Absence of implication of L-arginine/nitric oxide pathway on neuronal cell injury induced by L-glutamate or hypoxia.

L-glutamate, N-methyl-D-aspartate (NMDA), kainate, quisqualate and sodium nitroprusside increased cyclic GMP (cGMP) level on rat whole brain cell culture. The accumulation of cGMP evoked by L-glutamate was inhibited by a NMDA antagonist MK-801, an inhibitor of guanylate cyclase methylene blue and two nitric oxide (NO) synthase inhibitors NG-monomethyl-L-arginine (L-NMMA) and L-NG-nitroarginine (NO2Arg). The inhibition of L-NMMA on cGMP level was reversed partially by addition of L-arginine. Although MK-801 was able to protect cells from neuronal injury induced by L-glutamate or by 5 h hypoxia, L-NMMA and NO2Arg were ineffective. The present study suggests that cGMP elevation mediated by NO following activation by L-glutamate is not involved in neuronal cell injury.     

Postanoxic functional recovery of the developing heart is slightly altered by endogenous or exogenous nitric oxide

Nitric oxide synthase (NOS) is strongly and transiently expressed in the developing heart but its function is not well documented. This work examined the role, either protective or detrimental, that endogenous and exogenous NO could play in the functioning of the embryonic heart submitted to hypoxia and reoxygenation. Spontaneously beating hearts isolated from 4-day-old chick embryos were either homogenized to determine basal inducible NOS (iNOS) expression and activity or submitted to 30 min anoxia followed by 100 min reoxygenation. The chrono-, dromo- and inotropic responses to anoxia/reoxygenation were determined in the presence of NOS substrate (L-arginine 10 mM), NOS inhibitor L-NIO (1–5 mM), or NO donor (DETA NONOate 10–100 M). Myocardial iNOS was detectable by immunoblotting and its activity was specifically decreased by 53% in the presence of 5 mM L-NIO. L-Arginine, L-NIO and DETA NONOate at 10 M had no significant effect on the investigated functional parameters during anoxia/reoxygenation. However, irrespective of anoxia/reoxygenation, DETA NONOate at 100 M decreased ventricular shortening velocity by about 70%, and reduced atrio-ventricular propagation by 23%. None of the used drugs affected atrial activity and hearts of all experimental groups fully recovered at the end of reoxygenation. These findings indicate that (1) by contrast with adult heart, endogenously released NO plays a minor role in the early response of the embryonic heart to reoxygenation, (2) exogenous NO has to be provided at high concentration to delay postanoxic functional recovery, and (3) sinoatrial pacemaker cells are the less responsive to NO.     

Role of nitric oxide in antagonistic effects of transforming growth factor-beta and interleukin-1 beta on the beating rate of cultured cardiac myocytes.

We have recently shown that transforming growth factor-beta (TGF beta) acts in an autocrine manner to maintain the beating rate of neonatal rat cardiac myocytes cultured in serum-free medium on cardiac fibroblast matrix. Interleukin-1 beta (IL-1 beta) suppresses the myocyte-beating rate, and TGF beta antagonizes this effect. We now show that TGF beta and IL-1 beta also have antagonistic effects on the secretion of nitric oxide (NO) by these myocytes, and that NO secretion, the activity of NO synthase (NOS), and expression of the inducible form of NOS correlate inversely with the effects of these two agents on the beating rate. Western blot analysis shows that treatment of myocytes with TGF beta antagonizes the induction of NOS after treatment with IL-1 beta. Release of NO, induced by IL-1 beta, is dependent upon the availability of the substrate, L-arginine, and is suppressed by a competitive inhibitor, NG-monomethyl-L-arginine. L-Arginine (> 0.25 mM) also suppresses, and NG-monomethyl-L-arginine (> 0.5 mM) enhances the myocyte-beating rate. Treatment with IL-1 beta, but not TGF beta, increases cellular cGMP, presumably by activation of guanylate cyclase by NO. Methylene blue, an inhibitor of guanylate cyclase, reverses the suppression of beating caused by IL-1 beta. Bacterial lipopolysaccharide, present in the serum-free medium, is a coinducer of NO secretion. The suppressive effects of NO on the beating rate can be overcome by altering either the set of cytokines employed to induce NO or the matrix on which the myocytes are cultured, demonstrating that additional parameters are also involved in regulation of the beating rate.     

Ventricular but not atrial electro-mechanical delay of the embryonic heart is altered by anoxia-reoxygenation and improved by nitric oxide

BACKGROUND/AIM: Excitation-contraction coupling is modulated by nitric oxide (NO) which otherwise has either beneficial or detrimental effects on myocardial function during hypoxia-reoxygenation. This work aimed at characterizing the variations of electromechanical delay (EMD) induced by anoxia-reoxygenation within the developing heart and determining whether atrial and ventricular EMD are modulated by NO to the same extent. METHODS: Hearts of 4 or 4.5-day-old chick embryos were excised and submitted in vitro to normoxia (45 min), anoxia (30 min) and reoxygenation (60 min). Electrocardiogram and atrial and ventricular contractions were simultaneously recorded throughout experiment. Anoxia-reoxygenation-induced chrono-, dromo-and inotropic disturbances and changes in EMD in atrium (EMDa) and ventricle (EMDv) were investigated in control hearts and in hearts exposed to 0.1, 1, 10, 50 and 100 microM of DETA-NONOate (a NO donating agent) or to 50 microM of L-NAME (a NOS inhibitor). RESULTS: Under normoxia, heart rate, PR interval, ventricular shortening velocity, EMDa and EMDv were similar in control, L-NAME-treated and DETA-NONOate-treated hearts. Under anoxia, cardiac activity became markedly erratic within less than 10 min in all groups. At the onset of reoxygenation, EMDv was increased by about 300% with respect to the preanoxic value while EMDa did not vary significatively. Compared to control conditions, L-NAME or DETA-NONOate had no influence on the negative chrono-, dromo- and inotropic effects induced by anoxia-reoxygenation. However, L-NAME prolonged EMDv during anoxia and delayed EMDv recovery during reoxygenation while 100 microM DETA-NONOate had the opposite effects. EMDa was neither affected by NOS inhibitor nor NO donor. At the end of reoxygenation, all the investigated parameters returned to their basal values. CONCLUSION: This work provides evidence that a NO-dependent pathway is involved in regulation of the ventricular excitation-contraction coupling in the anoxic-reoxygenated developing heart.     

Nitric oxide, a biological double-faced janus--is this good or bad?

Nitric oxide (NO) is a biological messenger molecule produced by one of the essential amino acids L-arginine by the catalytic action of the enzyme NO synthase (NOS). The dual role of NO as a protective or toxic molecule is due to several factors, such as; the isoform of NOS involved, concentration of NO and the type of cells in which it is synthesised, the availability of the substrate L-arginine, generation of guanosine 3,5'-cyclic monophosphate (cGMP) from soluble guanylate cyclase and the overall extra and intracellular environment in which NO is produced. NOS activation as a result of trauma (calcium influx) or infection leads to NO production, which activates its downstream receptor sGC to synthesise cGMP and/or leads to protein nitrosylation. This may lead to one or more systemic effects including altered neurotransmission which can be protective or toxic, vaso/bronchodilatation in the cardiovascular and respiratory systems and enhanced immune activity against invading pathogens. In addition to these major functions, NO plays important role in thermoregulation, renal function, gastrointestinal motility, endocrine function, and various functions of the urogenital system ranging from renin secretion to micturation; spermatogenesis to penile erection; and ovulation to implantation and parturition. A schematic summary of the functions of NO and the various isoforms of NOS expressed in body systems is shown in figure 1. In this review, the historical background, biochemistry and biosynthesis of NO and its enzymes together with the mechanism of NO actions in physiology and pathophysiology are discussed.     

Twenty-four-hour variation of L-arginine/nitric oxide/cyclic guanosine monophosphate pathway demonstrated by the mouse visceral pain model

The L-arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway is known to be involved in central and peripheral nociceptive processes. This study evaluated the rhythmic pattern of the L-arginine/NO/cGMP pathway using the mouse visceral pain model. Experiments were performed at six different times (1, 5, 9, 13, 17, and 21 h after light on) per day in male mice synchronized to a 12 h:12 h light-dark cycle. Animals were injected s.c. with saline, 2 mg/kg L-arginine (a NO precursor), 75 mg/kg L-N(G)-nitroarginine methyl ester (L-NAME, a NOS inhibitor), 40 mg/kg methylene blue (a soluble guanylyl cyclase and/or NOS inhibitor), or 0.1 mg/kg sodium nitroprusside (a nonenzymatic NO donor) 15 min before counting 2.5 mg/kg (i.p.) p-benzoquinone (PBQ)-induced abdominal constrictions for 15 min. Blood samples were collected after the test, and the nitrite concentration was determined in serum samples. L-arginine or L-NAME caused both antinociception and nociception, depending on the circadian time of their injection. The analgesic effect of methylene blue or sodium nitroprusside exhibited significant biological time-dependent differences in PBQ-induced abdominal constrictions. Serum nitrite levels also displayed a significant 24 h variation in mice injected with PBQ, L-NAME, methylene blue, or sodium nitroprusside, but not saline or L-arginine. These results suggest that components of L-arginine/NO/cGMP pathway exhibit biological time-dependent effects on visceral nociceptive process.     

Endothelin-3 stimulates production of endothelium-derived nitric oxide via phosphoinositide breakdown.

Cultured bovine endothelial cells (EC) have specific receptors for endothelin (ET)-3 functionally coupled to phosphoinositide breakdown. We studied whether ET-3 stimulates synthesis of nitric oxide (NO), an endothelium-derived relaxing factor that activates soluble guanylate cyclase in EC, and whether the ET-3-induced NO formation involves G-proteins. ET-3 dose-dependently stimulated production of intracellular cGMP in EC, of which effects were abolished by pretreatment with NG-monomethyl L-arginine, an inhibitor of NO synthesis, and methylene blue, an inhibitor of soluble guanylate cyclase. The stimulatory effects of ET-3 on cGMP production, inositol trisphosphate formation and increase in cytosolic free Ca2+ concentration were similarly blocked by pretreatment with pertussis toxin (PTX). These data suggest that ET-3 induces synthesis of NO mediated by phosphoinositide breakdown via PTX-sensitive G-protein in EC.     

Cyclic guanosine-3',5'-monophosphate and biopteridine biosynthesis in Nocardia sp

Nocardia sp. strain NRRL 5646 contains a nitric oxide synthase (NOS) enzyme system capable of generating nitric oxide (NO) from arginine and arginine-containing peptides. To explain possible roles of the NOS system in this bacterium, guanylate cyclase (GC) and tetrahydrobiopterin (H(4)B) biosynthetic enzymes were identified in cell extracts and in culture media. Cell extracts contained GC activity, as measured by the conversion of GTP to cyclic guanosine-3',5'-monophosphate (cGMP) at 9.56 pmol of cGMP h(-1) mg of protein(-1). Concentrations of extracellular cGMP in culture media were significantly increased, from average control levels of 45 pmol cGMP liter(-1) to a maximum of 315 pmol liter(-1), in response to additions of GTP, L-arginine, H(4)B, and sodium nitroprusside to growing Nocardia cultures. On the other hand, the NOS inhibitor N(G)-nitro-L-arginine and the GC inhibitor 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one both dramatically decreased extracellular cGMP levels. Activities for GTP-cyclohydrase-1, 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase, enzymes essential for H(4)B biosynthesis, were present in Nocardia culture extracts at 77.5 pmol of neopterin and 45.8 pmol of biopterin h(-1) mg of protein(-1), respectively. In Nocardia spp., as in mammals, GTP is a key intermediate in H(4)B biosynthesis, and GTP is converted to cGMP by a GC enzyme system that is activated by NO.     

Decreased synthesis and vasodilation to nitric oxide in piglets with hypoxia-induced pulmonary hypertension

Nitric oxide (NO) is thought to play an important role in the regulation of neonatal pulmonary vasculature. It has been suggested that neonates with pulmonary hypertension have a defective NO pathway. Therefore, we measured in 1-day-old piglets exposed to hypoxia (fraction of inspired O(2) = 0.10) for 3 or 14 days to induce pulmonary hypertension 1) the activity of NO synthase (NOS) via conversion of L-arginine to L-citrulline and the concentration of the NO precursor L-arginine in isolated pulmonary vessels, 2) the vasodilator response to the NO donor 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1) and the cGMP analog 8-bromo-cGMP in isolated perfused lungs, and 3) the production of cGMP in response to SIN-1 in isolated perfused lungs. After 3 days of exposure to hypoxia, endothelial NOS (eNOS) activity was unaffected, whereas, after 14 days of hypoxia, eNOS activity was decreased in the cytosolic fraction of pulmonary artery (P < 0.05) but not of pulmonary vein homogenates. Inducible NOS activity was decreased in the cytosolic fraction of pulmonary artery homogenates after both 3 (P < 0.05) and 14 (P < 0.05) days of hypoxia but was unchanged in pulmonary veins. Pulmonary artery levels of L-arginine were unaffected by hypoxic exposure. After 3 days of exposure to hypoxia, the reduction in the dilator response to SIN-1 (P < 0.05) coincided with a decrease in cGMP production (P < 0.005), suggesting that soluble guanylate cyclase activity may be altered. When the exposure was prolonged to 14 days, dilation to SIN-1 remained decreased (P < 0.05) and, although cGMP production normalized, the dilator response to 8-bromo-cGMP decreased (P < 0.05), suggesting that, after prolonged exposure to hypoxia, cGMP-dependent mechanisms may also be impaired. In conclusion, neonatal hypoxia-induced pulmonary hypertension is associated with multiple disruptions in the NO pathway.     

The regulation of cardiac activity by nitric oxide (NO) in the Vietnamese stick insect, Baculum extradentatum

This study examines the role of the unconventional gaseous signaling molecule nitric oxide (NO) on the regulation of heart rate in the Vietnamese stick insect, Baculum extradentatum. Using nicotinamide dinucleotide hydrogen phosphate (NADPH)-diaphorase histochemistry, as well as immunohistochemistry and Western blotting with an antibody against NO synthetase (NOS), we identified the presence of NOS in hemocytes present throughout the lumen of the dorsal vessel. We propose that NO is delivered to heart muscle tissue via hemocytes circulating within the hemolymph. In the present study, stimulation of NO levels by the application of the NO donor MAHMA-NONOate and l-arginine led to a dose-dependent decrease in heart rate. Treatment of tissues with the NOS inhibitor, L-NAME, in equimolar concentrations with l-arginine, led to a recovery of heart rate, without modifying heart rate on its own. Finally guanosine 3',5'-cyclic monophosphate (cGMP) analog, 8-bromo-cGMP, elicited similar inhibitory effects on stick insect heart rate as did the guanylate cyclase activator, YC-1, and the phosphodiesterase inhibitor, dipyridamole, indicating that cGMP is most likely the second messenger in the stick insect NO signaling pathway. Contrary to the cardioexcitatory effect of NO on other insect hearts, we have found that NO inhibits stick insect heart rate independently from any nervous system input, in a similar inhibitory fashion as that of vertebrate hearts.     

NO increases permeability of cultured human cervical epithelia by cGMP-mediated increase in G-actin

Human cervicalepithelial cells express mRNA for the nitric oxide (NO) synthase (NOS)isoforms ecNOS, bNOS, and iNOS and release NO into the extracellularmedium. N^G-nitro-L-arginine methylester (L-NAME), an NOS inhibitor, and Hb, an NO scavenger,decreased paracellular permeability; in contrast, the NO donors sodiumnitroprusside (SNP) andN-(ethoxycarbonyl)-3-(4-morpholinyl)sydnonimine increasedparacellular permeability across cultured human cervical epithelia onfilters, suggesting that NO increases cervical paracellular permeability. The objective of the study was to understand the mechanisms of NO action on cervical paracellular permeability. 8-Bromo-cGMP (8-BrcGMP) also increased permeability, and the effect wasblocked by KT-5823 (a blocker of cGMP-dependent protein kinase), butnot by LY-83583 (a blocker of guanylate cyclase). In contrast, LY-83583and KT-5823 blocked the SNP-induced increase in permeability. Treatmentwith SNP increased cellular cGMP, and the effect was blocked by Hb andLY-83583, but not by KT-5823. Neither SNP nor 8-BrcGMP had modulatedcervical cation selectivity. In contrast, both agents increasedfluorescence from fura 2-loaded cells in theCa²⁺-insensitive wavelengths, indicating that SNP and8-BrcGMP stimulate a decrease in cell size and in the resistance of thelateral intercellular space. Neither SNP nor 8-BrcGMP had an effect ontotal cellular actin, but both agents increased the fraction ofG-actin. Hb blocked the SNP-induced increase in G-actin, and KT-5823blocked the 8-BrcGMP-induced increase in G-actin. On the basis of theseresults, it is suggested that NO acts on guanylate cyclase andstimulates an increase in cGMP; cGMP, acting via cGMP-dependent proteinkinase, shifts actin steady-state toward G-actin; this fragments thecytoskeleton and renders cells more sensitive to decreases in cell sizeand resistance of the lateral intercellular space and, hence, toincreases in permeability. These results may be important forunderstanding NO regulation of transcervical paracellular permeabilityand secretion of cervical mucus in the woman.

     

The Slow Cyclic GMP Increase Caused by Serotonin in NG108-15 Cells Is Not Inhibited by Antagonists of Known Serotonin Receptors: Possible Existence of a New Receptor Subtype Coupled with Membrane-Bound Guanylate Cyclase

Characterization of the serotonin (5-HT)-induced cyclic GMP (cGMP) elevation was investigated in comparison with bradykinin- and ANP-induced elevations in NG108-15 cells. At 20 s, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM, 100 microM), a membrane-permeabilized Ca2+ chelator, or N-monomethyl-L-arginine (NMMA, 300 microM), an inhibitor of L-arginine-derived nitric oxide (NO) synthesis, inhibited 5-HT-induced elevation by approximately 40%, and completely inhibited bradykinin-induced response. Neither 5-HT- nor ANP-induced cGMP elevation at 10 min was affected by BAPTA-AM or NMMA. The cGMP elevated by 5-HT as well as by ANP was effluxed to the extracellular medium. These results and our previous report suggest that 5-HT stimulates two subtypes of 5-HT receptors in NG108-15: first, 5-HT3 subtype stimulating Ca(2+)-sensitive cytosolic guanylate cyclase through NO derived from L-arginine and second, a probably novel 5-HT receptor subtype involved in activation of membrane-bound guanylate cyclase.     

Participation of the components of L-arginine/nitric oxide/cGMP cascade by chemically-induced abdominal constriction in the mouse

The purpose of this study was to investigate the role of the L-arginine/nitric oxide (NO)/cGMP pathway in p-benzoquinone-induced writhing model in mouse. L-arginine, a NO precursor, displayed antinociceptive effects at the doses of 0.125-1.0 mg/kg. When the doses of L-arginine were increased gradually to 10-100 mg/kg, a dose-dependent triphasic pattern of nociception-antinociception-nociception was obtained. The NO synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) (18.7515 mg/kg), possessed antinociceptive activity. Methylene blue (MB), a guanylyl cyclase and/or NOS inhibitor, (5-160 mg/kg) also produced a dose-dependent triphasic response. When L-arginine (50 mg/ kg) was combined with L-NAME (75 mg/kg). L-arginine-induced antinociception did not change significantly. Cotreatment of L-arginine with 5 mg/kg MB significantly decreased MB-induced antinociception and reversed the nociception induced by 40 mg/kg MB to antinociception. It is concluded that the components of L-arginine/nitric oxide/cGMP cascade may participate in nociceptive processes both peripherally and centrally by a direct effect on nociceptors or by the involvement of other related pathways of nociceptive processes induced by NO.     

Facilitatory role of NO in neural norepinephrine release in the rat kidney

We examined modulation by nitric oxide (NO) of sympathetic neurotransmitter release and vasoconstriction in the isolated pump-perfused rat kidney. Electrical renal nerve stimulation (RNS; 1 and 2 Hz) increased renal perfusion pressure and renal norepinephrine (NE) efflux. Nonselective NO synthase (NOS) inhibitors [N(omega)-nitro-L-arginine methyl ester (L-NAME) or N(omega)-nitro-L-arginine], but not a selective neuronal NO synthase inhibitor (7-nitroindazole sodium salt), suppressed the NE efflux response and enhanced the perfusion pressure response. Pretreatment with L-arginine prevented the effects of L-NAME on the RNS-induced responses. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), which eliminates NO by oxidizing it to NO(2), suppressed the NE efflux response, whereas the perfusion pressure response was less susceptible to carboxy-PTIO. 8-Bromoguanosine cGMP suppressed and a guanylate cyclase inhibitor [4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one] enhanced the RNS-induced perfusion pressure response, but neither of these drugs affected the NE efflux response. These results suggest that endogenous NO facilitates the NE release through cGMP-independent mechanisms, NO metabolites formed after NO(2) rather than NO itself counteract the vasoconstriction, and neuronal NOS does not contribute to these modulatory mechanisms in the sympathetic nervous system of the rat kidney.     

Effect of centrally administered nitric oxide modulators in Brewer's yeast-induced nociception in rats

Possible modulation of Brewer's yeast-induced nociception by centrally (icv) administered nitric oxide (NO) modulators, viz., NO synthase (NOS) inhibitors, NO precursor, donors, scavengers and co-administration of NO donor (SIN-1) with NOS inhibitor (L-NAME) and NO scavenger (Hb) was investigated in rats. Administration of NOS inhibitors and NO scavenger Hb increased the pain threshold capacity significantly, whereas NO donors SIN-1, SNP and NO precursor L-arginine were found to be hyperalgesic. D-arginine, the inactive isomer of L-arginine and methylene blue, inhibitor of soluble guanylate cyclase failed to alter the nociceptive behaviour in rats. Co-administration of SIN-1 with L-NAME and Hb found to increase the nociceptive threshold. The results indicate, that centrally administered NO modulators alter the nociceptive transmission induced by Brewer's yeast in rats.     

Nitric oxide: an unconventional messenger in the nervous system of an orthopteroid insect

Nitric oxide (NO) is a membrane-permeant messenger molecule generated from the amino acid L-arginine. NO can activate soluble guanylyl cyclase leading to the formation of cyclic GMP (cGMP) in target cells. In the nervous system, NO/cGMP signalling is thought to play essential roles in synaptic plasticity during development and also in the mature animal. This paper examines biochemical, cell biological, and physiological investigations of NO/cGMP signalling in the nervous system of the locust, a commonly used neurobiological preparation. Biochemical investigations suggest that an identical enzyme is responsible for both NO synthase (NOS) and NADPH-diaphorase activity after tissue fixation. Immunocytochemical staining of an olfactory center in the locust brain shows that NOS-immunoreactivity colocalizes with NADPH-diaphorase at the cellular level. The cytochemical staining of NO donor and target cells in adult animals suggests functions in olfaction, vision, and sensorimotor integration. During development, NO is implicated in axonal outgrowth and synaptogenesis. The cellular distribution of NO-responsive cells in neural circuits reflects potential functions of NO as a retrograde synaptic messenger, as an intracellular messenger, and as a lateral diffusible messenger independent of conventional synaptic connectivity.     

Opioid receptor-independent protection of ischemic rat hepatocytes by morphine

We studied the role of morphine in anoxia/reoxygenation injury to hepatocytes. Overnight cultured rat hepatocytes were incubated in anoxic buffer at pH 6.2 for 4h and reoxygenated at pH 7.4 for 2h to simulate anoxia/reoxygenation. Some hepatocytes were preincubated with 50 microM morphine for 10 min prior to onset of anoxia/reoxygenation. To study the effect of morphine on nitric oxide (NO), hepatocytes were loaded with 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM). Changes in NO concentration were assessed with a multi-well fluorescence reader and confocal microscopy. Morphine substantially improved cell viability after reoxygenation and increased NO generation, which was blocked by ATP-sensitive potassium channel blockers. Confocal images revealed that the increase in NO occurred mainly at the cytosol. However, treatment with opioid receptor antagonists did not reverse cytoprotection by morphine. These results indicate that morphine prevents anoxia/reoxygenation injury to hepatocytes. Protective mechanisms are associated with the potassium channels and NO, but are independent of opioid receptor-mediated signaling.     

Antiproliferative effect of nitric oxide on rat glomerular mesangial cells via inhibition of mitogen-activated protein kinase.

The effect of nitric oxide (NO) donors and lipopolysaccharide (LPS) on the proliferation of rat glomerular mesangial cells was characterized. Exogenous application of a NO donor inhibited serum-induced proliferation in a time- and dose-dependent manner. S-Nitrosoglutathione (GSNO) also increased cGMP generation and arachidonic acid release, but it did not cause any measurable increase in the cytosolic Ca2+ concentration. Chelation of cytosolic Ca2+ or inhibition of mitogen-activated protein kinase (MAPK) kinase had an inhibitory effect on proliferation, but neither enhanced the antiproliferative effect of GSNO. In contrast, inhibition of guanylate cyclase or phospholipase A2 had no effect on proliferation, but partially reversed GSNO-induced antiproliferation by approximately 98 and 65%, respectively. GSNO did not cause cell death. Incubation of cells with LPS induced endogenous NO generation and had an antiproliferative effect. LPS-induced antiproliferation was reversed completely by inhibition of nitric oxide synthase and partially by inhibition of guanylate cyclase or phospholipase A2. GSNO or LPS inhibited serum-induced MAPK activation, and both effects were partially reversed by inhibition of guanylate cyclase or phospholipase A2. Inclusion of 8-bromo-cGMP or arachidonic acid in the growth medium resulted in a similar antiproliferative effect. In conclusion, in rat glomerular mesangial cells, MAPK inhibition and an antiproliferative effect could be induced by either an increase in the cellular concentration of NO or exposure of the cells to LPS. Part of the effect of NO was attributable to the increased cellular cGMP generation and arachidonic acid release.     

Adrenergic Stimulation of Cyclic GMP Formation Requires NO-Dependent Activation of Cytosolic Guanylate Cyclase in Rat Pinealocytes

Abstract: Cyclic GMP (cGMP) formation in rat pinealocytes is regulated through a synergistic dual receptor mechanism involving β-and α₁-adrenergic receptors. The effects of N -monomethyl- l -arginine (NMMA), which inhibits nitric oxide (NO) synthase and NO-mediated activation of cytosolic guanylate cyclase, and methylene blue (MB), which inhibits cytosolic guanylate cyclase, were investigated in an attempt to understand the role of NO in adrenergic cGMP formation. Both NMMA and MB inhibited β-adrenergic stimulation of cGMP formation as well as α₁-adrenergic potentiation of β-adrenergic stimulation of cGMP formation, whereas they had no effect in unstimulated pinealocytes. The inhibitory action of NMMA was antagonized by addition of l -arginine. On the basis of these findings it can be concluded that the adrenergic stimulation of cGMP formation involves NO synthesis followed by activation of cytosolic guanylate cyclase.