base properties, as it is exerted also by ammonium chloride, another lysosomotropic agent. Both compounds activate NOS by limiting the availability of iron: their stimulating effects on NO synthesis and inhibiting action on cell proliferation are reverted by iron supplementation with ferric nitrilotriacetate, and are mimicked by incubation with desferrioxamine. Our results suggest that NO synthesis can be stimulated in endothelial cells by chloroquine via an impairment of iron metabolism.

Topics: Aconitate Hydratase; Animals; Calcium; Canavanine; Cell Division; Cell Line, Transformed; Chloroquine; Cycloheximide; Cytosol; Endothelium, Vascular; Enzyme Activation; Enzyme Inhibitors; Guanidines; Heme; Humans; Hydrogen-Ion Concentration; Iron; Mice; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Ornithine Decarboxylase; Protein Synthesis Inhibitors; Stimulation, Chemical; Swine

Despite intense investigation, the effector of the infarct-limiting protection observed during the late phase of ischemic preconditioning (PC) remains unknown. The goal of this study was to test the hypothesis that late PC against myocardial infarction is mediated by the activity of nitric oxide synthase (NOS).. Conscious rabbits underwent a 30-minute coronary occlusion followed by 3 days of reperfusion. In group I (control group, n= 10), infarct size (tetrazolium staining) averaged 56.8+/-5.3% of the risk region, which was decreased to 27.6+/-2.5% (P<0.05) in rabbits preconditioned 24 hours earlier with a sequence of six 4-minute occlusion/4-minute reperfusion cycles (group II, n= 10). When preconditioned rabbits were given the nonselective NOS inhibitor N(omega)-nitro-L-arginine (L-NA, 13 mg/kg i.v. [group III, n=8]) or the selective iNOS inhibitor aminoguanidine (AG, 150 mg/kg SC [group V, n=7]) before the 30-minute occlusion, the protective effect of late PC was completely abrogated; that is, infarct size (59.9+/-4.5% and 65.8+/-3.3%, respectively) was similar to that measured in the control group. Measurements of systolic wall thickening (sonomicrometry) demonstrated that L-NA and AG also abolished the improved recovery of myocardial function effected by late PC in group II. When rabbits were given L-NA or AG without prior PC (group IV [n=8] and group VI [n=6], respectively), infarct size did not differ from that observed in controls (53.8+/-4.3% and 59.8+/-4.3%, respectively), demonstrating that L-NA and AG do not increase the extent of cell death in nonpreconditioned myocardium.. Taken together, these results indicate that in the conscious rabbit, the infarct-sparing effect of the late phase of ischemic PC is mediated by the activity of NOS and suggest that the specific isoform primarily responsible for this cardioprotective phenomenon is iNOS. Thus, NO appears to be a pivotal component of the pathophysiological cascade of late PC.

Topics: Analysis of Variance; Animals; Consciousness; Disease Models, Animal; Enzyme Inhibitors; Guanidines; Heart; Ischemic Preconditioning, Myocardial; Male; Myocardial Infarction; Myocardium; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Rabbits; Time Factors

The expression and function of inducible nitric oxide synthase (iNOS) in the stomach is unclear. This study assessed the effects of endotoxin on rat gastric iNOS expression and its role in gastric injury from luminal irritants. In conscious rats, a 5-h treatment with intraperitoneal lipopolysaccharide (LPS; 1-20 mg/kg) dose dependently increased gastric mucosal iNOS immunoreactivity and increased gastric luminal nitrate and nitrite accumulation (Griess reaction). LPS also increased gastric luminal fluid accumulation and reduced macroscopic gastric injury from orogastric acidified ethanol. Aminoguanidine (45 mg/kg) did not prevent LPS-induced gastroprotection or gastric fluid accumulation. NG-nitro-L-arginine methyl ester increased gastric luminal fluid and caused macroscopic gastric injury when given with LPS. Using an anesthetized preparation followed by removal of luminal fluid, LPS reduced gastric mucosal blood flow and exacerbated gastric injury from either acidified ethanol or acidified taurocholate, an effect that was negated by aminoguanidine. These data indicate that in conscious rats, the gastroprotective effect of endotoxin is dependent on constitutive NOS but not iNOS activity. However, the inducible isoform participates in the ability of endotoxin to exacerbate gastric injury from luminal irritants in the anesthetized rat.

Topics: Animals; Drug Interactions; Endotoxins; Escherichia coli; Ethanol; Female; Gastric Mucosa; Gene Expression Regulation, Enzymologic; Guanidines; Irritants; Lipopolysaccharides; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Rats; Rats, Sprague-Dawley; Regional Blood Flow

To evaluate the effect of treatment with a combination of nitric oxide synthase inhibitors and inhaled nitric oxide on systemic hypotension during sepsis.. Prospective, randomized, controlled study on anesthetized animals.. A cardiopulmonary research laboratory.. Forty-seven male adult Sprague-Dawley rats.. Animals were anesthetized, mechanically ventilated with room air, and randomized into six groups: a) the control group (C, n=6) received normal saline infusion; b) the endotoxin-treated group received 100 mg/kg i.v. of Escherichia coli lipopolysaccharide (LPS, n=9); c) the third group received LPS, and 1 hr later the animals were treated with 100 mg/kg i.v. Nw-nitro-L-arginine (LNA, n=9); d) the fourth group received LPS, and after 1 hr, the animals were treated with 100 mg/kg i.v. aminoguanidine (AG, n=9); e) the fifth group received LPS and 1 hr later was treated with LNA plus 1 ppm inhaled nitric oxide (LNA+NO, n=7); f) the sixth group received LPS and 1 hr later was treated with aminoguanidine plus inhaled NO (AG+NO, n=7). Inhaled NO was administered continuously until the end of the experiment.. Systemic mean blood pressure (MAP) was monitored through a catheter in the carotid artery. Mean exhaled NO (ENO) was measured before LPS (T0) and every 30 mins thereafter for 5 hrs. Arterial blood gases and pH were measured every 30 mins for the first 2 hrs and then every hour. No attempt was made to regulate the animal body temperature. All the rats became equally hypothermic (28.9+/-1.2 degrees C [SEM]) at the end of the experiment. In the control group, blood pressure and pH remained stable for the duration of the experiment, however, ENO increased gradually from 1.3+/-0.7 to 17.6+/-3.1 ppb after 5 hrs (p< .05). In the LPS treated rats, MAP decreased in the first 30 mins and then remained stable for 5 hrs. The decrease in MAP was associated with a gradual increase in ENO, which was significant after 180 mins (58.9+/-16.6 ppb) and reached 95.3+/-27.5 ppb after 5 hrs (p< .05). LNA and AG prevented the increase in ENO after LPS to the level in the control group. AG caused a partial reversal of systemic hypotension, which lasted for the duration of the experiment. LNA reversed systemic hypotension almost completely but only transiently for 1 hr, and caused severe metabolic acidosis in all animals. The co-administration of NO with AG had no added benefits on MAP and pH. In contrast, NO inhalation increased the duration of the reversal in MAP after LNA, alleviated the degree of acidosis, and decreased the mortality rate (from 55% to 29%).. In this animal model, LPS-induced hypotension was alleviated slightly and durably after AG, but only transiently after LNA. Furthermore, co-administration of NO with AG had no added benefits but alleviated the severity of metabolic acidosis and mortality after LNA. We conclude that nitric oxide synthase (NOS) inhibitors, given as a single large bolus in the early phase of sepsis, can exhibit some beneficial effects. Administration of inhaled NO with NOS inhibitors provided more benefits in some conditions and therefore may be a useful therapeutic combination in sepsis. NO production in sepsis does not seem to be a primary cause of systemic hypotension. Other factors are likely to have a major role.

Topics: Administration, Inhalation; Animals; Disease Models, Animal; Drug Evaluation, Preclinical; Drug Therapy, Combination; Escherichia coli Infections; Free Radical Scavengers; Guanidines; Hemodynamics; Male; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Random Allocation; Rats; Rats, Sprague-Dawley; Shock, Septic; Time Factors

Topics: Animals; Aorta, Thoracic; Arginine; Enzyme Inhibitors; Guanidines; In Vitro Techniques; Kinetics; Male; Muscle Contraction; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Nitroarginine; omega-N-Methylarginine; Ornithine; Phenylephrine; Rats; Rats, Wistar

We investigated the enzymatic source, cellular production, and functional importance of nitric oxide (NO) in rat diaphragm. Neuronal and endothelial isoforms of constituitive nitric oxide synthase (nc-NOS, ec-NOS) were identified by immunostaining. NOS activity measured in diaphragm homogenates averaged 5.1 pmol/min/mg. Passive diaphragm fiber bundles produced NO derivatives (NOx) at the rate of 0.9 pmol/min/mg as measured by the cytochrome c reduction assay; NO production was confirmed by photolysis/ chemiluminescence measurements. Endogenous NO depressed diaphragm contractile function. The force of submaximal contraction was increased by NOS inhibitors, an effect that was stable for up to 60 min and was reversed by NO donors. We conclude that diaphragm muscle fibers express nc-NOS, ec-NOS, or both; passive myocytes produce NOx; and NO or NO-derivatives inhibit force production by modulating excitation-contraction coupling.

Topics: Animals; Diaphragm; Enzyme Inhibitors; Guanidines; Hindlimb; Indazoles; Male; Muscle Contraction; Muscle, Skeletal; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; Nitrites; Nitroarginine; Rats; Rats, Sprague-Dawley

The identification of human inflammatory cells that express inducible nitric oxide synthase and the clarification of the role of inducible nitric oxide synthase in human infectious or inflammatory processes have been elusive. In neutrophil-enriched fractions from urine, we demonstrate a 43-fold increase in nitric oxide synthase activity in patients with urinary tract infections compared with that in neutrophil-enriched fractions from noninfected controls. Partially purified inducible nitric oxide synthase is primarily membrane associated, calcium independent, and inhibited by arginine analogues with a rank order consistent with that of purified human inducible nitric oxide synthase. Molecular, biochemical, and immunocytochemical evidence unequivocally identifies inducible nitric oxide synthase as the major nitric oxide synthase isoform found in neutrophils isolated from urine during urinary tract infections. Elevated inducible nitric oxide synthase activity and elevated nitric oxide synthase protein measured in patients with urinary tract infections and treated with antibiotics does not decrease until 6-10 d of antibiotic treatment. The extended elevation of neutrophil inducible nitric oxide synthase during urinary tract infections may have both antimicrobial and proinflammatory functions.

Topics: Adult; Aged; Anti-Bacterial Agents; Arginine; Bacterial Infections; Blotting, Western; Canavanine; Cell Membrane; Citrulline; Female; Guanidines; Humans; Immunohistochemistry; Isoenzymes; Leukocyte Common Antigens; Male; Middle Aged; Neutrophils; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitroarginine; omega-N-Methylarginine; Ornithine; Polymerase Chain Reaction; RNA, Messenger; Sulfonamides; Thiourea; Trifluoperazine; Urinary Tract Infections

1. In a rat model of experimental ileus, the effect of blockade of adrenergic and nitrergic neurotransmission was studied on the intestinal transit of Evans blue. 2. Ether anaesthesia and skin incision had no influence on the transit. Laparotomy significantly inhibited the transit of Evans blue. This inhibition was even more pronounced when the small intestine was manipulated. 3. Reserpine (5 mg kg-1), a drug that blocks adrenergic neurotransmission, completely reversed the inhibition of the transit induced by laparotomy but only partially reversed that induced by laparotomy with manipulation of the small intestine. 4. N omega-nitro-L-arginine (L-NOARG, 5 mg kg-1), a nitric oxide synthase inhibitor, completely reversed the reserpine-resistant inhibition induced by laparotomy with manipulation of the small intestine. The effect of L-NOARG was prevented by concomitant administration of L-arginine. L-Arginine itself slightly, but significantly enhanced the inhibition. S-methylisothiourea and aminoguanidine, selective inhibitors of the inducible NO synthase, had no effect on the transit after the three operations. 5. Treatment of the rats with reserpine plus L-NOARG had no additional effect on the transit after laparotomy as compared to reserpine alone. However, reserpine plus L-NNA completely reversed the inhibition of the transit induced by laparotomy with manipulation of the small intestine. 6. These findings support the involvement of adrenergic pathways in the pathogenesis of ileus and suggest that the additional inhibitory effect of mechanical stimulation results from an enhanced release of NO by the constitutive NO synthase.

Topics: Adrenergic Antagonists; Adrenergic Uptake Inhibitors; Anesthesia, General; Animals; Arginine; Autonomic Pathways; Enzyme Inhibitors; Gastrointestinal Transit; Guanidines; Intestinal Obstruction; Isothiuronium; Male; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Rats; Rats, Wistar; Reserpine

We investigated the effects of aminoguanidine, a relatively selective inhibitor of inducible nitric oxide (NO) synthase, on the systemic inflammatory response syndrome induced by platelet activating factor (PAF) and by lipopolysaccharide in rats, with emphasis on NO production in vivo. Aminoguanidine treatment improved survival rates after lipopolysaccharide challenge, whereas it aggravated the lethality caused by PAF. Lipopolysaccharide induced a marked increase in the concentrations of nitrate and nitrite in plasma compared with vehicle administration, and the increase was prevented by aminoguanidine. In contrast, PAF challenge with or without aminoguanidine did not affect the concentrations of nitrate and nitrite in plasma compared with vehicle administration. These results suggest that NO derived from inducible NO synthase is not a major participant in the systemic inflammatory response syndrome induced by PAF. Aminoguanidine is not likely to provide beneficial effects in conditions where PAF is produced and the concentrations of nitrate and nitrite in plasma are not significantly increased.

Topics: Animals; Disease Models, Animal; Enzyme Induction; Enzyme Inhibitors; Guanidines; Injections, Intravenous; Lipopolysaccharides; Male; Nitrates; Nitric Oxide Synthase; Nitrites; Nitroarginine; Platelet Activating Factor; Rats; Systemic Inflammatory Response Syndrome

The mechanisms of hemolysate-induced cerebral injury following subarachnoid hemorrhage are just beginning to be clarified. This study examined the injurious effects of hemolysate on endothelial cells derived from bovine middle cerebral arteries, and evaluated the roles of lipid peroxidation and nitric oxide production in this type of damage. Cultured endothelial cells were grown to confluency on gelatin-coated plates. The cells were characterized as endothelial cells on the basis of morphology. Factor VIII-related antigen staining, and low density lipoprotein (LDL) uptake. Additional cells were grown to confluency on collagen-coated well inserts, and were treated with hemolysate for 24 hours. Prior to hemolysate exposure, cells were treated with: a) an inhibitor of iron-dependent lipid peroxidation (tirilazad mesylate 100 microM), or b) an inhibitor of nitric oxide synthase (either N-nitro-L-arginine: NLA 300 microM, or aminoguanidine: AG at 1.5, 7.5, 15 or 150 microM). Permeability of the tracer, U-14C-sucrose, across the layer of endothelial cells was examined over a 24 hour period. Hemolysate induced a significant increase in the permeability across the endothelial cell layer. Pretreatment with tirilazad mesylate, NLA, or AG attenuated significantly hemolysate-induced changes in the endothelial cell barrier. These findings indicate that free radical generation and lipid peroxidation are critical participants in hemolysate-induced injury to the barrier function of the cerebrovascular endothelium. In addition, the results indicate that endothelial cells provide an adequate source of nitric oxide to damage their own cellular function. Finally, these findings strongly implicate free radical mechanisms in endothelial damage associated with subarachnoid hemorrhage.

Topics: Animals; Antioxidants; Brain; Cattle; Cell Membrane Permeability; Cells, Cultured; Endothelium, Vascular; Enzyme Inhibitors; Free Radicals; Guanidines; Lipid Peroxidation; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Pregnatrienes; Rabbits; Reactive Oxygen Species; Subarachnoid Hemorrhage

To evaluate the role of the inducible and endothelial constitutive nitric oxide synthase in vascular hyporeactivity to vasopressors in portal hypertension, in vitro experiments were performed on intact and endothelium-denuded isolated thoracic aortic rings from portal vein-ligated and sham-operated rats in control conditions, in the presence of aminoguanidine alone, considered to be a selective inhibitor of the inducible nitric oxide synthase, and of aminoguanidine and the nonselective nitric oxide synthase inhibitor N(G)-nitro-L-arginine. In control conditions, hyporeactivity to noradrenaline was observed in both rings with and without endothelium from portal hypertensive versus sham-operated rats. In the rings with endothelium, aminoguanidine reverted this hyporeactivity in portal hypertensive rats. N(G)-Nitro-L-arginine caused an additional shift to the left of the concentration-response curves to noradrenaline in portal hypertensive and a similar shift in sham-operated rats. In the endothelium-denuded rings, aminoguanidine caused no significant changes in portal hypertensive rats, whereas a significant shift to the right in the sham-operated rats was noted, however similar as the shift in the time controls not preincubated with aminoguanidine. No significant further changes were observed after preincubation with the two inhibitors. The endothelium-dependent relaxations to acetylcholine were attenuated in portal hypertensive versus sham-operated rats; addition of aminoguanidine shifted the relaxation curves to the left in portal hypertensive but not in sham-operated rats. These results provide indirect evidence for an increased activity of the inducible nitric oxide synthase in the intact aortic rings but not in the endothelium-denuded rings from portal vein-ligated rats, where other factors seem to be responsible for the observed hyporeactivity to noradrenaline. The endothelial constitutive nitric oxide synthase in rings from portal vein-ligated rats shows a reduced activity which is alleviated after inhibition of the inducible enzyme by aminoguanidine.

Topics: Acetylcholine; Animals; Aorta, Thoracic; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Induction; Enzyme Inhibitors; Guanidines; Hypertension, Portal; In Vitro Techniques; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Norepinephrine; Rats; Rats, Wistar

Nitric oxide is an important cellular mediator produced in keratinocytes and macrophages from arginine by the enzyme nitric oxide synthase during inflammatory reactions in the skin. We found that gamma-interferon stimulated nitric oxide production and the expression of inducible nitric oxide synthase in both cell types. However, macrophages produced more nitric oxide and nitric oxide synthase protein, and at earlier times than keratinocytes. Keratinocytes treated with gamma-interferon took up more arginine than macrophages; however, they were less efficient in metabolizing this amino acid and exhibited reduced nitric oxide synthase enzyme activity. In both cell types, the nitric oxide synthase inhibitors, N(G)-monomethyl-L-arginine (NMMA), L-N5-(iminoethyl)ornithine, L-canavanine, and N(omega)-nitro-L-arginine, as well as lysine, ornithine, and homoarginine markedly reduced arginine uptake. In contrast, N(omega)-nitro-L-arginine methyl ester and N(omega)-nitro-L-arginine benzyl ester were poor inhibitors of arginine uptake, while aminoguanidine had no effect on uptake of arginine by the cells. Moreover, NMMA was found to inhibit simultaneously arginine uptake and nitric oxide synthase enzyme activity in both cell types, whereas aminoguanidine only affected nitric oxide synthase activity. No major differences were observed between keratinocytes and macrophages. Taken together, these data demonstrate that, although keratinocytes and macrophages both synthesize nitric oxide, its production is regulated distinctly in these two cell types. Furthermore, in these cells, nitric oxide synthase inhibitors such as NMMA exhibit at least two sites of action: inhibition of nitric oxide synthase and cellular uptake of arginine.

Topics: Animals; Arginine; Cell Line; Citrulline; Dose-Response Relationship, Drug; Guanidines; Interferon-gamma; Keratinocytes; Macrophages; Mice; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; omega-N-Methylarginine; Tumor Cells, Cultured

Seventy-four conscious rabbits undergoing a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles for 3 consecutive days (days 1, 2, and 3) were assigned to nine groups. In group I (controls, n = 8), the recovery of systolic wall thickening (WTh) after the sixth reperfusion was markedly improved on days 2 and 3 compared with day 1, indicating late preconditioning (PC) against myocardial stunning; the total deficit of WTh after the sixth reperfusion was reduced by 56% on day 2 and 50% on day 3 compared with day 1 (P < .01). Administration on day 2 of the nonselective NO synthase (NOS) inhibitor N omega-nitro-L-arginine (L-NA) (group II, n = 8) or of the selective inducible NOS inhibitors aminoguanidine (AG) (group IV, n = 8) and S-methylisothiourea sulfate (SMT) (group VI, n = 6) completely abrogated late PC against stunning on day 2. On day 3, the expected PC effect became manifest in all groups. Administration of L-NA, AG, or SMT on day 1 (groups III [n = 7], V [n = 6], and VII [n = 5], respectively) had no discernible effect on the deficit of WTh on day 1, indicating that these agents do not augment the severity of myocardial stunning in nonpreconditioned myocardium. In group VIII (n = 7), the abrogation of late PC by SMT on day 2 was completely reversed by the concomitant administration of L-arginine (595 mg/kg IV), indicating that it was not due to nonspecific NOS-unrelated actions. Administration of L-arginine alone on day 2 (group IX [n = 5]) had no effect on the deficit of WTh. Furthermore, administration of L-NA on day 1 (group III) prevented the appearance of the PC effect on day 2, whereas AG (group V) and SMT (group VI) did not, suggesting that the development of late PC on day 1 is triggered by the endothelial (type III) isoform of NOS. This study demonstrates that three structurally different NOS inhibitors (L-NA, AG, and SMT), given 24 hours after the PC ischemia, consistently abrogate late PC against myocardial stunning in conscious rabbits, indicating that this cardioprotective effect is mediated by the activity of NOS. The results obtained with AG and SMT specifically implicate the inducible (type II) isoform as the mediator of the protection on day 2. Previous studies have shown that NO triggers the development of late PC. The present results indicate that NO plays a dual role in late PC against stunning, acting initially as the trigger and subsequently as the mediator of the protection.

Topics: Animals; Guanidines; Ischemic Preconditioning, Myocardial; Isothiuronium; Male; Myocardial Stunning; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Rabbits

Nitric oxide is synthesized from L-arginine and is metabolized to nitrate and nitrite. This study evaluates the effects of a pharmacological blockade of NO synthesis on fluid transport by the inflamed gallbladder mucosa.. Experiments were performed in cats with cholecystitis and in control animals. NO synthase activity was measured in gallbladder tissue; the enzyme was characterized by immunoblotting techniques and localized by immunofluorescence. Fluid transport and release of nitrate and nitrite by the gallbladder mucosa and bile and bile salt secretion from the liver were registered simultaneously in vivo.. Fluid secretion in inflamed gallbladders was reversed to a net absorption in response to the NO synthase blockers N omega-nitro-L-arginine and aminoguanidine, and formation of nitrate was reduced. The effects were reversed by L-arginine. Increased levels of inducible NO synthase in inflamed gallbladders were shown by immunoblotting, by immunofluorescence (mainly in macrophages), and by Ca(2+)-independent [3H]citrulline formation from [3H]arginine. The NO synthase blockers had no effect on gallbladder fluid transport in normal gallbladders.. Increased levels of inducible NO synthase activity are shown in inflamed gallbladders, and a pharmacological blockade of this enzyme blocks fluid secretion and decreases nitrate release from the mucosa.

Topics: Analysis of Variance; Animals; Arginine; Bile; Bile Acids and Salts; Body Fluids; Cats; Cholecystitis; Enzyme Inhibitors; Gallbladder; Guanidines; Immunohistochemistry; Mucous Membrane; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine

Endothelial dysfunction is known to occur in chemically-induced animal models of diabetes. The BB diabetic rat is a genetic diabetes-prone model which more closely resembles Type I diabetes mellitus. In this study, we examined the role of superoxide anion radical and cyclooxygenase activity on endothelial dysfunction in aorta of the spontaneous diabetic BB rat. Vascular endothelial function was studied in vitro in aortic rings from 8-wk diabetic rats and age-matched nondiabetic littermates. There was no alteration in reactivity to norepinephrine as a result of diabetes. Relaxation to acetylcholine (but not nitroglycerin) was impaired in diabetic rings. Relaxation to acetylcholine was abolished by 100 microM L-nitroarginine but unaltered by an equimolar concentration of aminoguanidine (an inducible nitric oxide synthase inhibitor) in both control and diabetic rings. Incubation with 10 microM indomethacin did not alter relaxation to acetylcholine in either control or diabetic rings. In contrast, addition of 20 U/ml superoxide dismutase enhanced relaxation to acetylcholine in diabetic rings but had no effect on relaxation to acetylcholine in control rings. Thus, nitric oxide-mediated, endothelium-dependent relaxation is diminished in aortic rings of the genetic diabetic BB rat. Furthermore, superoxide anion radicals but not cyclooxygenase products play an important role in endothelial dysfunction in this genetic diabetic model.

Topics: Acetylcholine; Animals; Aorta, Thoracic; Arginine; Diabetes Mellitus, Type 1; Endothelium, Vascular; Enzyme Inhibitors; Guanidines; In Vitro Techniques; Indomethacin; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitroarginine; Rats; Rats, Inbred BB; Reference Values; Vasodilation

Nitric oxide (NO), an unstable radical, is synthesized from L-arginine by the constitutive (cNOS) and inducible (iNOS) forms of NOS. cNOS is present mainly in endothelial cells and plays a role in the regulation of blood flow. iNOS, the dominant enzyme in heart muscle during myocardial infarction, allograft rejection, and cardiomyopathy, is activated in macrophages. We recently described a significant increase of iNOS activity in macrophages of infarcted rabbit myocardium 24 hours after coronary occlusion, with peak activity occurring 3 days following coronary artery ligation. Inhibitors of NOS are L-arginine derivatives that inhibit both cNOS and iNOS; S-methylisothiourea (SMT) and aminoguanidine (AMG) are specific inhibitors of iNOS. Cyclosporin A and dexamethasone inhibit by interfering with protein synthesis. iNOS inhibition by SMT, NG-nitro-L-arginine (L-NNA), AMG, cyclosporin A and dexamethasone was examined in homogenates of normal, risk and infarcted myocardium. Three days after coronary artery ligation, the heart was excised and divided into normal, risk and infarcted regions. The inhibitory effect was calculated as IC50. Results shows that SMT was the most potent inhibitor with the lowest IC50; its effect, as well as the effects of L-NNA and AMG, depended on the location in the myocardium. Inhibition for SMT and AMG was greater in the normal area than in the risk and infarcted regions. AMG induced an initial rise of iNOS followed by gradual decline in the area of risk and infarction. No inhibitory effects in cyclosporin A and dexamethasone were noted.

Topics: Animals; Arginine; Cyclosporine; Dexamethasone; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanidines; Heart; Isothiuronium; Male; Myocardial Infarction; Myocardium; Nitric Oxide Synthase; Nitroarginine; Rabbits

The effect of three nitric oxide (NO) synthase inhibitors, L-NG-nitro-arginine (NO2Arg), NG-Nitro-L-arginine methyl ester (L-NAME) and aminoguanidine, on in vitro fertilization in the mouse was examined. Mouse epididymal spermatozoa were capacitated in a medium with or without NO synthase inhibitors. Oocytes were inseminated and the percentage of oocytes with two pronuclei was scored after an 8-h incubation. NO2Arg and L-NAME, but not aminoguanidine, inhibited fertilization. L-NAME inhibited fertilization in a dose-dependent manner, and its effects were stereospecific. The inhibitory effect was neutralized by L-arginine but not by D-arginine. Moreover, D-NAME did not inhibit fertilization. The results suggest that NO synthase activity (presumably of the constitutive type is necessary for spermatozoa to display their full fertilizing ability.

Topics: Animals; Arginine; Enzyme Inhibitors; Fertilization in Vitro; Guanidines; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitroarginine; Sperm Capacitation

Nitric oxide (NO) is produced by enzymes called nitric oxide synthases (NOS). At least three different isoforms of NOS have been identified in the kidney. This study examines the effects of selective inhibition of the inducible isoform (iNOS) and the neuronal isoform (bNOS) on the glomerular capillary pressure (PGC), through studies of the tubuloglomerular feedback (TGF) mechanism in anaesthetized rats. The proximal tubular stop-flow pressure (PSF) was measured to estimate changes in PGC obtained after activation of the TGF system by varying the loop of Henle perfusion rate with artificial ultrafiltrate including vehicle, NOS inhibition or L-arginine. Infusion of nonspecific NOS inhibition (N omega-Nitro-L-arginine) increased maximal TGF responses (delta PSF) by 84% and L-arginine decreased delta PSF by 37%. Aminoguanidine, a selective iNOS-inhibitor, failed to increase delta PSF, whereas the nonspecific NOS inhibitor methylguanidine increased delta PSF by 64%. 7-Nitro indazole (7-NI), a selective bNOS inhibitor, increased delta PSF by 57% when infused intratubularly, and intraperitoneal administration of 7-NI increased delta PSF by 78%, without any change in blood pressure. Since bNOS is exclusively located in the macula densa (MD) cells, these results confirm and strengthen the obligatory role of MD-produced NO in regulation of TGF and PGC, which has been suggested earlier. iNOS, widely expressed in the kidney, does not seem to play any important role in regulation of PGC.

Topics: Animals; Arginine; Capillary Resistance; Enzyme Inhibitors; Guanidines; Indazoles; Juxtaglomerular Apparatus; Kidney Glomerulus; Kidney Tubules, Proximal; Male; Methylguanidine; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Pressure; Rats; Rats, Sprague-Dawley; Time Factors; Vasoconstriction

Recent work indicates that nitric oxide (NO) plays an important role in the systemic and renal alterations of liver cirrhosis. This study used aminoguanidine (AG), a preferential inhibitor of inducible nitric oxide synthase (iNOS), to evaluate the role of this NOS isoform in the systemic and renal alterations of an experimental model of liver cirrhosis with ascites (carbon tetrachloride/ phenobarbital). Experiments have been performed in anesthetized cirrhotic rats and their respective control rats prepared for clearance studies. Administration of AG (10 to 100 mg/kg, iv) elevated dose-dependent mean arterial pressure (MAP, in mm Hg) in the cirrhotic rats from a basal level of 79.3 +/- 3.6 to 115.0 +/- 4.7, whereas in the control animals, MAP increased only with the highest dose of the inhibitor (from 121.8 +/- 3.6 to 133.3 +/- 1.4). In the cirrhotic group, AG also significantly increased sodium and water excretion, whereas these effects were very modest in the control group. Plasma concentration of nitrates+nitrites, measured as an index of NO production, were significantly increased in the cirrhotic animals in the basal period and decreased with AG to levels not significantly different from the control animals. Similar experiments performed with the nonspecific NOS inhibitor N omega-nitro-L-arginine (NNA) also demonstrated an increased pressor sensitivity of the cirrhotic rats, but the arterial hypotension was completely corrected. These results, in an experimental model of liver cirrhosis with ascites, show that AG exerts a beneficial effect as a result of inhibition of NO production, increasing blood pressure and improving the reduced excretory function. Because NNA, but not AG, completely normalized the arterial hypotension, it is suggested that the constitutive NOS isoform is also contributing in an important degree. It is concluded that the activation of both inducible and constitutive NOS isoforms plays an important role in the lower systemic blood pressure and associated abnormalities that characterize liver cirrhosis.

Topics: Animals; Ascites; Blood Pressure; Enzyme Inhibitors; Guanidines; Kidney; Liver Cirrhosis, Experimental; Male; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Rats; Rats, Sprague-Dawley

We have studied the effects of NG-nitro-L-arginine (NNA) a nitric oxide synthase (NOS) inhibitor, and aminoguanidine (AG) a diamine oxidase inhibitor, on hemodynamic parameters and plasma histamine level using a dog model in which a hyperdynamic circulatory response was elicited with a 2-hour infusion of a low dose (13.75 micrograms/kg) of E. coli 055:B5 endotoxin (ETX). AG (50 mg/kg) or NNA (0.5 mg/kg) was administered intravenously as pretreatment. Hemodynamic variables were studied for 4 hours after the beginning of the ETX infusion. The ETX-elicited hyperdynamic response was abolished by NNA and partially inhibited by AG. AG prevented the increases in cardiac output and heart rate and delayed the early decrease in total peripheral resistance (TPR). The plasma histamine concentration elevation was higher in animals receiving AG than in those receiving only ETX. In the group treated with ETX plus NNA the cardiac output was lower and the TPR was higher than in the ETX plus AG group. In future studies, AG should be considered as one of the possible therapeutic tools in sepsis, as its adverse effect on the compensatory hyperdynamic response is less than that of NOS inhibitors of the L-arginine analog type, while it may favourably influence the deleterious excessive activity of the inducible NOS in the later stages.

Topics: Animals; Blood Pressure; Cardiac Output; Dogs; Endotoxemia; Endotoxins; Escherichia coli; Guanidines; Heart Rate; Hemodynamics; Histamine; Histamine Release; Nitroarginine; Stroke Volume; Time Factors; Vascular Resistance

We evaluated the effect of in vivo and in vitro administration of nitro-containing and nitro-deficient L-arginine-derived nitric oxide (NO) synthase inhibitors on the measurement of NO in plasma, urine and HEPES buffered physiologic salt solution (PSS) by ozone chemiluminescence and by the modified Griess reaction. In vivo administration of 1, 5, 25, 40 or 50 mg/kg of NG-nitro-L-or D-arginine methyl ester (LNAME, DNAME), NG-nitro-L-arginine (LNA) or aminoguanidine (AG) to rats and mice increased NO in urine and plasma as determined by chemiluminescence using 2.3% vanadium chloride in 2N HCI at 100 degrees C as the redox reagent. In vivo administration of 1 and 10 mg/kg/day of NG-imino-ethyl-L-ornithine (LNIO) or 3 amino-1,2,4 triazine (AT) reduced plasma and urine NO. Addition of LNAME, DNAME and LNA (100 nM to 1 mM) to the redox solution produced a concentration response curve for NO in the chemiluminescence assay similar to that produced by standard solutions of sodium nitrite and nitrate. LNMMA produced a small NO signal but only at concentrations equal to or exceeding 0.1 mM. LNIO, AT and AG did not give any NO signal even at concentrations exceeding 1 mM. Conversion of plasma or urine nitrate to nitrite with cadmium gave elevated values of plasma nitrite by the Greiss assay when LNAME or LNA was the NO synthase inhibitor. We conclude that in vivo and in vitro use of LNAME and LNA and in vivo use of high doses of aminoguanidine interfere with the assay of NO2- and NO3- with the modified Griess reaction and with chemiluminescence. We suggest that LNAME and LNA not be used in vivo or in vitro when total RNI is measured with these assays.

Topics: Animals; Arginine; Enzyme Inhibitors; Evaluation Studies as Topic; Guanidines; Luminescent Measurements; Macrophages, Alveolar; Male; Mice; Mice, Inbred BALB C; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide Synthase; Nitrites; Nitroarginine; Ornithine; Rats; Rats, Sprague-Dawley; Triazines

Complementary DNAs encoding three human isoforms (neuronal, inducible, and endothelial) of nitric oxide synthase were cloned into the baculovirus expression vector pVL1392/1393. Transfection of Sf-9 insect cells with the recombinant baculovirus resulted in the expression of high levels of nitric oxide synthases. The expressed proteins of neuronal and inducible nitric oxide synthase were predominantly soluble, whereas the endothelial enzyme was for the most part, particulate. Recombinant enzymes were purified with 2',5'-ADP Sepharose affinity chromatography. The effects of reference enzymatic inhibitors (NG-methyl-L-arginine, NG-nitro-L-arginine and N-iminoethyl-L-ornithine) on recombinant expressed proteins were not significantly different from native nitric oxide synthase enzyme preparations. L-aminoguanidine was found to be much less potent in inhibiting recombinant or native human inducible nitric oxide synthase compared to the murine isoform. These findings indicate previously unappreciated interspecies differences in the action of nitric oxide synthase enzymatic inhibitors. The functional expression of human nitric oxide synthase isoforms in a heterologous expression system allowed screening of novel inhibitors. Studies indicated that S-ethylisothiourea and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine were potent novel inhibitors of human nitric oxide synthases.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Baculoviridae; Blotting, Western; Cell Line; Chromatography, Affinity; DNA, Complementary; Endothelium, Vascular; Enzyme Induction; Gene Expression; Guanidines; Humans; Isoenzymes; Kinetics; Mice; Neurons; Nitric Oxide Synthase; Nitroarginine; omega-N-Methylarginine; Ornithine; Recombinant Proteins; Spodoptera; Transfection

Enhanced production of nitric oxide has been implicated in cardiac and vascular dysfunction associated with septic and endotoxic shock. To test this hypothesis, conscious rats were administered endotoxin. 6 h later, the rats were anesthetized, arterial pressure was measured, and hearts were removed for Langendorff perfusion in the absence and presence of .01 microM isoproterenol. Left ventricular developed pressure was 61 +/- 6 mmHg in control rats 39 +/- 5 mmHg in endotoxin-treated rats. Inotropic responses to isoproterenol were unaffected by endotoxin treatment. Administration of nitric oxide synthase (NOS) inhibitors (NG-nitro-L-arginine and aminoguanidine) prior to endotoxin did not improve left ventricular function in endotoxin-treated rats. Dexamethasone pretreatment, however, prevented endotoxin-induced cardiac depression. These results suggest that cardiac depression during endotoxemia is not caused by NOS activation and increased nitric oxide production. Furthermore, the cardioprotectant actions of dexamethasone are not related to its ability to inhibit inducible NOS expression.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Cardiac Output, Low; Dexamethasone; Guanidines; Heart Rate; Hemodynamics; Hypotension; Isoproterenol; Male; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Rats; Rats, Sprague-Dawley; Shock, Septic

The nitric oxide (NO) synthase pathway is activated during experimental autoimmune inflammation of the central nervous system, and administration of aminoguanidine, an inhibitor of the cytokine-inducible NO synthase (NOS), ameliorated the disease course of autoimmune encephalomyelitis in the SJL mouse. We studied the role of nitric oxide synthase (NOS) in the pathogenesis of experimental autoimmune neuritis (EAN) and experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. NG-L-monomethyl-arginine (L-NMMA), a competitive inhibitor of NOS, partially suppressed T cell line-mediated EAN, but not myelin-induced EAN, myelin basic protein (MBP)-induced EAE, or T cell line-mediated EAE. Aminoguanidine (AG), a selective inhibitor of the cytokine-inducible NOS, enhanced MBP-induced EAE, but had no significant effects on myelin-induced EAN. Two other NOS inhibitors, nitro-arginine methyl-ester and N-nitro arginine, had only little or no effects in EAN and EAE. The administration of NOS inhibitors showed some striking effects in EAN and EAE, but the observed diversity of actions points to a much more complex role of the NO pathway than previously suggested.

Topics: Acetates; Amino Acid Oxidoreductases; Animals; Arginine; Encephalomyelitis, Autoimmune, Experimental; Female; Guanidines; Mice; Mice, Inbred Strains; Myelin Basic Protein; Neuritis, Autoimmune, Experimental; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitroarginine; omega-N-Methylarginine; Rats; Rats, Inbred Lew

Elevation of the ocular pressure in the anterior chamber of the rat eye caused major ischemic damage, manifested as changes in retinal morphology. The two most affected structures were the inner plexiform layer, which decreased in thickness by 90%, and the number of ganglion cells, which decreased by 80%. Pretreatment of the animals with N omega-nitro-L-arginine, a nitric oxide (NOS) inhibitor, almost completely abolished the ischemic damage. Administration of aminoguanidine, a NOS inhibitor selective for the inducible enzyme, partially abolished the ischemic damage. Moreover, administration of the NOS inhibitors 1 h after ischemia, also protected the retina from damage, suggesting that similarly acting drugs could be used clinically to limit ischemic injury in humans. We conclude that NOS, and therefore NO, may be involved in the mechanism of ischemic injury to the retina.

Topics: Animals; Arginine; Cell Count; Enzyme Inhibitors; Guanidines; Intraocular Pressure; Ischemia; Male; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Rats; Rats, Sprague-Dawley; Retinal Diseases; Retinal Ganglion Cells; Retinal Vessels

A new selective inhibitor of the inducible nitric oxide synthase in the treatment of pathogenesis characterized by overproduction of nitric oxide may be useful. Therefore, we have examined the effects of two L-arginine analogues, NG-methyl-L-arginine (L-NMA) and NG-nitro-L-arginine (L-NNA), and aminoguanidine (AG) on the constitutive and inducible nitric oxide synthase in rat aorta. L-NNA induced greater contractions to phenylephrine than L-NMA whereas AG had no effect on dose-response curves to this alpha 1-agonist in rat aorta with endothelium. Relaxations to acetylcholine, adenosine triphosphate, and A 23187 were fully abolished by L-NNA, while L-NMA partially inhibited and AG did not affect the relaxations to these three vasodilators. L-NNA, L-NMA, and AG were equipotent in inhibiting the vascular hyporeactivity to phenylephrine induced by endotoxin in rat aortic rings with endothelium; however, the rate of onset of the maximum inhibitory effects of AG was slower than that obtained with L-NNA and L-NMA. L-arginine completely abolished the effects of AG, but only partially reversed the effects of L-NNA and L-NMA in LPS-treated rings. These results suggest that AG selectively inhibits inducible nitric oxide synthase, whereas L-NNA and L-NMA exert their effects on both the constitutive and inducible nitric oxide synthase.

Topics: Amino Acid Oxidoreductases; Animals; Aorta; Arginine; Enzyme Induction; Guanidines; Lipopolysaccharides; Nitric Oxide Synthase; Nitroarginine; omega-N-Methylarginine; Rats; Structure-Activity Relationship

The roles of hemoglobin (Hb) in the pathogenesis of cerebral vasospasm remain a matter of discussion. Hemoglobin is known to be released from extravasated red blood cells in a variety of pathological conditions, including subarachnoid hemorrhage. These conditions are often accompanied by infiltration of inflammatory cells and an associated release of multiple cytokines. Certain of these cytokines, including interleukin-1 beta (IL-1 beta), are capable of increasing nitric oxide (NO) production via the inducible form of nitric oxide synthase (NOS), and excessive NO production under these conditions may contribute to cellular dysfunction. This study further examines these questions by investigating the effects of Hb on the induction of NOS by IL-1 beta. The effects of Hb on IL-1 beta-induced NO production were examined in cultured smooth-muscle cells of rat aorta (RA-SMC's). Production of NO was estimated from the accumulation of nitrite, an oxidative product of NO, in the culture medium. The synthesis of NO was induced by IL-1 beta in a concentration-dependent manner. This activation of NO production was inhibited by: 1) a general inhibitor of NOS (N omega-nitro-L-arginine); 2) a protein synthesis inhibitor (cycloheximide); and 3) two selective inhibitors of the inducible form of NOS (hydrocortisone and aminoguanidine). These results suggest that IL-1 beta promotes the expression of the inducible form of NOS in RA-SMC's. The effects of Hb on NO production were tested by adding purified human Hb to the culture medium of the cells in both the presence and absence of IL-1 beta. Nitrite accumulation was slightly but significantly increased by Hb in the absence of IL-1 beta. In contrast, Hb markedly augmented nitrite accumulation induced by IL-1 beta. This augmentation persisted even after the removal of Hb from the culture medium. The number of cells was not affected by Hb or IL-1 beta. The findings demonstrate that Hb can modify cytokine-induced production of NO in RA-SMC's by increasing the inducible form of NOS. These observations suggest that Hb can also modify the action of inflammatory cells by facilitating NO production in target cells.

Topics: Animals; Aorta, Thoracic; Arginine; Cell Hypoxia; Cells, Cultured; Cycloheximide; Guanidines; Hemoglobins; Humans; Hydrocortisone; Interleukin-1; Iron; Muscle, Smooth, Vascular; Nitric Oxide; Nitrites; Nitroarginine; Rats