Functional coupling of a Ca2+/calmodulin-dependent nitric oxide synthase and a soluble guanylyl cyclase in vertebrate photoreceptor cells.

Electrophysiological recordings on retinal rod cells, horizontal cells and on-bipolar cells indicate that exogenous nitric oxide (NO) has neuromodulatory effects in the vertebrate retina. We report here endogenous NO formation in mammalian photoreceptor cells. Photoreceptor NO synthase resembled the neuronal NOS type I from mammalian brain. NOS activity utilized the substrate L-arginine (Km = 4 microM) and the cofactors NADPH, FAD, FMN and tetrahydrobiopterin. The activity showed a complete dependence on the free calcium concentration ([Ca2+]) and was mediated by calmodulin. NO synthase activity was sufficient to activate an endogenous soluble guanylyl cyclase that copurified in photoreceptor preparations. This functional coupling was strictly controlled by the free [Ca2+] (EC50 = 0.84 microM). Activation of the soluble guanylyl cyclase by endogenous NO was up to 100% of the maximal activation of this enzyme observed with the exogenous NO donor compound sodium nitroprusside. This NO/cGMP pathway was predominantly localized in inner and not in outer segments of photoreceptors. Immunocytochemically, we localized NO synthase type I mainly in the ellipsoid region of the inner segments and a soluble guanylyl cyclase in cell bodies of cone photoreceptor cells. We conclude that in photoreceptors endogenous NO is functionally coupled to a soluble guanylyl cyclase and suggest that it has a neuromodulatory role in visual transduction and in synaptic transmission in the outer retina.     

Nitric oxide from L-arginine stimulates the soluble guanylate cyclase in adrenal glands

The formation of nitric oxide (NO) by an L-arginine:NO synthase and its stimulation of the soluble guanylate cyclase was studied in rat whole adrenal and bovine cortex and medulla cytosol. In the presence of L-arginine, the stimulation of soluble guanylate cyclase was accompanied by the formation of citrulline and NO2-, formed from NO. The NO synthase was NADPH- and Ca(2+)-dependent and was inhibited by several L-arginine analogues. These results indicate that rat and bovine adrenal cytosol contains an L-arginine:NO synthase.     

Endogenous ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase that is not regulated by nitric oxide in Dictyostelium discoideum

A 41,000 M_r cytosolic protein (p41) in Dictyostelium discoideum was shown to be modified by ADP-ribosylation that was not regulated by nitric oxide (NO). This endogenous ADP-riboxylation was optimal at conditions distinct from those optimal for the NO-stimulated ADP-ribosylation of p41. These two activities were also differentially sensitive to reducing agents and modified different amino acids. The addition of haemoglobin, which sequesters NO, and 3 the NO synthase inhibitors failed to block the endogenous ADP-ribosylation. P41 was purified to homogeneity. The N-terminal sequence of the purified protein was shown to be highly homologous to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both endogenous and NO-stimulated activities ADP-ribosylated three isoforms of the protein, with pI values of 6.6., 6.8 and 7.0. In each case, the isoform with pI 6.8 was preferentially modified. Experiments using purified GAPDH indicate that both the endogenous and NO-stimulated ADP-ribosylation are self-catalysed modifications.     

Ca2+/calmodulin-dependent cytochrome c reductase activity of brain nitric oxide synthase.

Nitric oxide acts as a widespread signal molecule and represents the endogenous activator of soluble guanylyl cyclase. In endothelial cells and brain tissue, NO is enzymatically formed from L-arginine by Ca2+/calmodulin-regulated NO synthases which require NADPH, tetrahydrobiopterin, and molecular oxygen as cofactors. Here we show that purified brain NO synthase binds to cytochrome c-agarose and exhibits superoxide dismutase-insensitive cytochrome c reductase activity with a Vmax of 10.2 mumol x mg-1 x min-1 and a Km of 34.1 microM. Cytochrome c reduction was largely dependent on Ca2+/calmodulin and cochromatographed with L-citrulline formation during gel filtration. When reconstituted with cytochrome P450, NO synthase induced a moderate Ca(2+)-independent hydroxylation of N-ethylmorphine. NO synthase also reduced the artificial electron acceptors nitro blue tetrazolium and 2,6-dichlorophenolindophenol. Cytochrome c, 2,6-dichlorophenolindophenol, and nitro blue tetrazolium inhibited NO synthase activity determined as formation of L-citrulline from 0.1 mM L-arginine in a concentration-dependent manner with half-maximal effects at 166, 41, and 7.3 microM, respectively. These results suggest that NO synthase may participate in cellular electron transfer processes and that a variety of electron-acceptors may interfere with NO formation due to the broad substrate specificity of the reductase domain of NO synthase.     

Biosynthesis of nitric oxide and citrulline from L-arginine by constitutive nitric oxide synthase present in rabbit corpus cavernosum.

The objective of this study was to determine whether a constitutive isoform of nitric oxide (NO) synthase is present in rabbit corpus cavernosum that could account for the involvement of the L-arginine-NO pathway in neurogenically-elicited relaxation of the corpus cavernosum and, therefore, penile erection. Citrulline was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine. NO was determined by monitoring the formation of total NO(x) (NO+nitrite [NO2-]+nitrate [NO3-]) by chemiluminescence after reduction of NO(x) to NO by acidic vanadium (III). Equimolar quantities of NO plus citrulline were generated from L-arginine and the formation of both products was time-dependent at 37 degrees C. NO synthase activity was distributed almost entirely to the cytosolic fraction. Enzymatic activity was completely dependent on NADPH, calmodulin, and calcium. Addition of tetrahydrobiopterin increased NO synthase activity by about 30 percent. The NO synthase inhibitor NG-nitro-L-arginine, abolished enzymatic activity. The Km for L-arginine was 17 microM and the Vmax of the reaction was 18 pmol/min/mg protein. These observations indicate that a cytosolic, constitutive isoform of NO synthase, like that found in brain neuronal tissue, is present in rabbit corpus cavernosum.     

NADPH: a stimulatory cofactor for nitric oxide-induced ADP-ribosylation reaction.

An endogenous ADP-ribosyltransferase is present in the cytosolic fraction of human platelets. Agents known to release nitric oxide activated this ADP-ribosylation reaction in a cGMP-independent fashion. This enzymatic activity was further enhanced by the addition of NADPH to the platelet cytosolic fraction. Interestingly, NADPH was unable to replace DTT, which has been described as an essential cofactor. Our results indicate that NADPH is a stimulatory factor of the endogenous ADP-ribosylation reaction. NADPH shifts the dose-response curve of NO to the left and possibly increases, in this way, the ADP-ribosylation reaction under physiological conditions.     

Elevated endogenous nitric oxide increases Ca2+ flux via L-type Ca2+ channels by S-nitrosylation in rat hippocampal neurons during severe hypoxia and in vitro ischemia

Nitric oxide (NO) mediates pathogenic changes in the brain subsequent to energy deprivation; yet the NO mechanism involved in the early events remains unclear. We examined the acute effects of severe hypoxia and oxygen-glucose deprivation (OGD) on the endogenous NO production and the NO-mediated pathways involved in the intracellular calcium ([Ca(2+)](i)) response in the rat hippocampal neurons. The levels of NO and [Ca(2+)](i) in the CA1 region of the slices rapidly elevated in hypoxia and were more prominent in OGD, measured by the electrochemical method and spectrofluorometry, respectively. The NO and [Ca(2+)](i) responses were enhanced by L-arginine and were reduced by NO synthase inhibitors, suggesting that the endogenous NO increases the [Ca(2+)](i) response to energy deprivation. Nickel and nifedipine significantly decreased the NO and [Ca(2+)](i) responses to hypoxia and OGD, indicating an involvement of L-type Ca(2+) channels in the NO-mediated mechanisms. In addition, the [Ca(2+)](i) responses were attenuated by ODQ or KT5823, inhibitors of the cGMP-PKG pathway, and by acivicin, an inhibitor of gamma-glutamyl transpeptidase for S-nitrosylation, and by the thiol-alkylating agent N-ethylmaleimide (NEM). Moreover, L-type Ca(2+) currents in cultured hippocampal neurons with whole-cell recording were significantly increased by L-arginine and were decreased by L-NAME. Pretreatment with NO synthase inhibitors or NEM but not ODQ abolished the effect of L-arginine on the Ca(2+) currents. Also, vitamin C, which decomposes nitrosothiol but not disulfide by reduction, reversed the change in the Ca(2+) current with L-arginine. Taken together, the results suggest that an elevated endogenous NO production enhances the influx of Ca(2+) via the hippocampal L-type Ca(2+) channel by S-nitrosylation during an initial phase of energy deprivation.     

Nitric oxide inhibits nociceptive transmission by differentially regulating glutamate and glycine release to spinal dorsal horn neurons

Nitric oxide (NO) is involved in many physiological functions, but its role in pain signaling remains uncertain. Surprisingly, little is known about how endogenous NO affects excitatory and inhibitory synaptic transmission at the spinal level. Here we determined how NO affects excitatory and inhibitory synaptic inputs to dorsal horn neurons using whole-cell recordings in rat spinal cord slices. The NO precursor L-arginine or the NO donor SNAP significantly increased the frequency of glycinergic spontaneous and miniature inhibitory postsynaptic currents (IPSCs) of lamina II neurons. However, neither L-arginine nor SNAP had any effect on GABAergic IPSCs. L-arginine and SNAP significantly reduced the amplitude of monosynaptic excitatory postsynaptic currents (EPSCs) evoked from the dorsal root with an increase in paired-pulse ratio. Inhibition of the soluble guanylyl cyclase abolished the effect of L-arginine on glycinergic IPSCs but not on evoked monosynaptic EPSCs. Also, inhibition of protein kinase G blocked the increase in glycinergic sIPSCs by the cGMP analog 8-bromo-cGMP. The inhibitory effects of L-arginine on evoked EPSCs and high voltage-activated Ca(2+) channels expressed in HEK293 cells and dorsal root ganglion neurons were abolished by blocking the S-nitrosylation reaction with N-ethylmaleimide. Intrathecal injection of L-arginine and SNAP significantly increased mechanical nociceptive thresholds. Our findings suggest that spinal endogenous NO enhances inhibitory glycinergic input to dorsal horn neurons through sGC-cGMP-protein kinase G. Furthermore, NO reduces glutamate release from primary afferent terminals through S-nitrosylation of voltage-activated Ca(2+) channels. Both of these actions probably contribute to inhibition of nociceptive transmission by NO at the spinal level.     

Vaso-occlusion in Schimke-immuno-osseous dysplasia: is the NO pathway involved?

Schimke-immuno-osseous dysplasia (SIOD) is an autosomal recessive disorder with the main clinical findings of spondyloepiphyseal dysplasia, nephrotic syndrome, and defective cellular immunity. Vaso-occlusive processes, especially generalized atherosclerosis, are a life-limiting complication in patients with severe SIOD. The nitric oxide synthase (NOS) oxidizes L-arginine to nitric oxide (NO). NO is a potent vasodilator with inhibitory effects on platelet aggregation and the development of atherosclerosis. We hypothesized that reduced NO production due to antagonism of NOS by asymmetric dimethylarginine (ADMA) would be a possible pathophysiological mechanism for vaso-occlusion in SIOD. We tested this hypothesis in 10 patients with SIOD and 10 age-matched healthy controls. Plasma and urine levels of nitrite and nitrate, the indicators of NO synthesis, and of ADMA, an endogenous NOS inhibitor, in children suffering from SIOD were not significantly different from those in the age-matched healthy controls. Our results suggest that the L-arginine/NO pathway is not altered in SIOD. Antagonism of NOS by ADMA does not seem to be the cause of premature general atherosclerosis in SIOD. The underlying pathology of vaso-occlusion in SIOD still remains unclear.     

L-arginine immunoreactive enteric glial cells in the enteric nervous system of rat ileum

L-arginine is a precursor of nitric oxide (NO) that may be involved in neuronal activity in the gastrointestinal tract. It is known that NO is formed from L-arginine by NO synthase which is localized in neurons in the enteric nervous system. The present study demonstrated that significant L-arginine immunoreactivity was present in the enteric ganglia. Ultrastructural examination showed that L-arginine immunoreactivity was present in the ganglionic glial cells but not in neurons. These findings suggest that enteric glial cells may represent the main reservoir of L-arginine, which may possibly be transferred to neurons when used.     

Nitric oxide synthase from cerebellum catalyzes the formation of equimolar quantities of nitric oxide and citrulline from L-arginine.

This study examined whether constitutive nitric oxide (NO) synthase from rat cerebellum catalyzes the formation of equimolar amounts of NO plus citrulline from L-arginine under various conditions. Citrulline was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine. NO was determined by monitoring the formation of total NOx (NO+nitrite [NO2-] + nitrate [NO3-]) by chemiluminescence after reduction of NOx to NO by acidic vanadium (III). Equal quantities of NO plus citrulline were generated from L-arginine and the formation of both products was linear for about 20 min at 37 degrees C provided L-arginine was present in excess to maintain a zero order reaction rate. Deletion of NADPH, addition of the calmodulin antagonist calmidazolium, or addition of NO synthase inhibitors (NG-methyl-L-arginine, NG-amino-L-arginine) abolished or markedly inhibited the formation of both NO and citrulline. The Km for L-arginine (14 microM; 18 microM) and the Vmax of the reaction (0.74 nmol/min/mg protein; 0.67 nmol/min/mg protein) were the same whether NO or citrulline formation, respectively, was monitored. These observations indicate clearly that NO and citrulline are formed in equimolar quantities from L-arginine by the constitutive isoform of NO synthase from rat cerebellum.     

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.     

Influence of nitric oxide on the activity of cuneate neurons in the rat

Some neurons of main and external cuneate nuclei are immunoreactive for nitric oxide (NO) synthase, suggesting a role for endogenous NO in the early stages of somatosensory processing. We tested this hypothesis by investigating the possibility that NO modulates cuneate discharge. We observed that both spontaneous and N-methyl-D-aspartate-evoked activities of cuneate neurons were decreased by NO precursor L-arginine. The inhibition of NO synthase, by application of N-nitro-L-arginine methyl ester, instead, abolished the depressant effect induced by L-arginine. Our data suggest a NO modulation of cuneate neurons and provide support for a physiologic role not only in increasing the signal-to-noise ratio in the excited cells but also in a form of surround inhibition.     

Role of nitric oxide in lower esophageal sphincter relaxation to swallowing.

Studies were performed in the opossum to define the role of the L-arginine-nitric oxide (NO) pathway in lower esophageal sphincter (LES) relaxation to swallowing and vagal stimulation in viv and intramural nerve stimulation in vitro. In vivo, L-NAME, a water soluble NO synthase (NOS) inhibitor, caused antagonism of LES relaxation due to reflex-induced swallowing. L-NAME (20 mg/kg i.v.) reduced the amplitude of swallow induced relaxation from 88% to 28%. LES relaxation due to electrical stimulation of peripheral end of decentralized vagus nerve was also antagonized. The effects of L-NAME were reversed by L-arginine, but not by D-arginine. L-NAME treatment did not antagonize LES relaxation to intravenous administration of isoproterenol. In vitro, NO and sodium nitroprusside (SNP) caused a decrease in the sphincter tone. The relaxing effect caused by NO and SNP was not antagonized by tetrodotoxin or omega-conotoxin. Inhibitors of NO synthase, L-NMMA and L-NNA, caused slight increase in the spontaneous resting LES tone and concentration-dependent antagonism of electrical field stimulation (EFS) induced LES relaxation. L-NNA (10(-4)M) abolished EFS induced LES relaxation at low frequencies (less than 5 Hz) and antagonized the relaxation to a value 20% of the control at 20 Hz. The antagonistic action of L-NMMA and L-NNA was unaffected by D-arginine but was reversed by L-arginine. The inhibitory effect of NO, SNP, or two other putative inhibitory neurotransmitters (VIP and CGRP) on the LES was not antagonized by L-NNA. These studies show that inhibitors of NO synthase selectively antagonize LES relaxation to all three modes of intramural inhibitory nerve stimulation including physiological swallowing. These studies suggest that the L-arginine-nitric oxide pathway is involved in physiological relaxation of the LES.     

Opposite effects of galectin-1 on alternative metabolic pathways of L-arginine in resident,inflammatory, and activated macrophages

Recent evidence has implicated galectins and their carbohydrate ligands as master regulators of the inflammatory response. Galectin-1, a member of this family, has shown specific anti-inflammatory and immunoregulatory effects. To gain insight into the potential mechanisms involved in these effects, we investigated the effects of galectin-1 in L-arginine metabolism of peritoneal rat macrophages. Pretreatment of macrophages with galectin-1 resulted in a dose- and time-dependent inhibition of lipopolysaccharide-induced nitric oxide (NO) production, accompanied by a decrease in inducible nitric oxide synthase (iNOS) expression (the classic pathway of L-arginine). On the other hand, galectin-1 favored the balance toward activation of L-arginase, the alternative metabolic pathway of L-arginine. Inhibition of NO production was not the result of increased macrophage apoptosis because addition of this beta-galactoside-binding protein to macrophages under the same experimental conditions did not affect the apoptotic threshold of these cells. To understand how endogenous galectin-1 is regulated in macrophages under inflammatory stress, we finally explored the ultrastructural distribution, expression, and secretion of galectin-1 in resident, inflammatory, and activated macrophages. This study provides an alternative cellular mechanism based on the modulation of L-arginine metabolism to understand the molecular basis of the anti-inflammatory properties displayed by this carbohydrate-binding protein.     

Effect of Nitroprusside (Nitric Oxide) on Endogenous Dopamine Release from Rat Striatal Slices

It is becoming apparent that the synthesis of nitric oxide (NO) from L-arginine not only explains endothelium-dependent vascular relaxation, but is a widespread mechanism for the regulation of cell function and communication. We examined the role of NO on the endogenous dopamine (DA) release from rat striatum. Nitroprusside, in the concentration range of 3-100 microM, induced a dose-dependent increase in the endogenous DA release from rat striatal slices. The maximal response was 330% over the baseline release. A higher concentration of nitroprusside (300 microM) produced an inhibitory effect on the spontaneous release of DA. L-Arginine (10 and 100 microM), a substrate in the NO-forming enzyme system, also produced an elevation of DA release. L-Arginine-induced DA release was attenuated by NG-monomethyl-L-arginine, an inhibitor of NO synthase. NADPH (1 microM), a cofactor of NO synthase, enhanced L-arginine-induced DA release. These results suggest a possible involvement of NO in the DA release process in rat striatum.     

The nitric oxide pathway in the cardiovascular system

The present review analyzes the role nitric oxide (NO) plays in the homeostasis of the cardiovascular system. By regulating vascular smooth muscle cell and myocyte contractility, myocardial oxygen consumption and renal tubular transport, this simple molecule plays a central role in the control of vascular tone, cardiac contractility and short and long term regulation of arterial pressure. Fifteen years ago, all we knew about NO is that it had very similar properties as those of endothelium-derived relaxing factor and that its action was probably mediated by cGMP. An enormous amount of knowledge has since been amassed on the biochemical pathways that NO follows from the moment it is synthesized from L-arginine until the physiological or pathological actions take place in the effector cells. This review intends to organize this knowledge in a fashion that is easy to understand. We will dissect the NO pathway in different steps, focusing on the physiological and pathophysiological actions of the isoenzymes which synthesize NO, the molecules involved in this synthesis such as caveolins, protein kinases and cofactors, the situations in which endogenous inhibitors of NO synthase are formed from L-arginine instead of NO, the way in which NO exerts its physiological actions through cGMP-dependent protein kinases and finally, the pathological routes NO may follow when the oxidative status of the cell is high.     

Methods using stable isotopes to measure nitric oxide (NO) synthesis in the L-arginine/NO pathway in health and disease

Nitric oxide (NO) is an important gaseous radical involved in many physiological processes. It is produced from the amino acid L-arginine by the action of nitric oxide synthases (NOS) in what is called the L-arginine/NO pathway. Tracking its metabolic fate in biological fluids is of particular interest as it may indicate how the human body responds in health and disease. However, due to its short life span (a few seconds) it is very difficult to accurately monitor any up- or down-regulation in body fluids in vivo. As a consequence, methods have been developed based on the measurement of the NO-derived products nitrite and nitrate or on the substrate of NO, L-arginine and its simultaneously generated product, L-citrulline. Considering only a fraction of the endogenous L-arginine pool is used for the synthesis of NO, NO-production cannot be estimated by measuring changes in the concentrations of L-arginine and/or L-citrulline alone. Instead, to estimate NO-related changes in the L-arginine and/or L-citrulline pools a form of tagging these metabolites for the NOS-mediated reaction is required. The application of stable isotopes is an elegant way to track NOS-mediated changes. The present paper is focussed on the application of various combinations of chromatography and mass spectrometry to measure isotopic enrichments resulting from the conversion of L-arginine to NO and L-citrulline in a one-to-one stoichiometry. In addition, the various aspects and principles involved in the application of stable isotopes in metabolic studies in general and the study of the activity of NOS in particular are discussed.     

Induction of nitric oxide synthase is a necessary precondition for expression of tumor necrosis factor-independent tumoricidal activity by activated macrophages.

Various bacteria and bacterial products induce in pure, lymphocyte-free bone marrow-derived mononuclear phagocytes (BMM?) the generation of tumor necrosis factor, nitric oxide (NO) synthase, NO and nitrite (NO2-), the flow of L-arginine to citrulline, and tumoricidal activity. The flow of L-arginine to citrulline and formation of NO/NO2- on the one hand and expression of tumoricidal activity were not always closely related; however, these parameters were suppressed in a dose-dependent manner by the flavoprotein inhibitor, diphenyleneiodonium (DPI) and the L-arginine analogue, NG-monomethyl-L-arginine (NMMA). The findings support the concept of a central role of the NO synthase pathway in the generation of tumor necrosis factor-independent tumoricidal activity by activated macrophages but the exact conditions which enable the transfer of the lytic principle from the effector to the target cell remain to be elucidated.     

Bradykinin and ATP stimulate L-arginine uptake and nitric oxide release in vascular endothelial cells.

The effects of bradykinin and ATP on L-arginine transport and nitric oxide (NO) production were studied in porcine aortic endothelial cells cultured and perfused on microcarriers and deprived of L-arginine for 24 h. Stimulation of cells with bradykinin (100 nM) or ATP (100 microM) resulted in a rapid increase in L-arginine uptake and NO release. In the presence of nitro-L-arginine (100 microM), an inhibitor of NO synthase, the stimulatory effect of bradykinin on L-arginine uptake was partially inhibited while NO release was completely abolished. Nitro-L-arginine alone was not an inhibitor of basal L-arginine transport, suggesting that its inhibitory action was not directly on the L-arginine transporter but a result of the inhibition of NO generation. These data indicate that during agonist-stimulated NO production there is a concomitant increase in the transport of L-arginine into endothelial cells providing a mechanism for the continual generation of NO.