Regulation Of Nitric Oxide Synthesis Research Articles

Effect of Hypoxia on Nitric Oxide Production and Its Synthase Gene Expression in Rat Smooth Muscle Cells

It has not been clarified yet as to whether hypoxia and inflammation affect NO synthesis. In this study, we investigated the transcription of inducible nitric oxide synthase (iNOS) mRNA and the production of nitric oxide (NO) in rat smooth muscle cells (SMCs) cultured under hypoxic conditions in the presence and absence of proinflammatory cytokine interferon-γ (IFN-γ) and lipopolysaccharide (LPS). We found that hypoxia inhibited the production of NO but did not affect the transcription of iNOS mRNA in rat SMCs treated with IFN-γ, LPS, or both. These results indicate that O2 is involved in the regulation of NO synthesis in inflammatory tissues.

Nitric oxide (NO) upregulates NO synthase expression in fetal intrapulmonary artery endothelial cells.

Endothelium-derived nitric oxide (NO) generated by endothelial NO synthase (eNOS) is critically involved in pulmonary vasodilation during cardiopulmonary transition at birth. Inhaled NO therapy has recently been considered for patients with persistent pulmonary hypertension of the newborn (PPHN). To better understand the mechanisms regulating NO synthesis in the developing pulmonary circulation and the possible ramifications of NO therapy, studies were performed with early passage ovine fetal intrapulmonary artery endothelial cells (PAEC) to determine whether NO directly modulates eNOS expression. To examine the effects of exogenous NO, PAEC were treated with the NO donor spermine NONOate or the parent compound spermine. Exogenous NO caused increases in eNOS protein expression and NOS enzymatic activity that were detectable within 16 h of exposure. In contrast, the inhibition of endogenous NO production with nitro-L-arginine-methyl ester (L-NAME) caused a reduction in eNOS protein expression that was evident within 8 h. Paralleling the changes in eNOS protein, eNOS messenger RNA (mRNA) abundance was upregulated by exogenous NO and downregulated by L-NAME, suggesting that NO modulation of eNOS expression involves processes at the level of gene transcription or mRNA stability. Thus, in fetal PAEC there is positive-feedback regulation of eNOS expression by both exogenous and endogenous NO. These findings suggest that difficulties with transient effectiveness or prolonged requirements for NO therapy in certain PPHN patients are not due to declines in eNOS expression. Further, conditions such as fetal hypoxemia that impair PAEC NO production may attenuate eNOS expression through this mechanism, thereby contributing to the pathogenesis of PPHN.

Regulation of macrophage nitric oxide synthesis by endothelial cells: a role for NG,NG-dimethylarginine.

NG,NG-dimethylarginine is an endogenous inhibitor of nitric oxide synthesis produced by endothelial cells and found in the plasma and urine of normal adults. We have examined the ability of NG, NG-dimethylarginine, produced by endothelial cells (SGHEC-7), to regulate the production of nitric oxide by lipopolysaccharide-stimulated mouse macrophage cells (J774.2). Stimulation of SGHEC-7 or J774.2 cells with lipopolysaccharide had no effect on their release of NG,NG-dimethylarginine into the culture supernatant. Stimulation of J774.2 cells with lipopolysaccharide for 24 h significantly stimulated nitric oxide production by J774.2 but not SGHEC-7 cells. When lipopolysaccharide-stimulated J774.2 cells were co-cultured with endothelial cells for 24 h, there was a significant inhibition of nitrite accumulation. The inhibition observed was dependent on the endothelial cell number (12 +/- 5% [mean +/- SEM] following incubation with 0.6 x 105 cells, up to 47 +/- 8% with 4.8 x 105 cells). The inhibitory effect of endothelial cells was prevented by incubation with increasing concentrations of L-arginine; the IC50 was 2.9 +/- 0.6 mM arginine. Western blot analysis indicated that the expression of inducible nitric oxide synthase was not inhibited by co-culture with SGHEC-7 cells. The results presented here demonstrate that NG,NG-dimethylarginine synthesized by endothelial cells may inhibit nitric oxide synthase in adjacent cells and play a role in the regulation of nitric oxide synthesis by macrophages.

Inducible nitric oxide synthase and nitric oxide production in Leishmania infantum-infected human macrophages stimulated with interferon-gamma and bacterial lipopolysaccharide.

Nitric oxide produced by an inducible nitric oxide synthase constitutes one of the main microbicidal mechanisms of murine macrophages and its importance is now being recognized for human macrophages. In this study we evaluated inducible nitric oxide synthase expression, nitric oxide release, and parasitocidal ability of Leishmania infantum-infected monocyte-derived human macrophages. The inducible nitric oxide synthase was detected by immunofluorescence and western blotting and nitric oxide production was measured by the Griess reaction for nitrites. Parasite killing was microscopically evaluated by fluorescent dyes. Experiments were performed on macrophages with or without previous stimulation with recombinant human interferon-gamma and bacterial lipopolysaccharide. Inducible nitric oxide synthase expression and nitric oxide release were higher in Leishmania-infected stimulated macrophages than in uninfected cells or infected cells without previous stimulation. Nitric oxide production and parasitocidal activity against Leishmania infantum were reduced in macrophages treated with the nitric oxide synthase inhibitor L-N(G) monomethylarginine. These results suggest a microbicidal role for nitric oxide in human leishmaniasis, with the possible practical application of immunological or pharmacological regulation of nitric oxide synthesis in the treatment of this infection.

Heat shock and 5-azacytidine inhibit nitric oxide synthesis and tumor necrosis factor-alpha secretion in activated macrophages.

To elucidate the role of stress response during macrophage activation, the effects of heat shock and the amino acid analog, 5-azacytidine on nitric oxide (NO) production, tumor necrosis factor-alpha (TNF-alpha) secretion, and heat shock protein (HSP) synthesis have been studied in murine peritoneal macrophages (C57BL/6). Heat shock (1 hr at 43 degrees C) or 5-azacytidine markedly inhibited the release of NO into the medium from interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS)-stimulated macrophages. Although heat shock significantly decreased TNF-alpha secretion only at the initiation stage of macrophage stimulation, 5-azacytidine treatment resulted in a more prolonged reduction in the secretion of TNF-alpha. When heat-shocked cells were stimulated with IFN-gamma plus LPS under normal culture conditions at 37 degrees C, the heat shock-induced inhibition of NO release reversed progressively with increasing recovery time. Although the total amount of cellular HSP72 measured by Western blot increased time-dependently over 7 hr, newly synthesized HSP72 measured by [35S]methionine incorporation was evident only after 1 and 3 hr of recovery time after heat shock treatment. At these time points, the lowest nitrite accumulation and TNF-alpha secretion into the medium was evident. It is concluded that signaling pathways related to newly synthesized HSP such as HSP72 are implicated in the down regulation of NO synthesis and TNF-alpha secretion in macrophages.

Alphav integrin regulates TNF-alpha-induced nitric oxide synthesis in rat mesangial cells--possible role of osteopontin.

Tumour necrosis factor-alpha (TNF-alpha) induces nitric oxide (NO) synthesis in rat mesangial cells (MCs). We previously demonstrated that osteopontin (OP), a matrix protein that mainly interacts with the alphav integrin family, increased time-dependently by TNF-alpha stimulation at gene and protein levels. The regulation of NO synthesis by integrins or matrix proteins is unclear. We examined whether integrin, especially alphav integrin, regulates NO synthesis in rat MCs and whether OP, an alphav integrin ligand, has an effect on TNF-alpha-induced NO synthesis. Furthermore, OP and inducible NO synthase (iNOS) gene expression was examined by Northern blotting. TNF-alpha increased NO synthesis in MCs in a time-dependent manner. Synthetic GRGDSP peptide, which is known to inhibit various integrins that interact with RGD-containing extracellular matrices, increased TNF-alpha-induced NO levels in a dose-dependent manner. Cyclical RGD peptide, the specific inhibitor of alphav integrin, also exhibited a dose-dependent effect of increasing NO levels, while GRGESP peptide, which has very low affinity to integrins, had no effect. In addition, NO synthesis was found to be significantly reduced when MCs were plated on OP-coated dishes compared to type I or IV collagen-coated dishes. Furthermore, anti-OP antibody increased NO synthesis in MCs. iNOS mRNA levels were increased by TNF-alpha, and were abruptly diminished after OP mRNA was significantly induced. The present study demonstrated the involvement of alphav integrin in TNF-alpha-induced NO synthesis in rat MCs, and the possible role of OP was suggested in the mechanism. TNF-alpha and extracellular matrices can co-operate to regulate the behaviour of MCs at least partly through NO synthesis, which may participate in the course of glomerular diseases.

Hepatic and splanchnic nitric oxide activity in patients with cirrhosis

BACKGROUNDIn animal models of cirrhosis, altered activity of nitric oxide (NO) has been implicated in the pathogenesis of increased intrahepatic portal vascular resistance and abnormal mesenteric vasodilatation.AIMSTo investigate NO activity...

Cholinergic and GABAergic regulation of nitric oxide synthesis in the guinea pig ileum.

Nitric oxide (NO) synthesis was examined in intact longitudinal muscle-myenteric plexus preparations of the guinea pig ileum by determining the formation of [3H]citrulline during incubation with [3H]arginine. Spontaneous [3H]citrulline production after 30 min was 80-90 dpm/mg, which constituted approximately 1% of the tissue radioactivity. Electrical stimulation (10 Hz) led to a threefold increase in [3H]citrulline formation. Removal of calcium from the medium or addition of NG-nitro-L-arginine strongly inhibited both spontaneous and electrically induced production of [3H]citrulline. TTX reduced the electrically induced but not spontaneous [3H]citrulline formation. The electrically induced formation of [3H]citrulline was diminished by (+)-tubocurarine and mecamylamine and enhanced by scopolamine, which suggests that endogenous ACh inhibits, via muscarinic receptors, and stimulates, via nicotinic receptors, the NO synthesis in the myenteric plexus. The GABAA receptor agonist muscimol and GABA also reduced the electrically evoked formation of [3H]citrulline, whereas baclofen was without effect. Bicuculline antagonized the inhibitory effect of GABA. It is concluded that nitrergic myenteric neurons are equipped with GABAA receptors, which mediate inhibition of NO synthesis.

Regional and subcellular distribution of a neutral and basic amino acid transporter in forebrain neurons containing nitric oxide synthase.

The neutral and basic amino acid transporter (NBAT) facilitates sodium-independent transport of L-amino acids in renal and intestinal epithelial cells and has been postulated to play a similar role in neurons. In previous studies, NBAT has been detected within enteric and brainstem autonomic neurons in a distribution similar to that of constitutive nitric oxide synthase (cNOS). Furthermore, L-arginine, the required precursor for nitric oxide synthesis, is an excellent NBAT substrate. Together, these findings suggest that NBAT may play a role in the regulation of nitric oxide synthesis, through the control of precursor availability. To gain insight into the potential physiological role of NBAT in central neurons, we used an antipeptide antiserum to examine the light and electron microscopic immunocytochemical localization of NBAT in the rat forebrain and to compare this distribution with that of cNOS. Immunolabeling for NBAT was detected within perikarya and dendrite-like processes that were most numerous in the frontal and cingulate cortex, the ventral striatum, the central amygdala, and the bed nucleus of the stria terminalis. Labeled varicose axonal processes were distributed most densely in the agranular insular cortex and the paraventricular nuclei of the thalamus and hypothalamus (PVH). Electron microscopy showed that immunogold labeling for NBAT was distributed along plasmalemmal and vacuolar membranes within somata, dendrites, and axonal profiles. Many of the NBAT-containing somata and dendrites contained detectable cNOS. Our results suggest that expression of NBAT may provide specific populations of cNOS-containing forebrain neurons with a unique mechanism for regulating somatodendritic synthesis of nitric oxide.

Expression and regulation of protein inhibitor of neuronal nitric oxide synthase in ventilatory muscles.

In skeletal muscle fibers, nitric oxide (NO) is synthesized by neuronal NO synthase (nNOS) and regulates excitation-contraction coupling, glucose uptake, and mitochondrial respiration. Recently, a novel 89-amino acid protein, designated protein inhibitor of nNOS (PIN), has been shown to interact with and specifically inhibit nNOS activity. In this study, we investigated the distribution, localization, and regulation of PIN expression in ventilatory and limb muscles of various species. Amplified PIN cDNA from the rat diaphragm revealed an open reading frame identical to that of human PIN. Among muscles of adult rats, PIN mRNA was strongly expressed in muscles rich in type I fibers, whereas much weaker expression was evident in muscles rich in type II fibers. By comparison, PIN protein expression was not related to fiber-type distribution. Similarly, PIN protein was equally expressed among rat, mouse, and human diaphragms. Both PIN mRNA and PIN protein were expressed at much higher levels in the embryonic rat diaphragm than in adult muscle. Immunohistochemistry revealed that PIN protein was localized in close proximity to the sarcolemma and nuclei. PIN protein was also abundant in muscle spindles and axons of nerves supplying skeletal muscle fibers. We conclude that PIN is expressed in various skeletal muscle fibers and that its expression is regulated during muscle development. The localization of PIN in muscle regions containing abundant nNOS protein suggests that it plays a role in the regulation of NO synthesis in skeletal muscle fibers.

Regulation of M1 muscarinic receptor-mediated signaling in intact cells by exogenous, but not endogenously produced, nitric oxide.

Regulation of nitric oxide (NO) formation is critical to ensure maintenance of appropriate cellular concentrations of this labile, signaling molecule. This study investigated the role exogenous and endogenously produced NO have in feeding back to regulate NO synthesis in intact cells. Two NO donors inhibited activation of neuronal NO synthase (nNOS) in response to the muscarinic receptor agonist carbachol in Chinese hamster ovary (CHO) cells stably transfected with the M1 muscarinic receptor and nNOS. The presence of the NO scavenger [2-(4-Carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide potassium salt] (C-PTIO) potentiated carbachol-induced activation of nNOS in transfected CHO cells. C-PTIO also potentiated nNOS activity in response to the Ca2+ ionophore ionomycin. In contrast, the NO scavenger oxyhemoglobin depressed carbachol- and ionomycin-induced NO formation. These discrepant results suggest that it is unlikely that endogenously produced NO induces feed back inhibition at the level of nNOS activation itself. Exogenous sources of NO inhibited carbachol-induced inositol phosphates formation. However, endogenously produced NO did not appear to feed back to regulate phosphoinositide hydrolysis as there was no difference in [3H]inositol phosphates formation between cells that do or do not express nNOS. There was also no change in carbachol-induced [3H]inositol phosphates formation in the presence or absence of a NOS inhibitor or the NO scavenger C-PTIO. A decrease in the carbachol-mediated transient Ca2+ peak was observed in cells that express nNOS as compared to cells lacking the enzyme, suggesting that endogenous NO might inhibit receptor mediated Ca2+ signaling. This conclusion, however, was not supported by the lack of ability of a NOS inhibitor to modulate carbachol-induced Ca2+ elevations. Taken together, these results highlight differences in the regulation of the nNOS activation cascade by endogenous vs. exogenous sources of NO.

Interaction between nitric oxide and prostaglandin E pathways in rat smooth muscle myometrial cells

Previously, we demonstrated the presence of a nitric oxide (NO) prostaglandin (PG) pathway in myometrial cells obtained from uterine rat tissue. This pathway was modulated by estrogen and one possible function could be to modulate uterine relaxation. In the present study, we investigated the role of progesterone in the regulation of NO synthesis and the uterotonic PGE production by myometrial cells from uterine rat tissue. We worked with two groups of rats: (i) ovariectomizcd (OV) rats, without influence of sex hormones and (ii) OV rats injected with progesterone (4 mg) s.c. Myometrial uterine cells were obtained by a selective enzymatic digestion. In the incubation medium of these cells, nitrite concentration (as a measure of NO production) and PGE production were evaluated. To ensure a specific response, a competitive NOs inhibitor, N G-monomethyl-L-arginine; L-NMMA (300 μM) was used. We found that at 48 h of the incubation period, cells obtained from progesterone-primed uterine tissue presented an increase in the nitrite concentration concomitant with a decrease in the PGE production. When L-NMMA was added to the cells, nitrite production and PGE synthesis returned to control values. The fact that this effect had not been observed in the group of cells obtained from OV rats suggests that progesterone was responsible for it. These data provide strong evidence that in spite of the fact that estrogen and progesterone modulate the NO-PG pathway in the uterine rat tissue, the two hormones have opposite effects.

Cell-specific effects of pentoxifylline on nitric oxide production and inducible nitric oxide synthase mRNA expression.

Cytokine-stimulated astrocytes and macrophages are potent producers of nitric oxide (NO), a free radical proposed to play an important role in organ-specific autoimmunity, including demyelinating diseases of the central nervous system. The aim of this study was to investigate effects of pentoxifylline (PTX), a phosphodiesterase inhibitor with immunomodulatory properties, on NO production and inducible NO synthase (iNOS) mRNA expression in rat astrocytes and macrophages. We have shown that PTX affects cytokine (interferon-gamma, IFN-gamma; interleukin-1, IL-1; tumour-necrosis factor-alpha, TNF-alpha)-induced NO production in both cell types, but in the opposite manner--enhancing in astrocytes and suppressive in macrophages. While PTX did not have any effect on enzymatic activity of iNOS in activated cells, expression of iNOS mRNA was elevated in astrocytes and decreased in macrophages treated with cytokines and PTX. Treatment with PTX alone affected neither NO production nor iNOS mRNA levels in astrocytes or macrophages. This study indicates involvement of different signalling pathways associated with iNOS induction in astrocytes and macrophages, thus emphasizing complexity of regulation of NO synthesis in different cell types.

Role of Nuclear Factor κB in Cytokine-Induced Nitric Oxide and Tetrahydrobiopterin Synthesis in Rat Neonatal Cardiac Myocytes

The nitric oxide (NO) signalling pathway is thought to play a direct role in regulating the contractile properties of cardiac muscle bothin vitroandin vivo. The inducible isoform of NO synthase (iNOS) mediates a sustained increase in NO production in response to cytokines in the cardiac myocytes; however, the regulation of NO synthesis in these cells remains poorly understood. Tetrahydrobiopterin (BH4) is an essential cofactor for NO formation. Cytokines induce thede novosynthesis of BH4 in cardiac myocytes, an event that is essential for the induction of NO synthesis. Activation of NO formation by cytokines in cardiac myocytes requires transcriptional induction of the genes that encode iNOS and guanosine triphosphate cyclohydrolase I (GTPCH), the first and rate-limiting enzyme inde novoBH4 synthesis. Given that nuclear factorκB (NF-κB) mediates the induction of iNOS gene expression in various cell types, the role of NF-κB in the induction of iNOS in cytokine-stimulated rat neonatal cardiac myocytes was assessed by examining the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB activation, on the abundance of iNOS mRNA and NO synthesis. The effects of PDTC on GTPCH mRNA abundance and biopterin synthesis were also investigated. PDTC inhibited in a dose-dependent manner both NO and BH4 synthesis induced by a combination of interleukin-1α (IL-1α) and interferon-γ(IFNγ), with a half-maximal inhibitory concentration of 22 μm. PDTC also prevented the accumulation of iNOS and GTPCH mRNAs induced by IL-1α and IFNγ. Cytokine-induced NO and BH4 synthesis was also inhibited bytosyl-lysine-chloromethyl ketone, another inhibitor of NF-κB activation. Results suggest that PDTC inhibits cytokine-induced NO and BH4 synthesis by inhibiting the expression of iNOS and GTPCH genes. Thus, the induction of both genes necessary for NO synthesis in cardiac myocytes appears to be regulated, at least in part, by a common mechanism: NF-κB activation.

Regulation of nitric oxide synthesis by oxygen in vascular endothelial cells.

Vascular endothelial cells synthesize nitric oxide (NO) in response to agonists that elevate cytosolic free Ca2+ concentrations. Once activated, NO synthase (NOS) requires arginine, NADPH, and O2 as cosubstrates. In this study, we investigated the role of O2 in regulating endothelial NOS activity in intact bovine aortic endothelial cells by measuring the rate of nitrite (NO2-) and nitrate (NO3-) production after conversion of NO2- to S-nitrosoglutathione before analysis or after reduction of NO2- and NO3- to NO using acidic vanadium chloride. The basal rate of NO2- production was 6.5 +/- 0.8 pmol.min-1.mg protein-1. Thapsigargin (TG, 1 microM) elevated free cytosolic Ca2+ concentration and increased the rate of NO2- synthesis. At maximal concentrations of TG, the rate of stimulated NO2- production was linear for at least 20 min and was eightfold higher than the basal rate (53.5 +/- 1.8 pmol.min-1.mg protein-1). Incubation of cells in gas mixtures chosen to produce PO2 values in the physiological range led to a progressive fall in the rate of TG-stimulated NO2- production, as O2 concentrations were reduced from that of room air. The half-maximal effective concentration for NO2- production by intact cells was found to occur at 38 Torr. PO2 values higher than that of room air did not lead to a change in the rate of TG-stimulated NO2- production. To confirm that measurement of NO2- accurately reflects total NO production, both NO2- plus NO3- were measured in buffer samples from cells incubated in either room air or N2. The sum of these NO oxidation products was inhibited similarly by hypoxia. These findings suggest that O2 is an important determinant of NOS activity in hypoxic tissues or in vascular beds such as the pulmonary arterial or fetal circulation where PO2 values in the range of 40 Torr are encountered normally.

Roles of tyrosine kinases in the regulation of nitric oxide synthesis in murine liver cells: modulation of NF-kappa B activity by tyrosine kinases.

Nitric oxide (NO) synthesis is upregulated during chronic hepatic inflammation. The present study characterized the mechanisms involved in the induction of NO production and inducible NO synthase (iNOS) messenger RNA (mRNA) expression in murine embryonic liver cell line, BNL CL.2 cells. No production by BNL CL.2 cells was induced by interferon-r (IFN-r) plus lipopolysaccharide (LPS). However, other inflammatory cytokines such as interleukin (IL)-beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 had no additional effects on it. The stimulatory effects of IFN-r and LPS were time- and dose-dependent. NO secretion was inhibited by treatment with inducible NOS inhibitors such as NG-monomethyl L-arginine, NG-amino-L-arginine, and diphenylene iodonium. iNOS mRNA was induced 3 hours after IFN-r plus LPS treatment, and iNOS expression was maximal in the presence of IFN-r and LPS. The protein tyrosine kinase inhibitors such as genistein and tyrphostin reduced IFN-r plus LPS-induced iNOS mRNA expression and NO production. In contrast, the inhibitors of protein kinase C, protein kinase A, and protein phosphatases did not affect iNOS expression induced by IFN-r plus LPS. In addition, iNOS mRNA expression was completely blocked by treatment with tyrphostin. However, mRNA expression of an early response gene, JunB, and constitutively expressed genes beta-actin and GAPDH were not inhibited by tyrphostin. Furthermore, tyrphostin inhibited the promoter activation of iNOS gene induced by IFN-gamma plus LPS, and it also suppressed IFN-gamma plus LPS-induced nuclear factor-kappa B-binding activity but not AP-1-binding activity. These results suggest that NO production and iNOS mRNA expression in this cell line is dependent on protein tyrosine kinases but does not require protein kinase C, protein kinase A, or protein phosphatases.

Heavy physical exercise decreases nitric oxide levels in the nasal airways in humans.

A continuous production of nitric oxide (NO) takes place in human nasal airways. NO in the nasal airways is mainly derived from the paranasal sinuses. The factors that regulate NO synthesis in the upper airways are presently not known. We have investigated the effects of physical exercise on NO levels in the nasal airways. Nasal cavity NO levels were measured by chemiluminescence technique in five healthy non-smoking male subjects before, during and after 5 min of maximal exercise (245 W) on an ergometer cycle. In addition, in one subject NO levels were measured directly in the maxillary sinus during exercise. Nasal cavity NO levels were decreased by 47% after only 1 min of exercise compared with the control situation. A maxima 76% reduction was found at the end of the exercise period and thereafter NO levels slowly increased, reaching basal levels again in about 15-20 min. NO levels in the sinus decreased in a similar manner during exercise. The decrease in nasal cavity NO levels cannot be explained merely by dilution of nasal air due to changes in nasal cavity volume or increased ventilation. We conclude that the excretion of NO in the nasal airways is decreased acutely during heavy short term physical exercise.

Regulation of nitric oxide synthesis by dimethylarginine dimethylaminohydrolase.

1. Dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes the endogenous nitric oxide synthase inhibitors NG-monomethyl-arginine and NG,NG-dimethy-L-arginine to citrulline, was identified by Western blotting in rat and human tissue homogenates. 2. S-2-amino-4(3-methylguanidino)butanoic acid (4124W) inhibited the metabolism of [14C]-NG-monomethyl-L-arginine to [14C]-citrulline by rat liver homogenates (IC50 416 +/- 66 microM; n = 9), human cultured endothelial cells (IC50 250 +/- 34 microM; n = 9) and isolated purified dimethylarginine dimethylaminohydrolase. 3. Addition of 4124W to culture medium increased the accumulation of endogenously-generated NG,NG-dimethy-L-arginine in the supernatant of human cultured endothelial cells from 3.1 +/- 0.3 to 5 +/- 0.7 microM (n = 15; P < 0.005). 4. 4124W (1 microM - 1 mM) had no direct effect on endothelial nitric oxide synthase activity but caused endothelium-dependent contraction of rat aortic rings (1 mM 4124W increased tone by 81.5 +/- 9.6% of that caused by phenylephrine 100 nM). This effect was reversed by L-arginine (100 microM). 4124W reversed endothelium-dependent relaxation of human saphenous vein (19.2 +/- 6.7% reversal of bradykinin-induced relaxation at 1 mM 4124W). 5. These data suggest that inhibition of dimethylarginine dimethylaminohydrolase increases the intracellular contraction of NG,NG-dimethyl-L-arginine sufficiently to inhibit nitric oxide synthesis. Inhibiting the activity of DDAH may provide an alternative mechanism for inhibition of nitric oxide synthases and changes in the activity of DDAH could contribute to pathophysiological alterations in NO generation.

Alternative Splicing of Human Inducible Nitric-Oxide Synthase mRNA: TISSUE-SPECIFIC REGULATION AND INDUCTION BY CYTOKINES

Human inducible nitric-oxide synthase (iNOS) is responsible for nitric oxide synthesis in response to inflammatory mediators. The human iNOS gene, containing 26 exons, encodes a protein of 131 kDa. This study was aimed at investigating the presence of alternative splicing of human iNOS mRNA. Total RNA from human alveolar macrophages, nasal and bronchial epithelial cells, and several human tissues was transcribed to cDNA and analyzed using polymerase chain reaction with specific primers for segmental analysis of the iNOS gene. Four sites of alternative splicing were identified by sequence analysis; these included deletion of: (i) exon 5; (ii) exons 8 and 9; (iii) exons 9, 10, and 11; and (iv) exons 15 and 16. The deduced amino acid sequences of the novel iNOS cDNAs predict one truncated protein (resulting from exon 5 deletion) and three iNOS proteins with in-frame deletions. Southern analyses of polymerase chain reaction products were consistent with tissue-specific regulation of alternative splicing. In cultured cells, iNOS induction by cytokines and lipopolysaccharide was associated with an increase in alternatively spliced mRNA transcripts. Because iNOS is active as a dimer, the novel forms of alternatively spliced iNOS may be involved in regulation of nitric oxide synthesis.

Increased expression of inducible nitric oxide synthase in psoriatic skin and cytokine-stimulated cultured keratinocytes.

Since nitric oxide (NO) has been implicated in the pathogenesis of various hyperproliferative and inflammatory diseases, the mRNA expression of constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) were investigated in psoriatic skin by reverse transcriptase coupled to the polymerase chain reaction (PCR). The study showed that the mRNA expression of brain nitric oxide synthase (bNOS), one of two isoforms of cNOS, was weak in both psoriatic plaques lesions and uninvolved skin, while mRNA transcripts for the second isoform, endothelial nitric oxide synthase (eNOS), were not detectable using the present method. In contrast, the mRNA expression of iNOS was markedly increased in lesional skin as compared to uninvolved skin. Cultured human keratinocytes exposed to a combination of interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha) for 4 h, showed strong gene expression of iNOS, while in 24 h, the expression had returned to baseline expression. In summary, the study demonstrates that mRNA for the inducible form of NOS is over-expressed in psoriatic lesions. The cause of this may be the local presence of inflammatory cytokines. These findings imply that iNOS may play an important part in local regulation of NO synthesis in psoriasis and other inflammatory dermatoses.