cyclic-gmp and pimagedine

Considering the pivotal role of nitric oxide (NO) pathway in depressive disorders, the aim of the present study was to investigate the antidepressant-like effect of selegiline in mice forced swimming test (FST), and possible involvement of NO-cyclic guanosine monophosphate (cGMP) pathway in this action.. After assessment of locomotor activity in open-field test, mice were forced to swim individually and the immobility time of the last 4min was evaluated. All drugs were given intraperitoneally (ip).. Selegiline (10mg/kg) decreased the immobility time in the FST similar to fluoxetine (20mg/kg). Pretreatment with l-arginine (NO precursor, 750mg/kg) or sildenafil (a phosphodiesterase 5 inhibitor, 5mg/kg) significantly reversed the selegiline anti-immobility effect. Sub-effective dose of selegiline (1mg/kg) showed a synergistic antidepressant effect with NG-nitro-l-arginine methyl ester (L-NAME, inhibitor of NO synthase, 10mg/kg) or 7-nitroindazole (specific neuronal NO synthase inhibitor, 30mg/kg), but not with aminoguanidine (specific inducible NO synthase inhibitor, 50mg/kg). Pretreatment of mice with methylene blue (an inhibitor of NO synthase and soluble guanylyl cyclase, 10mg/kg) significantly produced a synergistic response with the sub-effective dose of selegiline. Neither of the drugs changed the locomotor activity. Also, hippocampal and prefrontal cortex (PFC) nitrite content was significantly lower in selegiline-injected mice compared to saline-administrated mice. Also, co-injection of 7-nitroindazole with selegiline produced a significant reduction in hippocampal or PFC nitrite contents.. It is concluded that selegiline possesses antidepressant-like effect in mice FST through inhibition of l-arginine-NO-cyclic guanosine monophosphate pathway.

Topics: Animals; Antidepressive Agents; Arginine; Cyclic GMP; Drug Synergism; Fluoxetine; Guanidines; Hippocampus; Immobility Response, Tonic; Indazoles; Male; Methylene Blue; Mice; Motor Activity; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitrites; Prefrontal Cortex; Selegiline; Signal Transduction; Sildenafil Citrate; Swimming

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

Inflammation, oxidative and nitrosative stress underlie depression being assessed in rodents by the systemic administration of lipopolysacharide (LPS). There is an increasing body of evidence of an involvement of nitric oxide (NO) pathway in depression, but this issue was not investigated in LPS-induced model. Thus, herein we evaluated the effects of NO-pathway-modulating drugs, named aminoguanidine, l-NAME, sildenafil and l-arginine, on the behavioral (forced swimming test [FST], sucrose preference [SPT] and prepulse inhibition [PPI] of the startle) and neurochemical (glutathione [GSH], lipid peroxidation, IL-1β) alterations in the prefrontal cortex, hippocampus and striatum as well as in BDNF levels in the hippocampus 24h after LPS (0.5mg/kg, i.p.) administration, a time-point related to depressive-like behavior. Twenty-four hours post LPS there was an increase in immobility time in the FST, decrease in sucrose preference and PPI levels accompanied by a decrease in GSH levels and an increase in lipid peroxidation, IL-1β and hippocampal BDNF levels suggestive of a depressive-like state. The pretreatment with the NOS inhibitors, l-NAME and aminoguanidine as well as sildenafil prevented the behavioral and neurochemical alterations induced by LPS, although sildenafil and l-NAME were not able to prevent the increase in hippocampal BDNF levels induced by LPS. The iNOS inhibitor, aminoguanidine, and imipramine prevented all behavioral and neurochemical alterations induced by LPS. l-arginine did not prevent the alterations in immobility time, sucrose preference and GSH induced by LPS. Taken together our results show that the NO-cGMP pathway is important in the modulation of the depressive-like alterations induced by LPS.

Topics: Animals; Antidepressive Agents; Arginine; Behavior, Animal; Brain; Brain-Derived Neurotrophic Factor; Cyclic GMP; Depressive Disorder; Disease Models, Animal; Enzyme Inhibitors; Guanidines; Imipramine; Interleukin-1beta; Lipopolysaccharides; Male; Mice; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Oxidative Stress; Piperazines; Purines; Signal Transduction; Sildenafil Citrate; Sulfones

The present study was designed to investigate the involvement of the nitric oxide (NO)/cyclic guanylate monophosphate pathway in pilocarpine-induced seizures in mice. Male Swiss mice (26-32 g) were used as the in vivo model. The following pharmacological tools were utilized: the non-selective NO synthase (NOS) inhibitor L-NAME (10 mg/kg, i.p.), a preferential inducible NOS (iNOS) inhibitor aminoguanidine (25 mg/kg, i.p.), a highly specific iNOS inhibitor 1400W (2.5 mg/kg, i.p.), the NO donor L-arginine (150 mg/kg, i.p.), and the soluble guanylyl cyclase inhibitor ODQ (10 mg/kg, i.p.). The animals were divided into groups (n = 8) and pretreated for 30 min before receiving pilocarpine (400 mg/kg, i.p.), while the control group received only pilocarpine. They were observed for 60 min to measure initial seizure latency, latency till death, and mortality. An administration of L-NAME or ODQ delayed the onset of initial seizure, increased latency till death, and produced a 25% survival rate. Aminoguanidine increased the initial seizure and latency until death, and administration of 1400W did not have an effect. Incremental increases of NO by L-arginine were capable of decreasing the seizure and death latency. These results support the idea that the constitutive NOS, probably neuronal NOS, followed by soluble guanylyl cyclase activation is involved in the convulsive responses caused by pilocarpine administration.

Topics: Amidines; Animals; Arginine; Benzylamines; Cyclic GMP; Disease Models, Animal; Enzyme Inhibitors; Guanidines; Guanylate Cyclase; Male; Mice; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Oxadiazoles; Pilocarpine; Receptors, Cytoplasmic and Nuclear; Seizures; Soluble Guanylyl Cyclase

The barnacle Balanus amphitrite is among the most dominant fouling species on intertidal rocky shores in tropical and subtropical areas and is thus a target organism in antifouling research. After being released from adults, the swimming nauplius undertakes six molting cycles and then transforms into a cyprid. Using paired antennules, a competent cyprid actively explores and selects a suitable substratum for attachment and metamorphosis (collectively known as settlement). This selection process involves the reception of exogenous signals and subsequent endogenous signal transduction. To investigate the involvement of nitric oxide (NO) and cyclic GMP (cGMP) during larval settlement of B. amphitrite, we examined the effects of an NO donor and an NO scavenger, two nitric oxide synthase (NOS) inhibitors and a soluble guanylyl cyclase (sGC) inhibitor on settling cyprids. We found that the NO donor sodium nitroprusside (SNP) inhibited larval settlement in a dose-dependent manner. In contrast, both the NO scavenger carboxy-PTIO and the NOS inhibitors aminoguanidine hemisulfate (AGH) and S-methylisothiourea sulfate (SMIS) significantly accelerated larval settlement. Suppression of the downstream guanylyl cyclase (GC) activity using a GC-selective inhibitor ODQ could also significantly accelerate larval settlement. Interestingly, the settlement inhibition effects of SNP could be attenuated by ODQ at all concentrations tested. In the developmental expression profiling of NOS and sGC, the lowest expression of both genes was detected in the cyprid stage, a crucial stage for the larval decision to attach and metamorphose. In summary, we concluded that NO regulates larval settlement via mediating downstream cGMP signaling.

Topics: Animals; Benzoates; Cyclic GMP; Guanidines; Guanylate Cyclase; Imidazoles; Isothiuronium; Larva; Metamorphosis, Biological; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Oxadiazoles; Quinoxalines; Signal Transduction; Thoracica

Sepsis induces production of inflammatory mediators such as nitric oxide (NO) and causes physiological alterations, including changes in body temperature (Tb). We evaluated the involvement of the central NO-cGMP pathway in thermoregulation during sepsis induced by cecal ligation and puncture (CLP), and analyzed its effect on survival rate. Male Wistar rats with a Tb probe inserted in their abdomen were intracerebroventricularly injected with 1 microL NG-nitro-L-arginine methyl ester (L-NAME, 250 microg), a nonselective NO synthase (NOS) inhibitor; or aminoguanidine (250 microg), an inducible NOS inhibitor; or 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 0.25 microg), a guanylate cyclase inhibitor. Thirty minutes after injection, sepsis was induced by cecal ligation and puncture (CLP), or the rats were sham operated. The animals were divided into 2 groups for determination of Tb for 24 h and assessment of survival during 3 days. The drop in Tb seen in the CLP group was attenuated by pretreatment with the NOS inhibitors (p < 0.05) and blocked with ODQ. CLP rats pretreated with either of the inhibitors showed higher survival rates than vehicle injected groups (p < 0.05), and were even higher in the ODQ pretreated group. Our results showed that the effect of NOS inhibition on the hypothermic response to CLP is consistent with the role of nitrergic pathways in thermoregulation.

Topics: Animals; Body Temperature; Body Temperature Regulation; Cecum; Cyclic GMP; Guanidines; Injections, Intraperitoneal; Ligation; Male; Nitric Oxide; Nitric Oxide Synthase; Punctures; Random Allocation; Rats; Rats, Wistar; Sepsis; Survival Rate

Endothelial dysfunction is a feature of hypertension and diabetes. Methylglyoxal (MG) is a reactive dicarbonyl metabolite of glucose and its levels are elevated in spontaneously hypertensive rats and in diabetic patients. We investigated if MG induces endothelial dysfunction and whether MG scavengers can prevent endothelial dysfunction induced by MG and high glucose concentrations.. Endothelium-dependent relaxation was studied in aortic rings from Sprague-Dawley rats. We also used cultured rat aortic and human umbilical vein endothelial cells. The MG was measured by HPLC and Western blotting and assay kits were used.. Incubation of aortic rings with MG (30 µM) or high glucose (25 mM) attenuated endothelium-dependent, acetylcholine-induced relaxation, which was restored by two different MG scavengers, aminoguanidine (100 µM) and N-acetyl cysteine (NAC) (600 µM). Treatment of cultured endothelial cells with MG or high glucose increased cellular MG levels, effects prevented by aminoguanidine and NAC. In cultured endothelial cells, MG and high glucose reduced basal and bradykinin-stimulated nitric oxide (NO) production, cGMP levels, and serine-1177 phosphorylation and activity of endothelial NO synthase (eNOS), without affecting threonine-495 and Akt phosphorylation or total eNOS protein. These effects of MG and high glucose were attenuated by aminoguanidine or NAC.. Our results show for the first time that MG reduced serine-1177 phosphorylation, activity of eNOS and NO production. MG caused endothelial dysfunction similar to that induced by high glucose. Specific and safe MG scavengers have potential to prevent endothelial dysfunction induced by MG and high glucose concentrations.

Topics: Acetylcholine; Acetylcysteine; Animals; Aorta; Cells, Cultured; Cyclic GMP; Endothelial Cells; Glucose; Guanidines; Humans; Male; Nitric Oxide; Nitric Oxide Synthase; Phosphorylation; Pyruvaldehyde; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Vascular Diseases; Vasodilation

Common bile duct ligation (CBDL) in the guinea pig is a well-defined model of acalculous cholecystitis. Nitric oxide (NO) mediates smooth muscle relaxation by stimulating the activity of soluble guanylate cyclase. The aim of this study was to determine whether the NO/cyclic guanosine monophosphate pathway plays a role in gallbladder relaxant response after CBDL.. Relaxant response of gallbladder muscle strips from CBDL and sham-operated guinea pigs was studied in vitro. Animals were treated with saline, aminoguanidine or an aminoguanidine + L-arginine combination in vivo. Concentration-response curves of papaverine, diethylamine/NO, YC-1, sildenafil and amrinone were obtained and relaxations in each group were calculated as the percent of the contractions induced by carbachol (10(-6) M).. There was a significant decrease in the gallbladder muscle relaxant responses to these substances in CBDL and aminoguanidine groups compared with sham surgical controls. The decreased relaxant response was reversed by aminoguanidine + L-arginine but not by aminoguanidine alone.. Decreased relaxant responses might be due to the reduced guanylate cyclase enzyme activity, but further studies are required.

Topics: Amrinone; Animals; Arginine; Bile Ducts; Cyclic GMP; Gallbladder; Guanidines; Guinea Pigs; Hydrazines; In Vitro Techniques; Indazoles; Ligation; Male; Muscle Relaxation; Nitric Oxide; Nitric Oxide Donors; Papaverine; Piperazines; Purines; Sildenafil Citrate; Sulfones

In the present study, effect of aminoguanidine (12.5, 25 and 50mg/kg, i.p.), a selective inhibitor of inducible nitric oxide synthase, was evaluated for its anti-anxiety activity in stressed mice employing elevated plus maze, open field test, light/dark test and social interaction test. Restraint stress induced by immobilizing for 6h enhanced an anxiety-like behavior and increased plasma nitrite levels in mice. Only the highest dose (50mg/kg) employed of aminoguanidine attenuated the stress-induced anxiety-like behavior and decreased plasma nitrite levels. There was no significant anxiolytic effect of aminoguanidine in unstressed mice. Sildenafil (1mg/kg i.p.), was used to explore the probable mechanism of anti-anxiety activity of aminoguanidine through NO-cGMP signaling. Aminoguanidine (50mg/kg) attenuated the anxiogenic effect of sildenafil. Aminoguanidine and sildenafil per se and in combination did not affect the locomotor activity of stressed and unstressed mice as compared to their respective control groups. Thus, aminoguanidine produced anti-anxiety activity in stressed mice through iNOS-NO-cGMP pathway.

Topics: Adaptation, Ocular; Analysis of Variance; Animals; Anti-Asthmatic Agents; Cyclic GMP; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Exploratory Behavior; Female; Guanidines; Interpersonal Relations; Male; Maze Learning; Mice; Nitric Oxide; Nitrites; Piperazines; Purines; Restraint, Physical; Signal Transduction; Sildenafil Citrate; Stress, Psychological; Sulfones

The central complex of acridid grasshoppers integrates sensory information pertinent to reproduction-related acoustic communication. Activation of nitric oxide (NO)/cyclic GMP-signaling by injection of NO donors into the central complex of restrained Chorthippus biguttulus females suppresses muscarine-stimulated sound production. In contrast, sound production is released by aminoguanidine (AG)-mediated inhibition of nitric oxide synthase (NOS) in the central body, suggesting a basal release of NO that suppresses singing in this situation. Using anti-citrulline immunocytochemistry to detect recent NO production, subtypes of columnar neurons with somata located in the pars intercerebralis and tangential neurons with somata in the ventro-median protocerebrum were distinctly labeled. Their arborizations in the central body upper division overlap with expression patterns for NOS and with the site of injection where NO donors suppress sound production. Systemic application of AG increases the responsiveness of unrestrained females to male calling songs. Identical treatment with the NOS inhibitor that increased male song-stimulated sound production in females induced a marked reduction of citrulline accumulation in central complex columnar and tangential neurons. We conclude that behavioral situations that are unfavorable for sound production (like being restrained) activate NOS-expressing central body neurons to release NO and elevate the behavioral threshold for sound production in female grasshoppers.

Topics: Acoustic Stimulation; Animal Communication; Animals; Auditory Threshold; Behavior, Animal; Brain; Citrulline; Cyclic GMP; Enzyme Inhibitors; Female; Ganglia, Invertebrate; Grasshoppers; Guanidines; Immunohistochemistry; Nitrergic Neurons; Nitric Oxide; Nitric Oxide Synthase; Sexual Behavior, Animal

The objective of this study was to examine the effects of manipulating the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway on bovine oocyte nuclear maturation in vitro. Cumulus-enclosed oocytes (CEO) were recovered from abattoir-derived ovaries and cultured in M199+FCS for 7 or 21h in the presence of various molecules affecting the NO/cGMP pathway, and then fixed and stained for evaluation of the stage of nuclear maturation. Cyclic GMP levels were also measured in cumulus-oocyte complexes after 3 and 6 h of culture. The iNOS inhibitor, aminoguanidine (AG, 10 and 50 mM) and the NO donor sodium nitroprusside (SNP, 100 and 500 microM) significantly inhibited GVBD after 7h of culture. However, a lower concentration of SNP (0.01 microM) stimulated GVBD. The inhibitory effects of AG and SNP were reversible, indicating that they were not toxic effects. Although SNP (500 microM) increased cGMP levels in cumulus-oocyte complexes after 3 h of culture, the inhibitor of soluble guanylate cyclase ODQ and the protein kinase G (PKG) inhibitor KT5823 did not reverse the inhibitory effect of SNP on meiosis, suggesting that SNP does not inhibit meiosis through the cGMP/PKG pathway. Similarly, an analogue of cGMP (8-Bromo-cGMP 0.5, 1, 3, and 6 mM), as well as activation of guanylate cyclase with Protoporphyrin IX or atrial natriuretic peptide, or inhibition of the enzyme with ODQ, did not have any significant effect on GVBD after 7 h of culture, supporting the idea that the effects of AG and SNP were not due to altered cGMP levels. Atrial natriuretic peptide, Protoporphyrin IX and SNP 500 microM increased cGMP levels after 3 h but not 6 h of culture. In conclusion, soluble and particulate guanylate cyclases could be activated in bovine cumulus-oocyte complexes, but accumulation of cGMP was probably not responsible for the effects of NO on meiosis.

Topics: Animals; Cattle; Cells, Cultured; Cyclic GMP; Female; Guanidines; Meiosis; Models, Biological; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; Oocytes; Signal Transduction

Activating the nitric oxide (NO)-cyclic guanosine 3',5'-monophosphate (cGMP) pathway improves haemodynamics following acute pulmonary thromboembolism (APT). However, the role of NO synthase (NOS) isoforms in the responses to APT has not been determined. We examined the effects of selective and non-selective inducible NOS (iNOS) inhibition.. Haemodynamic evaluations were performed in non-embolized dogs treated with saline (control group; n = 4), L-NAME (NAME group; n = 3), or aminoguanidine (AG group; n = 3), and in dogs that received the same drugs and were embolized with 5 mL kg(-1) of clots made with autologous blood (Emb group, n = 9; NAME + Emb group, n = 4 and AG + Emb group, n = 7). The lung concentrations of nitrite/nitrate (NOx) and cGMP were determined by chemiluminescence and ELISA respectively.. Acute pulmonary thromboembolism increased mean pulmonary arterial pressure (MPAP) and pulmonary vascular resistance index (PVRI) by 21.4 +/- 1.7 mmHg and by 843 +/- 34 dyn s cm(-5) m(-2), respectively, in Emb group. MPAP and PVRI increased to higher levels in the NAME + Emb group 15 min after APT and all dogs in this group died 15-30 min after APT. Conversely, lower MPAP and PVRI levels were found in the AG + Emb group 2 h after APT compared with the Emb group (both P < 0.05). Higher NOx concentrations were found in the Emb group compared with the other groups (all P < 0.05). Higher cGMP concentrations were found in the Emb and AG + Emb groups compared with the other groups (all P < 0.05).. These results indicate that endogenous NO protects against APT-induced cardiovascular responses. Moreover, iNOS-derived NO possibly produces unfavourable effects, which are counteracted by aminoguanidine. However, non-NO-related mechanisms may also be involved.

Topics: Acute Disease; Animals; Blood Pressure; Cyclic GMP; Dogs; Female; Guanidines; Lung; Male; Models, Animal; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide; Nitric Oxide Synthase Type II; Pulmonary Embolism; Vascular Resistance

Inducible NO-synthase inhibitor aminoguanidine (AG) was used for investigation into enhanced nitric oxide (NO) production influence on elevated pressure in the pulmonary circulation (pulmonary hypertension, PH) under endothelial dysfunction. PH was simulated by subcutaneous injection of 60 mg/kg MCT to Wistar rats. Experimental groups were given AG in drinking water (15 mg/(kg x day)), and control groups were given drinking water. Rate of nitrite/nitrate excretion (RENOx) with urine was measured. The RENOx was elevated since second week as long as through the PH development. Chronic AG administration led to RENOx and soluble guanylate cyclase (sGC) NO-dependent activity restoration, and also it led to partial restoration of the right ventricular pressure. AG administration restored the perfusion pressure responses of isolated pulmonary arteries to acetylcholine. These results suggest that chronic inducible NO-synthase inhibition restores the impaired endothelium-dependent and sGC-dependent relaxation of pulmonary artery in MC-induced PH.

Topics: Animals; Cyclic GMP; Disease Models, Animal; Endothelium, Vascular; Enzyme Inhibitors; Guanidines; Hypertension, Pulmonary; Lung; Monocrotaline; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Pulmonary Circulation; Rats; Rats, Wistar; Vasodilation

Based on the role of inducible nitric oxide synthase (iNOS) in heart failure, we hypothesized that the elevated expression of iNOS compared to young mice in the myocardium contributes to the age-related decline of left ventricular (LV) function. Cardiac iNOS mRNA and protein expression was singularly identified in old, wild type (WT) male mice (I6-month) and not in young WT male mice (6-month). Characterized with in vivo pressure-volume loops analysis, an age-related LV dysfunction was found in the old WT mice. The LV dysfunction of the aged mice was modified to that of the younger mice by the specific iNOS inhibitors, aminoguanidine (AMG, 10 mg/Kg, i.v. or infusion, n = 15) and S-methyl-isothiourea (MITU, 3 mg/Kg, i.v. n = 7), and declined with L-arginine (10 mg/Kg, i.v. n = 7). All three drugs had no effects on the LV function of young WT mice or old iNOS knockout (KO) mice. The NOx and cGMP levels were significantly higher only in the old WT mice (n = 6) and cGMP levels decreased to normal with AMG administration. In conclusion, these results suggested that the iNOS/NO/cGMP pathway may contribute to ventricular dysfunction during the aging process and that inhibition of iNOS activity significantly improved heart function in aged mice.

Topics: Aging; Animals; Arginine; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fluorescent Antibody Technique, Indirect; Guanidines; Isothiuronium; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; RNA, Messenger; Ventricular Dysfunction, Left

To identify the novel mechanism by which nitric oxide (NO) suppresses flavin-containing monooxygenase (FMO) activity in endotoxemic rat livers, NO-overproducing conditions were induced in primary cultured rat hepatocytes by treatment with a mixture (LCM) of lipopolysaccharide and proinflammatory cytokines (IL-1beta, TNF-alpha, and IFN-gamma), or by the addition of a pure NO donor, spermine-NONOate. mRNA levels of the major hepatic form, FMO1, decreased via a cGMP-independent destabilizing effect of NO rather than by decreased transcription. The decrease in the mRNA levels caused by LCM-induced inducible NO synthase (iNOS) was completely blocked by co-treatment with aminoguanidine, a selective iNOS inhibitor. Furthermore, spermine-NONOate, but not the cGMP analog, 8-bromo-cGMP, dose- and time-dependently attenuated FMO1 mRNA stability in actinomycin-D-pretreated cells, resulting in decreases in protein levels and biochemical activity. These results suggest that NO acts directly in a cGMP-independent mechanism by decreasing the half-life of FMO1 mRNA, thereby inducing impairment of FMO-related functions in endotoxemia.

Topics: Animals; Cells, Cultured; Cyclic GMP; Cytokines; Dactinomycin; Guanidines; Hepatocytes; Lipopolysaccharides; Liver; Male; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrogen Oxides; Oxygenases; Protein Modification, Translational; Protein Synthesis Inhibitors; Rats; Rats, Sprague-Dawley; RNA Stability; Spermine

Lung surfactant is secreted from epithelial type II cells into alveolar airspace in response to airborne and circulating stimuli. Nitric oxide (NO) can be generated by constitutive and inducible nitric oxide synthases (cNOS and iNOS) in pulmonary endothelial and epithelial cells. The authors therefore examined the effects of NO on lung surfactant secretion using an isolated perfused rat lung model and primary culture of type II cells. Infusion of L-N(G)-nitroarginine methyl ester (L-NAME) (100 micro M), an inhibitor of cNOS and iNOS, via pulmonary circulation for 90 minutes resulted in a decrease of lung surfactant secretion (1.55%+/-0.15% in control versus 0.79%+/-0.16% in L-NAME-treated lungs, P <.05). However, aminoguanide, an inhibitor of iNOS, had no effect, indicating that the decline of lung surfactant secretion is due to the specific blockage of cNOS rather than iNOS activity in perfused lungs. A reduction of cGMP level by 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (25 micro M), a specific inhibitor of guanylyl cyclase, inhibited surfactant secretion by 64%. Furthermore, KT5823 (1 micro M), an inhibitor of protein kinase G, depressed surfactant secretion by 40%. These results suggest that physiological concentrations of NO are required for lung surfactant secretion and NO-mediated secretion is at least partly via a rise of cGMP level and activation of protein kinase G. In primary culture of alveolar type II cells, spermine NONOate (SPER/NO), a NO donor, increased basal phosphatidylcholine (PC) secretion in a dose-dependent manner. Maximal stimulation was observed at 1 micro M. However, in the ATP-stimulated type II cells, SPER/NO displayed a biphasic effect on PC secretion. At low concentrations (0.1 to 1 micro M), SPER/NO increased ATP-stimulated PC secretion, whereas at a high concentration (100 micro M), SPER/NO inhibited the secretion. The results suggest that NO may play an important role in lung surfactant secretion.

Topics: Animals; Carbazoles; Cells, Cultured; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epithelial Cells; Guanidines; Indoles; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitrogen Oxides; Oxadiazoles; Perfusion; Phosphatidylcholines; Pulmonary Alveoli; Pulmonary Surfactants; Quinoxalines; Rats; Rats, Sprague-Dawley; Spermine

Recently, we have found that the nitrate/nitrite concentrations in preovulatory follicles significantly decrease after hCG injection and that inducible nitric oxide synthase (iNOS) plays a main role in the decrease of the intrafollicular nitric oxide (NO) concentration. The purpose of the present study was to investigate the role of NO on oocyte meiotic maturation and to consider the physiological means of the decrease in intrafollicular NO concentration. Immature rats received 15 IU of eCG, and ovaries were removed under ether anesthesia 48 h later. Each ovary was bluntly divided into five or six pieces containing from four to seven preovulatory follicles under the microscope and then incubated with hCG, aminoguanidine (AG; an iNOS inhibitor), or S-nitroso-L-acetyl penicillamine (SNAP; an NO donor) for 5 h. After incubation, preovulatory follicles were punctured, and germinal vesicle breakdown (GVBD) was observed. Also, cGMP concentrations in these follicles were measured. Next, denuded oocytes were recovered from preovulatory follicles at 48 h after injection of 15 IU of eCG and incubated with SNAP with or without ferrous hemoglobin. Every 30 min up to 12 h, GVBD was observed. Both AG and hCG promoted GVBD, and SNAP prevented this effect. In addition, AG decreased intrafollicular cGMP levels, and the concomitant addition of SNAP prevented this decrease. Finally, SNAP dose-dependently inhibited GVBD in denuded oocyte, and this effect of SNAP was reversed by the addition of hemoglobin. We conclude that the iNOS-NO-(cGMP) axis may play an important role in oocyte meiotic maturation.

Topics: Animals; Cells, Cultured; Chorionic Gonadotropin; Cyclic GMP; Enzyme Inhibitors; Female; Guanidines; Meiosis; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oocytes; Organ Culture Techniques; Ovarian Follicle; Ovulation Induction; Rats; Rats, Sprague-Dawley; S-Nitroso-N-Acetylpenicillamine

Aminoguanidine inhibits the development of retinopathy in diabetic animals, but the mechanism remains unclear. Inasmuch as aminoguanidine is a relatively selective inhibitor of the inducible isoform of nitric oxide synthase (iNOS), we have investigated the effects of hyperglycemia on the retinal nitric oxide (NO) pathway in the presence and absence of aminoguanidine. In vivo studies utilized retinas from experimentally diabetic rats treated or without aminoguanidine for 2 months, and in vitro studies used bovine retinal endothelial cells and a transformed retinal glial cell line (rMC-1) incubated in 5 mm and 25 mm glucose with and without aminoguanidine (100 microg/mL). NO was detected as nitrite and nitrate, and nitrotyrosine and iNOS were detected using immunochemical methods. Retinal homogenates from diabetic animals had greater than normal levels of NO and iNOS (p < 0.05), and nitrotyrosine was greater than normal, especially in one band immunoprecipitated from retinal homogenates. Oral aminoguanidine significantly inhibited all of these increases. Nitrotyrosine was detected immunohistochemically only in the retinal vasculature of non-diabetic and diabetic animals. Retinal endothelial and rMC-1 cells cultured in high glucose increased NO and NT, and aminoguanidine inhibited both increases in rMC-1 cells, but only NT in endothelial cells. Hyperglycemia increases NO production in retinal cells, and aminoguanidine can inhibit this abnormality. Inhibition of diabetic retinopathy by aminoguanidine might be mediated in part by inhibition of sequelae of NO production.

Topics: Animals; Cattle; Cells, Cultured; Cyclic GMP; Diabetes Complications; Diabetes Mellitus; Diabetic Retinopathy; Endothelium, Vascular; Glucose; Guanidines; Hyperglycemia; Immunohistochemistry; Male; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidative Stress; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Retina; Streptozocin; Tyrosine

The role of nitric oxide (NO) in the stimulation of soluble guanylyl cyclase (sGC) is well established, but the mechanism by which the enzyme is inactivated during the prolonged NO stimulation has not been characterized. In this paper we studied the interactions between NO and intracellular Ca(2+) in the control of sGC in rat anterior pituitary cells. Experiments were done in cultured cells, which expressed neuronal and endothelial NO synthases, and in cells with elevated NO levels induced by the expression of inducible NO synthase and by the addition of several NO donors. Basal sGC-dependent cGMP production was stimulated by the increase in NO levels in a time-dependent manner. In contrast, depolarization of cells by high K(+) and Bay K 8644, an L-type Ca(2+) channel agonist, inhibited sGC activity. Depolarization-induced down-regulation of sGC activity was also observed in cells with inhibited cGMP-dependent phosphodiesterases but not in cells bathed in Ca(2+)-deficient medium. This inhibition was independent from the pattern of Ca(2+) signaling (oscillatory versus nonoscillatory) and NO levels, and was determined by averaged concentration of intracellular Ca(2+). These results indicate that inactivation of sGC by intracellular Ca(2+) serves as a negative feedback to break the stimulatory action of NO on enzyme activity in intact pituitary cells.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcium; Calcium Channel Blockers; Calcium Signaling; Cells, Cultured; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Female; Guanidines; Guanylate Cyclase; Isoenzymes; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; Pituitary Gland, Anterior; Potassium; Rats; Rats, Sprague-Dawley; Solubility; Vinca Alkaloids

Nitric oxide (NO)-dependent soluble guanylyl cyclase (sGC) is operative in mammalian cells, but its presence and the role in cGMP production in pituitary cells have been incompletely characterized. Here we show that sGC is expressed in pituitary tissue and dispersed cells, enriched lactotrophs and somatotrophs, and GH(3) immortalized cells, and that this enzyme is exclusively responsible for cGMP production in unstimulated cells. Basal sGC activity was partially dependent on voltage-gated calcium influx, and both calcium-sensitive NO synthases (NOS), neuronal and endothelial, were expressed in pituitary tissue and mixed cells, enriched lactotrophs and somatotrophs, and GH(3) cells. Calcium-independent inducible NOS was transiently expressed in cultured lactotrophs and somatotrophs after the dispersion of cells, but not in GH(3) cells and pituitary tissue. This enzyme participated in the control of basal sGC activity in cultured pituitary cells. The overexpression of inducible NOS by lipopolysaccharide + interferon-gamma further increased NO and cGMP levels, and the majority of de novo produced cGMP was rapidly released. Addition of an NO donor to perifused pituitary cells also led to a rapid cGMP release. Calcium-mobilizing agonists TRH and GnRH slightly increased basal cGMP production, but only when added in high concentrations. In contrast, adenylyl cyclase agonists GHRH and CRF induced a robust increase in cGMP production, with EC(50)s in the physiological concentration range. As in cells overexpressing inducible NOS, the stimulatory action of GHRH and CRF was preserved in cells bathed in calcium-deficient medium, but was not associated with a measurable increase in NO production. These results indicate that sGC is present in secretory anterior pituitary cells and is regulated in an NO-dependent manner through constitutively expressed neuronal and endothelial NOS and transiently expressed inducible NOS, as well as independently of NO by adenylyl cyclase coupled-receptors.

Topics: 1-Methyl-3-isobutylxanthine; Adenylyl Cyclases; Animals; Calcium; Cells, Cultured; Corticotropin-Releasing Hormone; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gonadotropin-Releasing Hormone; Growth Hormone-Releasing Hormone; Guanidines; Guanylate Cyclase; Immunoblotting; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Phosphodiesterase Inhibitors; Pituitary Gland, Anterior; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Thyrotropin-Releasing Hormone

The current study was designed to evaluate the endotoxin-induced alterations of the mechanisms involved in Ca(2+)handling within the rat thoracic aorta and further to examine whether in vitro inhibition of inducible nitric oxide synthase (iNOS) by aminoguanidine would account for this effect or not. Endothelium denuded aortic rings from rats injected with lipopolysaccharide (LPS) (5 mg kg(-1), i.p. 18 h prior to functional studies) or saline were mounted in isolated organ baths. Various experimental conditions were studied on paired rings of the same animal which were incubated in the presence or absence of aminoguanidine (100 microM). Phenylephrine contractility in Ca(2+)-containing buffer or in Ca(2+)-free buffer, contractions induced by K(+)depolarization and CaCl(2)in depolarized muscle and by caffeine exposure were significantly decreased in LPS-treated rings and were reversed by aminoguanidine exposure. Aminoguanidine also improved the contractions recorded while switching the Ca(2+)-free buffer to Ca(2+)-containing buffer. We conclude that endotoxin induces a generalized contractile defect in vascular smooth muscle including impairment in the influx of extracellular Ca(2+)and release of Ca(2+)from intracellular stores. An increase in iNOS activation leading to excessive nitric oxide synthesis, possibly non-endothelial in origin, may account for this defect.

Topics: Animals; Aorta, Thoracic; Calcium; Cyclic GMP; Drug Interactions; Endotoxins; Enzyme Inhibitors; Guanidines; In Vitro Techniques; Lipopolysaccharides; Male; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Wistar; Vascular Resistance

The present study was undertaken to investigate relaxant effect of L-citrulline in phenylephrine precontracted endothelium intact thoracic aortic rings obtained from control or lipopolysaccharide (1 mg/kg)-treated rats. L-citrulline produced 40+/-3% (n=36) and 60+/-5% (n=24) relaxations in control and lipopolysaccharide-treated rings, respectively. Nitric oxide (NO) release and cyclic guanosine-3',5'-monophosphate levels from the rings were also increased following treatment with L-citrulline. Inhibition of guanylate cyclase, L-citrulline recycling to L-arginine or denudation of the endothelium, significantly reduced L-citrulline-induced relaxations both in control and lipopolysaccharide-treated rings. Treatment of rings with protein synthesis inhibitors prevented relaxations to L-citrulline. Inhibitor of Ca2+-activated K+ channels, tetrabutylammonium or precontraction of the rings with KCl (80 mM), significantly attenuated L-citrulline mediated relaxations in control and lipopolysaccharide-treated rings. Thus, L-citrulline seems to exert significant relaxation by supplementing the release of NO due to its recycling to L-arginine, which gets further augmented after lipopolysaccharide treatment.

Topics: Animals; Aorta; Arginine; Citrulline; Corticosterone; Cyclic GMP; Cycloheximide; Glutamine; Guanidines; In Vitro Techniques; Lipopolysaccharides; Male; Muscle, Smooth, Vascular; Nitric Oxide; Nitrites; Potassium Channels; Rats; Rats, Sprague-Dawley; Vasodilation

beta-adrenergic hyporesponsiveness in many cardiomyopathies is linked to expression of inducible nitric oxide synthase (iNOS) and increased production of NO. The purpose of this study was to examine whether iNOS expression alters the function of the sarcoplasmic reticulum (SR) Ca(2+) release channel (ryanodine receptor, RyR) during beta-adrenergic stimulation.. Expression of iNOS was induced by lipopolysaccharide (LPS) injection (10 mg/kg) 6 hours before rat myocyte isolation. Confocal microscopy (fluo-3) was used to measure Ca(2+) spark frequency (CaSpF, reflecting resting RyR openings) and Ca(2+) transients. CaSpF was greatly increased by the adenylate cyclase activator forskolin (100 nmol/L) in normal myocytes (iNOS not expressed), but this effect was suppressed (by 77%) in LPS myocytes (iNOS expressed). When NO production by iNOS was inhibited by aminoguanidine (1 mmol/L), there was a further increase in the forskolin-induced CaSpF in LPS myocytes (to levels similar to the forskolin-stimulated CaSpF in normal myocytes). This effect was also seen in myocytes isolated from a failing human heart. There was no effect of aminoguanidine on forskolin-stimulated CaSpF in normal myocytes. ODQ (10 micromol/L), an inhibitor of NO stimulation of guanylate cyclase, did not restore the forskolin-induced rise in CaSpF in LPS myocytes. Aminoguanidine also increased twitch Ca(2+) transient amplitude in LPS myocytes after forskolin application (independent of changes in SR Ca(2+) load).. iNOS/NO depresses beta-adrenergic-stimulated RyR function through a cGMP-independent pathway (eg, NO- and/or peroxynitrite-dependent redox modification). This mechanism limits beta-adrenergic responsiveness and may be an important signaling pathway in cardiomyopathies, including human heart failure.

Topics: Animals; Calcium; Colforsin; Cyclic GMP; Enzyme Inhibitors; Guanidines; Guanylate Cyclase; Heart Failure; Heart Ventricles; Humans; Lipopolysaccharides; Male; Middle Aged; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxadiazoles; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, beta; Sarcoplasmic Reticulum

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

We have previously reported that leukotoxin, 9,10-epoxy-12-octadecenoate (Lx) dilates rat pulmonary arteries by means of nitric oxide synthase (NOS) activation. In this study, we investigated if Lx stimulates constitutive and/or inducible NOS. We studied the effect of the NOS inhibitors, N(G)-monomethyl-L-arginine and aminoguanidine, as well as endothelium denudation on Lx-induced rat pulmonary arterial dilation and that of aminoguanidine on Lx-induced endothelium denuded lipopolysaccharide (LPS)-treated rat pulmonary arterial dilation and tissue cGMP content. Furthermore, we assessed the effect of aminoguanidine, an inducible NOS (iNOS) inhibitor, on the cGMP content increase induced by Lx in LPS-treated human pulmonary artery smooth muscle cells (HPASMC). The NOS inhibitors and endothelium denudation significantly attenuated Lx-induced vasodilation. Aminoguanidine also significantly attenuated Lx-induced vasodilation in LPS-treated rat denuded pulmonary arteries, and attenuated Lx-induced cGMP content increase in denuded pulmonary arterial rings from LPS-treated rats and in LPS-treated HPASMC. These results suggest that Lx causes pulmonary vasodilation by stimulation of vascular endothelial NOS (eNOS) and iNOS.

Topics: Animals; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Enzyme Inhibitors; Exotoxins; Guanidines; Humans; Linoleic Acids; Lipopolysaccharides; Male; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; omega-N-Methylarginine; Pulmonary Artery; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Vasodilation

To investigate the roles of nitric oxide and adenosine triphosphate (ATP)-sensitive potassium channels (KATP) in the shortening of cardiac action potential in endotoxic shock.. Prospective animal study with concurrent controls.. University animal research laboratory.. Adult Hartley guinea pigs, weighing 300-400 g.. Guinea pigs were anesthetized and mechanically ventilated for 6 hrs. Lipopolysaccharide (LPS) or saline (sham group) were given intravenously. Drug effects were examined at the end of 6 hrs.. Plasma nitrate concentration was measured hourly, while guanosine 3',5'-cyclic monophosphate (cGMP) content and action potential duration at 90% of repolarization (APD90) of papillary muscle were examined every 2 hrs in the 6-hr endotoxemia in both the sham and the LPS-treated groups. The basal levels of these three variables showed no difference in the two groups. In the sham group, these variables did not change significantly (n = 14 for plasma nitrate determination; n = 5 for cGMP content measurement; n = 5-14 for APD90 measurement; all p > .05). But in the LPS-treated group, both plasma nitrate concentration and cGMP content of papillary muscle showed time-dependent increases and they were significantly higher than those in the sham group (at the 6th hr, plasma nitrate: 42.6 +/- 7.7 vs. 21.8 +/- 3.1 micromol/L, both n = 14, p < .01; cGMP: 1.52 +/- 0.15 vs. 0.73 +/- 0.08 pmol/mg protein, both n = 5, p < .01). In contrast, APD90 revealed a time-dependent decrease compared with that in the sham group (at the 6th hr, 137.1 +/- 52 vs. 188.2 +/- 4.8 msecs, both n = 14, p < .001). In the following 60-min in vitro recording of action potentials after the end of 6-hr endotoxemia, the shortened APD90 in the LPS-treated group did not recover and remained shorter compared with that in the sham group, in which the APD90 showed no significant changes (at the 60th min, 165.1 +/- 5.7 vs. 200.2 +/- 3.8 msecs, each n = 14, p < .01). However, in the presence of glibenclamide, a specific KATP blocker (100 micromol/L; n = 10), the APD90 could be reversed almost completely to the same value as that in the sham group (n = 14) (196.6 +/- 3.5 vs. 200.2 +/- 3.8 msecs; p > .05), despite glibenclamide having no effect on the APD90 in the sham group. In the LPS-treated group, NG-nitro-L-arginine methyl ester (1 mmol/L; n = 4), methylene blue (10 micromol/L; n = 5), and aminoguanidine (100 micromol/L; n = 4) significantly prolonged the shortened APD90 (192.5 +/- 3.1, 195.0 +/- 3.3, and 176.5 +/- 3.3 msecs, respectively; p < .01, p < .01, and p < .05, respectively, compared with that without these agents, 165.1 +/- 5.7 msecs, n = 14). These agents had negligible effects on the APD90 in the sham group (all p > .05). Furthermore, 8-bromoguanosine-3',5'-cyclic monophosphate (500 micromol/L; n = 5) decreased APD in intact papillary muscle (mean reduction of APD90, 13.5 +/- 3.5%, n = 5; p < .05), an effect abolished by pretreatment with glibenclamide (100 micromol/L; n = 5) that did not. In this experimental model, we provide reasonably convincing evidence to suggest that in endotoxic shock, an increase in nitric oxide activity may activate KATP, which plays a major role in the shortening of APD, presumably through a cGMP-dependent pathway.

Topics: Action Potentials; Adenosine Triphosphate; Animals; Cyclic GMP; Enzyme Inhibitors; Glyburide; Guanidines; Guinea Pigs; Lipopolysaccharides; Methylene Blue; NG-Nitroarginine Methyl Ester; Nitric Oxide; Papillary Muscles; Potassium Channels; Shock, Septic; Time Factors

This paper aimed to study the mechanism of vascular hyporeactivity during severe hemorrhagic shock. Rats were divided into control and shock group. Membrane potential of arteriolar strips was measured with intracellular recording method and membrane potential changes in arteriolar smooth muscle cells (ASMC) were recorded with membrane potential sensitive fluorescent dye (DiBAC4) and confocal microscopy. Hyperpolarization of ASMC membrane appeared at the late stage of shock, which correlated to low vasoreactivity. Glybenclamide, an inhibitor of K(ATP) channel reversed the hyperpolarizing effect. S-nitroso-N-acetylpenicillamine (SNAP), a donor of NO, in a higher concentration (400 mol/l) caused membrane hyperpolarization in control and shock group, which was completely reversed by application of Tiron, a scavenger of O2-. The hyperpolarizing effect of SNAP was decreased by ODQ, glybenclamide and (or) charybdotoxin. It is concluded that hyperpolarization of ASMC leads to vascular hyporeactivity. Peroxynitrite (OONO-) involves in the development of hyperpolarization in severe shock. The production of cGMP and activation of K(ATP) and K(Ca) channel contribute to the hyperpolarizing effect of OONO-*.

Topics: 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; Animals; Arginine; Arterioles; Charybdotoxin; Cyclic GMP; Drug Interactions; Female; Free Radical Scavengers; Glyburide; Guanidines; Ion Transport; Male; Membrane Potentials; Mesenteric Arteries; Microscopy, Confocal; Microscopy, Fluorescence; Muscle, Smooth, Vascular; Nitrates; Nitric Oxide Donors; Norepinephrine; Oxadiazoles; Penicillamine; Potassium; Potassium Channels; Quinoxalines; Rats; Rats, Sprague-Dawley; Shock, Hemorrhagic; Vasodilator Agents

Although ACE inhibitors can protect myocardium against ischemia/reperfusion injury, the mechanisms of this effect have not yet been characterized at the cellular level. The present study was designed to examine whether an ACE inhibitor, cilazaprilat, directly protects cardiac myocytes against hypoxia/reoxygenation (H/R) injury.. Neonatal rat cardiac myocytes in primary culture were exposed to hypoxia for 5.5 hours and subsequently reoxygenated for 1 hour. Myocyte injury was determined by the release of creatine kinase (CK). Both cilazaprilat and bradykinin significantly inhibited CK release after H/R in a dose-dependent fashion and preserved myocyte ATP content during H/R, whereas CV-11974, an angiotensin II receptor antagonist, and angiotensin II did not. The protective effect of cilazaprilat was significantly inhibited by Hoe 140 (a bradykinin B2 receptor antagonist), NG-monomethyl-L-arginine monoacetate (L-NMMA) (an NO synthase inhibitor), and methylene blue (a soluble guanylate cyclase inhibitor) but not by staurosporine (a protein kinase C inhibitor), aminoguanidine (an inhibitor of inducible NO synthase), or indomethacin (a cyclooxygenase inhibitor). Cilazaprilat significantly enhanced bradykinin production in the culture media of myocytes after 5.5 hours of hypoxia but not in that of nonmyocytes. In addition, cilazaprilat markedly enhanced the cGMP content in myocytes during hypoxia, and this augmentation in cGMP could be blunted by L-NMMA and methylene blue but not by aminoguanidine.. The present study demonstrates that cilazaprilat can directly protect myocytes against H/R injury, primarily as a result of an accumulation of bradykinin and the attendant production of NO induced by constitutive NO synthase in hypoxic myocytes in an autocrine/paracrine fashion. NO modulates guanylate cyclase and cGMP synthesis in myocytes, which may contribute to the preservation of energy metabolism and cardioprotection against H/R injury.

Topics: Adrenergic beta-Antagonists; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Animals; Antihypertensive Agents; Benzimidazoles; Biphenyl Compounds; Bradykinin; Cell Hypoxia; Cells, Cultured; Cilazapril; Creatine Kinase; Cyclic GMP; Cyclooxygenase Inhibitors; Enzyme Inhibitors; Guanidines; Indomethacin; Methylene Blue; Muscle Fibers, Skeletal; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Oxygen; Rats; Rats, Wistar; Staurosporine; Tetrazoles

The pleiotropic cytokine interleukin-1 (IL-1) is an inducer of the inducible nitric oxide synthase (iNOS). It was surprising to find that treatment of normal mice with an iNOS inhibitor resulted in detectable IL-1beta mRNA in colon and spleen, suggesting feedback regulation. When mouse peritoneal exudate cells (PEC) or RAW 264.7 cells were stimulated with lipopolysaccharide (LPS), concomitant inhibition of iNOS resulted in an increase of IL-1beta and IL-1alpha protein secretion. Conversely, after addition of the NO-generating compound NOC-18, IL-1beta and IL-1alpha concentrations in supernatants were dose-dependently reduced. Costimulation with interferon-gamma (IFN-gamma) reversed the NOC-18-mediated suppression of IL-1alpha protein concentration into an almost fivefold increase in RAW 264.7 cells. This effect was specific for IL-1alpha and was also seen in PEC. The mRNA expression for IL-1beta and IL-1alpha in RAW 264.7 cells correlated with the protein levels, suggesting transcriptional regulation by NO. Dysregulated IL-1/NO cross-regulation may play a role in inflammatory diseases.

Topics: Animals; Cell Line; Cyclic GMP; Enzyme Inhibitors; Feedback; Female; Guanidines; Interferon-gamma; Interleukin-1; Macrophages; Male; Mice; Mice, Inbred BALB C; Nitric Oxide Synthase; Nitric Oxide Synthase Type II

High concentrations of nitric oxide (NO) inhibit bone resorption by mature osteoclasts. We examined the effects of low NO concentrations on osteoclast formation in mouse bone marrow cultures. The NO releasers sodium nitroprusside (SNP) and S-nitroso-N-acetyl-DL-penicillamine inhibited the formation of multinucleated cells expressing tartrate-resistant acid phosphatase (a marker for osteoclasts) when administered during the last 3 days of 6-day cultures (differentiation stage) but not during the first 3 days (proliferation stage). SNP (1 microM) completely inhibited pit formation on dentine wafers when added to cultures during osteoclast formation, but 100 microM SNP was required to inhibit pitting by mature osteoclasts. Conversely, the NO synthase inhibitors aminoguanidine and nitro-L-arginine methyl ester both increased osteoclast formation. Inhibition of osteoclast formation by NO likely was guanosine 3',5'-cyclic monophosphate (cGMP) dependent, as SNP increased cGMP in marrow cultures, and 1 mM 8-bromo-cGMP or dibutyryl-cGMP reduced osteoclast formation when administered during the differentiation stage. The cGMP-specific type V phosphodiesterase inhibitor, zaprinast (M & B 22948) also inhibited osteoclast formation (half-maximal inhibitory constant, 100 microM) only when added during the differentiation stage. We conclude that the differentiation stage of osteoclast formation is inhibited by increases in cGMP levels elicited by NO.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Bone Marrow Cells; Bone Resorption; Bucladesine; Calcitriol; Cells, Cultured; Cyclic GMP; Dentin; Dibutyryl Cyclic GMP; Guanidines; In Vitro Techniques; Mice; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Osteoclasts; Penicillamine; Purinones; S-Nitroso-N-Acetylpenicillamine; Whales

To investigate whether nitric oxide (NO) contributed to a higher mortality induced by lipopolysaccharide (LPS) in spontaneously hypertensive rats (SHR), NO synthase inhibitors were used to examine the mortality from LPS in SHR and normotensive Wistar-Kyoto (WKY) rats. We evaluated the mortality from LPS in a series of doses (5, 10, or 20 mg/kg, i.v.) in the anesthetized rat. Plasma nitrite was measured before and at 1, 2, and 3 h after treated rats with LPS (5 mg/kg, i.v.). Pressure responses to N omega-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine (AG) were performed in rats treated with or without LPS for 3 h. Thoracic aortic cyclic guanosine 3',5'-monophosphate (cGMP) levels were also assessed. Our results demonstrated that injection of LPS caused a dose-dependent mortality in both strains, having a more marked effect in SHR. The survival time of rats after injection of LPS (5 mg/kg, i.v.) was much shorter in SHR. A higher basal level of plasma nitrite was observed in SHR and this difference was further augmented by LPS. The administration of L-NAME (3 mg/kg, i.v.) and AG (15 mg/kg, i.v.) 3 h after LPS had no significant effects on the survival time of WKY rats, but significantly prolonged that of SHR to a similar time of WKY rats. The injecton of L-NAME prior to LPS increased blood pressure of WKY rats by 28+/-5 mmHg and increased that of SHR by 38+4 mmHg. At 3 h after LPS, L-NAME had a greater pressor effect in SHR than in WKY rats. By contrast, before rats injected with LPS, AG slightly increased blood pressure of SHR by 7+/-3 mmHg but not of WKY rats (3+/-2 mmHg), whereas it also had a greater pressor effect in SHR than in WKY rats after treated rats with LPS for 3 h. In addition, LPS induced a higher level of cGMP in SHR than in WKY rats, which was attenuated by in vitro treatment of aortic rings from LPS-rats with L-NAME or AG to a similar level in SHR and WKY rats. These results suggest that a higher level of NO evoked by LPS is associated with a higher mortality in SHR and we propose that the elevated NO synthesis in SHR may play an important role in the compensatory mechanisms activated to combat the hypertensive state.

Topics: Animals; Aorta, Thoracic; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Escherichia coli; Guanidines; Hypertension; Lipopolysaccharides; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Survival Rate

Topics: Animals; Cells, Cultured; Cyclic GMP; Cytokines; Dinoprostone; Enzyme Inhibitors; Fetus; Glucose; Guanidines; Indomethacin; Insulin; Insulin Secretion; Interleukin-1; Islets of Langerhans; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Rats; Rats, Wistar; Somatostatin; Tumor Necrosis Factor-alpha

Nitric oxide (NO), identified as the mediator of endothelium-dependent relaxation of vascular smooth muscle, is known to cause a number of inflammatory conditions, especially in ischemia/reperfusion injury. This experimental study, using a rabbit epigastric island flap, was designed to investigate whether skin flap ischemia followed by reperfusion-influenced serum NO and c-GMP concentrations in the flap. In addition, we also investigated the premedicated effects of the NO synthase inhibitor and heparin on serum NO and c-GMP concentrations in skin flap ischemia/reperfusion. Serum NO concentration after 15, 30, 45, and 60 minutes of ischemia followed by reperfusion significantly increased compared with that in nonischemic control and elevated flaps. On the contrary, serum NO concentration was suppressed in L-NAME or aminoguanidine pretreated animals with ischemic group. Administration of heparin increased the serum NO concentration in elevated flaps, but suppressed it in ischemic flaps followed by reperfusion. The changes in serum c-GMP and NO concentrations were related in all of the experimental groups. These results suggest that NO may be derived from vascular endothelial cells and dilate peripheral vessels in compensation for ischemia.

Topics: Animals; Cyclic GMP; Endothelium, Vascular; Epigastric Arteries; Guanidines; Heparin; Ischemia; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Rabbits; Reperfusion; Surgical Flaps

This study investigates the mechanism of the production of nitric oxide (NO) caused by lipopolysaccharide (LPS) in spontaneously hypertensive rats (SHR) or Wistar-Kyoto (WKY) rats. The injection of LPS (5 mg/ kg, i.v.) caused a mild hypotension in WKY rats, while it induced a more severe hypotensive effect in SHR. The basal level of plasma nitrite was slightly higher in SHR than in WKY rats. At 3 h after injection of LPS, the increment in plasma nitrite was more significant in SHR. Prior to the treatment of rats with LPS, the plasma level of tumor necrosis factor-alpha (TNF alpha) was also higher in SHR than in WKY rats, and LPS induced a more significant increase of TNF alpha level (at 1 h) in SHR. In rats treated with LPS, acetylcholine-induced relaxation was significantly impaired in thoracic aortic rings obtained from WKY rats, but not in those from SHR. By contrast, L-arginine (1 mM) did not cause any relaxations in rings without the endothelium obtained from WKY rats while it slightly relaxed those from SHR, and this difference was further augmented by treatment of rats with LPS for 3 h. In addition, the basal cGMP level was higher in SHR, which was inhibited by aminoguanidine (AG, 1 mM). The treatment of rats with LPS further increased the formation of cGMP in both strains and this increment was greater in SHR than in WKY rats, which was also attenuated by AG to a similar level between both strains. Interestingly, an expression of inducible NO synthase (NOS II) protein was only observed in SHR, and further enhanced by treated rats with LPS. We conclude that an increased production of NO in SHR, which was further enhanced by LPS, is attributed to a basal expression of NOS II.

Topics: 1-Methyl-3-isobutylxanthine; Acetylcholine; Animals; Aorta, Thoracic; Arginine; Blood Pressure; Cyclic GMP; Enzyme Induction; Guanidines; In Vitro Techniques; Isoenzymes; Lipopolysaccharides; Muscle Contraction; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitrites; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Tumor Necrosis Factor-alpha

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

Topics: Animals; Cyclic GMP; Enzyme Inhibitors; Guanidines; Heparin; Ischemia; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Rabbits; Reperfusion Injury; Skin; Surgical Flaps

Our aim was to demonstrate increased NO activity from inducible NO synthase (iNOS) in pulmonary arteries (PA) from rats treated with endotoxin [lipopolysaccharide (LPS), 20 mg/kg ip]. LPS treatment diminished the contractile response of PA to potassium chloride (KCl) and phenylephrine (PE) and increased levels of guanosine 3',5'-cyclic monophosphate (cGMP) in endothelium-denuded vessels. Both the NO synthase (NOS) antagonists NG-monomethyl-L-arginine (L-NMMA; nonselective) and aminoguanidine (selective for iNOS) enhanced PE-induced contraction in endothelium-denuded vessels from LPS-treated rats. Furthermore, L-NMMA-induced contraction of endothelium-denuded vessels from LPS-treated rats was stereospecifically antagonized by L-arginine and associated with decreased cGMP levels. These data suggest that NO is produced in increased amounts from PA smooth muscle after LPS treatment. LPS treatment caused increased expression of mRNA for iNOS in PA. This effect of LPS was attenuated by pretreatment with dexamethasone, suggesting that induction of NOS in PA smooth muscle underlies the increased NO activity associated with LPS administration.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Base Sequence; Cyclic GMP; Dexamethasone; Endothelium, Vascular; Guanidines; Lipopolysaccharides; Male; Molecular Sequence Data; Muscle Contraction; Nitric Oxide Synthase; omega-N-Methylarginine; Phenylephrine; Phosphatidylethanolamines; Potassium Chloride; Pulmonary Artery; Rats; Rats, Wistar; RNA, Messenger

Lipopolysaccharide (LPS; endotoxin) produces dilatation of cerebral arterioles in vivo which may be due, in part, to expression of inducible nitric oxide (NO) synthase. We tested the hypothesis that aminoguanidine, an inhibitor of inducible NO synthase, would reduce endotoxin-induced dilatation of cerebral arterioles. Because mechanisms other than expression of inducible NO synthase may contribute to endotoxin-induced dilatation of cerebral arterioles, we also tested the hypothesis that calcitonin gene-related peptide (CGRP) contributes to vascular responses to endotoxin. Cerebral arteriolar diameter was measured using a closed cranial window in anesthetized rabbits under control conditions [77 +/- 3 (SE) microns] and during topical application of endotoxin (100 micrograms/ml). After 4 h, diameter of cerebral arterioles increased by 41 +/- 5%. Coapplication of aminoguanidine (0.3 mM) with endotoxin reduced vasodilatation at all time points (30 min to 4 h). Relative to control values, endotoxin treatment increased guanosine 3',5'-cyclic monophosphate (cGMP) concentration in cerebrospinal fluid (CSF) by approximately 20 fold at 4 h. Aminoguanidine attenuated the endotoxin-induced increased in CSF cGMP concentration. Aminoguanidine (0.3 mM) did not alter acetylcholine-mediated dilatation of cerebral arterioles. Coapplication of CGRP-(8-37) (0.5 microM), a specific blocker of CGRP receptors, with endotoxin significantly reduced vasodilatation in response to endotoxin at 2, 3, and 4 h. Thus 1) aminoguanidine inhibits endotoxin- but not acetylcholine-mediated dilatation of cerebral arterioles, and 2) activation of CGRP receptors mediates a portion of endotoxin-induced dilation of cerebral arterioles.

Topics: Acetylcholine; Animals; Arteries; Arterioles; Biomechanical Phenomena; Blood Pressure; Calcitonin Gene-Related Peptide; Cerebrovascular Circulation; Cyclic GMP; Endotoxins; Female; Gases; Guanidines; Male; Nitroprusside; Peptide Fragments; Rabbits; Vasodilation

Overproduction of nitric oxide (NO) following induction of NO synthase in vascular smooth muscle by endotoxin and certain cytokines contributes to the vasodilation and hyporesponsiveness to vasopressors that characterize the septic circulation. Guanosine 3',5'-cyclic monophosphate (cGMP) mediates the effects of NO in vascular smooth muscle. Vessels from animals treated with endotoxin have elevated cGMP levels compared with control animals. Aminoguanidine has been proposed as a selective inhibitor of the inducible form of NO synthase. This study compares the effects of aminoguanidine on phenylephrine-induced contractions and cGMP levels in thoracic aortic rings from endotoxin treated (20 mg/kg intraperitoneally) with sham-treated (1 ml saline intraperitoneally) rats. Endotoxin-treatment depressed phenylephrine-induced contraction and raised tissue levels of cGMP. Aminoguanidine (100 microM and 1 mM) increased phenylephrine-induced tension and decreased cGMP levels in a dose-dependent manner in intact and endothelium-denuded aortas from endotoxin-treated rats but had no effect on vessels from sham-treated rats. These findings are consistent with the hypothesis that endotoxin treatment causes increased vascular production of endothelium-independent NO, which is associated with a diminished response to vasoconstrictors. Aminoguanidine decreases indices of NO production only after endotoxin treatment, providing further evidence that it is a selective inhibitor of inducible NO synthase.

Topics: Animals; Aorta, Thoracic; Cyclic GMP; Dose-Response Relationship, Drug; Endothelium, Vascular; Endotoxins; Enzyme Induction; Guanidines; Male; Muscle Contraction; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Phenylephrine; Rats; Rats, Wistar; Salmonella enteritidis

Recent reports suggest that endothelial-dependent relaxant factor, recognized as nitric oxide (NO), reduces myocardial contractility. Here, we showed that both exposures to acetylcholine and bradykinin for 30 min increased cyclic guanylate monophosphate (cyclic GMP) in isolated rat cardiomyocytes. These increases in cyclic GMP were blunted by NW-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase. Hypoxia augmented the cyclic GMP accumulation due to exposures to acetylcholine and bradykinin, which were blunted by L-NAME. The increases in cyclic GMP due to acetylcholine and bradykinin during normoxic and hypoxic conditions were not blunted by aminoguanidine, an inhibitor of inducible NO synthase. These findings revealed that NO is produced in cardiomyocytes due to stimulation of NO synthase and modulates their own guanylate cyclase, which was augmented by hypoxia. NO production, through NO synthase in cardiomyocytes, may constitute autocrine regulations of myocardial contractility and paracrine regulations of coronary vasodilation and platelet aggregation.

Topics: Acetylcholine; Animals; Arginine; Bradykinin; Cell Hypoxia; Cyclic AMP; Cyclic GMP; Guanidines; Guanylate Cyclase; Heart; Male; Myocardium; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Rats; Rats, Wistar; Stimulation, Chemical

As nitric oxide reduces gut epithelial permeability, we designed a study to determine if chronic nitric oxide synthase inhibition predisposes the gut to inflammation. Nitric oxide synthase (NOS) inhibitors were administered in the drinking water ad libitum, for seven days: aminoguanidine (10 micrograms/ml), a selective inhibitor of the inducible form of nitric oxide synthase; and NG-nitro-L-arginine methyl ester (L-NAME, 1, 10, and 100 micrograms/ml), which inhibits both the constitutive and inducible forms. Control animals drank tap water only or water with D-NAME, the inactive enantiomer. After one week, circulating leukocyte count and tissue myeloperoxidase activity were measured. L-NAME (100 micrograms/ml), but not D-NAME or aminoguanidine, caused a twofold increase in a circulating leukocyte numbers. This increase in leukocyte numbers was time- and dose-dependent, but the differential count was unaltered. Tissue myeloperoxidase (MPO) activity as an index of granulocyte infiltration was comparable in all groups in the stomach, jejunum, colon, liver, lung, kidney, heart, and skeletal muscle. However, ileal MPO activity was elevated threefold in the L-NAME-(100 micrograms/ml) treated group (P < 0.05). Results in the D-NAME and aminoguanidine groups were similar to controls. L-NAME administration resulted in a reduction in NOS activity ([14C]citrulline formation) in the ileum but not jejunum, whereas cGMP levels were elevated in both ileum and jejunum. We conclude that chronic inhibition of the constitutive form of nitric oxide synthase predisposes the ileum to inflammation and leads to a progressive leukocytosis.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Citrulline; Cyclic GMP; Female; Guanidines; Guinea Pigs; Ileitis; Leukocyte Count; Leukocytosis; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Peroxidase

Overproduction of the free radical nitric oxide (NO) has been implicated in the pathogenesis of a variety of inflammatory and immunologically mediated diseases as well as complications of diabetes. In the present study we have demonstrated that aminoguanidine selectively inhibits the cytokine-inducible isoform of NO synthase which appears to be responsible for the excess production of NO linked to these disease states. By using organ, cell, and enzyme-based measurements we have shown that aminoguanidine is equipotent to NG-monomethyl-L-arginine (L-NMA) as an inhibitor of the cytokine-induced isoform of NO synthase but is 10 to 100-fold less potent as an inhibitor of the constitutive isoform. Thus, aminoguanidine may be useful as a selective inhibitor of the inducible NO synthase in the treatment of disease states characterized by the pathological overproduction of NO.

Topics: Amino Acid Oxidoreductases; Animals; Aorta, Thoracic; Arginine; Cells, Cultured; Citrulline; Cyclic GMP; Guanidines; Macrophages; Male; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Nitrites; Rats; Rats, Sprague-Dawley; Spectrometry, Fluorescence

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

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Cell Line; Cyclic GMP; Dinoprostone; Enzyme Activation; Guanidines; Humans; Indomethacin; Mice; Nitrates; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Prostaglandin-Endoperoxide Synthases; Recombinant Proteins

Nitric oxide has recently been implicated as a cellular molecule that mediates interleukin-1 beta (IL-1 beta)-induced inhibition of glucose-stimulated insulin secretion by islets of Langerhans. In this study evidence is presented which demonstrates that islets contain both the cytokine inducible and the constitutive isoforms of nitric oxide synthase as determined by NADPH diaphorase staining and immunohistochemical localization. Untreated islets contain NADPH diaphorase activity, and the intensity of NADPH diaphorase staining is dramatically increased after culture for 18 hrs with IL-1 beta. Both control and IL-1 beta-induced NADPH diaphorase staining of islets is inhibited by the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (NMMA). Importantly, approximately 60-70% of islet cells stained positive for NADPH diaphorase (under both IL-1 beta treated and control conditions), suggesting that a subset of islet cells contain nitric oxide synthase. The beta-cell appears to be the endocrine cell type which contains constitutive nitric oxide synthase as demonstrated by immunohistochemical co-localization of constitutive nitric oxide synthase and insulin. IL-1 beta is believed to stimulate the expression of cytokine inducible nitric oxide synthase because the synthetic glucocorticoid, dexamethasone, prevents IL-1 beta induced inhibition of glucose stimulated insulin secretion and cGMP accumulation by islets. Both dexamethasone, and the nitric oxide synthase inhibitors NMMA and aminoguanidine also prevent IL-1 beta induced islet degeneration. These results indicate that nitric oxide produced by the inducible isoform of nitric oxide synthase mediates cytokine induced islet dysfunction and destruction, and that the beta-cell is the islet endocrine cellular source of constitutive nitric oxide synthase.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Cyclic GMP; Dexamethasone; Guanidines; Insulin; Insulin Secretion; Interleukin-1; Islets of Langerhans; Male; NADPH Dehydrogenase; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Rats; Rats, Sprague-Dawley