Arginine pathways and the inflammatory response: Interregulation of nitric oxide and polyamines: Review article

Summary. An early response to an acute inflammatory insult, such as wound healing or experimental glomerulonephritis, is the conversion of arginine to the cytostatic molecule nitric oxide (NO). This anti-bacterial phase is followed by the conversion of arginine to ornithine, which is the precursor for the pro-proliferative polyamines as well as proline for the production of extracellular matrix. This latter, pro-growth phase constitutes a repair phase response. The temporal switch of arginine as a substrate for the cytostatic iNOS/NO axis to the pro-growth arginase/ ornithine/polyamine and proline axis is subject to regulation by inflammatory cytokines as well as interregulation by the arginine metabolites themselves. Arginine is also the precursor for another biogenic amine, agmatine. Here we describe the capacity of these three arginine pathways to interregulate, and propose a model whereby agmatine has the potential to serve in the coordination of the early and repair phase pathways of arginine in the inflammatory response by acting as a gating mechanism at the transition from the iNOS/NO axis to the arginase/ODC/polyamine axis. Due to the pathophysiologic and therapeutic potential, we will further examine the antiproliferative effects of agmatine on the polyamine pathway.     

Expression of the kynurenine enzymes in macrophages and microglial cells: regulation by immune modulators

Summary The regulation of the expression of indoleamine 2,3-dioxygenase (IDO) was studied in cloned murine macrophages (MT2) and microglial (N11) cells. Both cell lines express IDO and inducible nitric oxide synthase activity after interferon- (IFN-) stimulation. The regulation of IDO expression appears to differ in the two cell lines. Nitric oxide (NO) production negatively modulates the expression of IDO activity in IFN--primed macrophages, thereby indicating a cross-talk between the kynurenine and nitridergic pathways in these cells. Conversely, this down-regulation of IDO activity by NO does not occour in microglial cells. A differential regulation of IDO expression in the two cell lines was also observed with LPS and picolinic acid. Together with previous findings, these results indicate the existence of marked differences in the regulation of the expression of the kynurenine pathway enzymes between macrophages and microglial cells.Abbreviation used IFN- interferon- - IDO indoleamine 2,3-dioxygenase - NO nitric oxide - iNOS inducible nitric oxide synthase - NAME N-())-nitro-L-arginine methyl ester - SMTC S-methyl-L-thiocitrulline - BNI 3-bromo-7-nitroindazole - PIC picolinic acid - IL interleukin     

Arginine catabolism in Aphanocapsa 6308

The catabolic products of arginine metabolism were observed in Aphanocapsa 6308, a unicellular cyanobacterium, by thin layer chromatography of growth media, by limiting growth conditions, and by enzymatic analysis. Of the organic, nitrogenous compounds examined, only arginine supported growth in CO₂-free media. The excretion of ornithine at a concentration level greater than citrulline suggested the existence in Aphanocapsa 6308 of the arginine dihydrolase pathway which produced ornithine, CO₂, NH₄, + adenosine 5-triphosphate. Its existence was confirmed by enzymatic analysis. Although cells could not grow on urea as a sole carbon source a very active urease and subsequently an arginase were also demonstrated, indicating that Aphanocapsa can metabolize arginine via the arginase pathway. The level of enzymes for both pathways indicates a lack of genetic control. It is suggested that the arginase pathway provides only nitrogen for the cells whereas the arginine dihydrolase pathway provides not only nitrogen, but also CO₂ and adenosine 5-triphosphate.Nonstandard Abbreviations CCCP carbonylcyanide mchlorophenyl hydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - CGP cyanophycin granule protein - PS II photosystem II - PSI photosystem I - TLC thin layer chromatography - TCA trichloroacetic acid - DPM disintegrations per min     

A role for guanidino compounds in the brain

Guanidino compounds of guanidinoethanesulfonic acid, guanidinoacetic acid, guanidinosuccinic acid, N-acetylarginine, -guanidinopropionic acid, creatinine, -guanidinobutyric acid, arginine, guanidine, methylguanidine, homoarginine and -guanidinoglutaric acid are present in the mammalian brain. These guanidino compounds except for arginine and guanidine induce seizures and convulsions in rat, rabbit and cat by intracisternal injection.Hirudonine, audonine, -keto--guanidinovaleric acid, N,N-dibenzoylguanidine and phenylethylguanidine are also convulsants. Levels of creatinine, guanidinoethanesulfonic acid, creatinine, guanidinoacetic acid and methylguanidine in animal brain were changed at pre- and during convulsions induced by pentylentetrazol, amygdala kindling, iron-induced epileptogenesis and so on. These convulsions are thought to be due to depressed functions of serotonergic neurons and accumulated free radicals.Arginine is a substrate of nitric oxide production by nitric oxide synthase. -Guanidinoglutaric acid is a generator of superoxide, hydroxyl radicals and nitric oxide, and induced C6 glial cell death. On the other hand, aminoguanidine is a free radical scavenger. Energy formation by creatine metabolism may inhibit apoptosis induced by pathogenesis. Free radical generation/reaction and energy generation by guanidino compounds must be important key role in the brain.     

Comparative studies on the localization of esteroproteases and kallikrein-like activity in primate organs

Summary Various organs of three species of monkey were screened histochemically for esteroproteases usingN-acethyl-l-methionine--naphthylester ( N-O-met) as the substrate and also for enzymes with kallikrein-like activity usingd-Val-Leu-Arg-4-methoxy-2-naphthylamide as the substrate. Characteristic differences were found in the localization of the reaction products obtained with both substrates. In the main salivary glands, esteroproteases ( N-O-met reactivity) were found in mucous cells (submandibular gland), intercalated duct cells (parotid gland), acinar cells (sublingual gland), striated and interlobular duct cells (all glands). They were also localized in superficial lining epithelial cells of the digestive system, in liver cells, and acinar cells of the pancreas.Enzymes with kallikrein-like activity were found only in the striated and interlobular duct cells of salivary glands, in acinar cells of the pancreas, and in proximal tubular cells of the kidney. Free cells (including mast cells) normally distributed in the connective tissue of various organs showed reactivity towards N-O-met. Some of these cells were also reactive against Val-Leu-Arg-4-MNA.     

Utilization of 11β-hydroxysteroids by the salivary gland ducts

Summary Parotid, submandibular and sublingual glands were removed from rats and investigated histochemically. Pyruvate oxidase, iso-citric dehydrogenase, -ketoglutarate oxidase, succinic dehydrogenase, malate dehydrogenase and furfuryl alcohol dehydrogenase activity were observed in the salivary ducts which may be interpreted as significant of high metabolic activity.The 11 -hydroxysteroid dehydrogenase in these ducts displayed marked substrate specificity utilizing 11-hydroxyandrostenedione and cortisol but not 11 -hydroxyoestrone or 11 -hydroxyprogesterone. The relationship between corticosteroids and salivary electrolyte concentrations is discussed.     

Effects of nitric oxide synthase inhibitors on systemic hypotension, cytokines and inducible nitric oxide synthase expression and lung injury following endotoxin administration in rats

Endotoxin shock is characterized by systemic hypotension, hyporeactiveness to vasoconstrictors and acute lung edema. A nitric oxide synthase (NOS) inhibitor, N^G-monomethyl-L-arginine (L-NMMA) has been shown to be effective in reversing acute lung injury. In the present study, we evaluated the effects of NOS blockade by different mechanisms on the endotoxin-induced changes. In anesthetized rats, lipopolysaccharide (LPS,Klebsiella pneumoniae) was administered intravenously in a dose of 10 mg/kg. LPS caused sustained systemic hypotension accompanied by an eightfold increase of exhaled NO during an observation period of 4 h. After the experiment, the lung weight was obtained and lung tissues were taken for the determination of mRNA expressions of inducible NOS (iNOS), interleukin-1 (IL-1) and tumor necrosis factor--(TNF-). Histological examination of the lungs was also performed. In the control group injected with saline solution, mRNA expressions of iNOS, IL-1 and TNF- were absent. Four hours after LPS, the mRNA expressions of iNOS and IL-1 were still significantly enhanced, but TNF- was not discernibly expressed. LPS also caused a twofold increase in lung weight. Pathological examination revealed endothelial damage and interstitial edema. Various NOS inhibitors were given 1 h after LPS administration. These agents included N-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg), a constitutive NOS and iNOS inhibitor; S,S-1,4-phenylene-bis-(1,2-ethanedinyl) bis-isothiourea dihydrobromide (1,4-PBIT, 10 mg/kg), a relatively specific iNOS inhibitor, and dexamethasone (3 mg/kg), an inhibitor of iNOS expression. These NOS inhibitors all effectively reversed the systemic hypotension, reduced the exhaled NO concentration and prevented acute lung injury. The LPS-induced mRNA expressions of iNOS and IL-1 were also significantly depressed by these NOS inhibitors. Our results suggest that NO production through the iNOS pathway is responsible for endotoxin-induced lung injury. Certain cytokines such as IL-1 are possibly involved. These changes are minimized by NOS inhibitors through different mechanisms.     

Effect of α-amanitine on puffing and intranuclear RNA synthesis in Chironomus salivary glands

Incubation of Chironomus salivary glands with -amanitine in concentrations from 1 to 10 /ml results in the suppression of puffing and chromosomal ³H-uridine incorporation after 30 to 60 min in 80% of the cells. Nucleolar ³H-uridine incorporation remains completely unaffected. Even 4 h after the injection of high doses of -amanitine into living larvae, nucleolar incorporation is still pronounced. The distribution of resistant cells within the salivary glands suggests that the uptake of -amanitine is subject to physiological restrictions.—A puff typically induced during in vitro incubation of salivary glands was found to be less sensitive to -amanitine than the Balbiani rings.     

Inhibition of nitric oxide synthase activity in cerebral cortical synaptosomes by nitric oxide donors: Evidence for feedback autoregulation

Despite evidence which supports a neurotransmitter-like role for nitric oxide (NO) in the CNS, relatively little is known regarding mechanisms which control NO formation within CNS neurons. In this study, isolated nerve endings (synaptosomes) from rat cerebral cortex were used to ascertain whether NO can autoregulate its own formation within neurons through feedback inhibition of the NO biosynthetic enzyme nitric oxide synthase (NOS). Under the conditions described here, N-nitro-l-arginine methyl ester-sensitive conversion ofl-[³H]arginine intol-[³H]citrulline (i.e., NOS activity) was found to be highly calcium-dependent and strongly inhibited (up to 60 percent) by NO donors, including sodium nitroprusside, hydroxylamine and nitroglycerin. The inhibitory effect of sodium nitroprusside was concentration-dependent (IC₅₀100 M) and prevented by the NO scavenger oxyhemoglobin.l-Citrulline, the other major end-product from NOS, had no apparent effect on synaptosomal NOS activity. Taken together, these results indicate that neuronal NOS can be inhibited by NO released from exogenous donors and, therefore, may be subject to end-product feedback inhibition by NO that is formed locally within neurons or released from proximal cells.     

The Inhibitory Effects of Nω-Nitro-L-Arginine Methyl Ester on Nitric Oxide Synthase Activity Vary Among Brain Regions in Vivo but Not in Vitro

We studied the effects of intracerebroventricular and intraperitoneal injection and the in vitro effects of N-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, on the nitric oxide synthase activities of the cerebellum, brainstem, hypothalamus, hippocampus, and the remainder of the brain after dissections. Male rats were chronically implanted with lateral icv guide cannula. L-NAME was injected in doses of 0.2, 1, and 5 mg intracerebroventricularly, and 50 mg/kg intraperitoneally. L-NAME induced dose-dependent suppression of NOS activities in each brain region. The threshold dose was 0.2 mg; 1 mg L-NAME completely abolished brain nitric oxide synthase activity 90 min after the injection. Brain NOS activities returned to baseline level 48 h after the injection of 5 mg L-NAME. There were significant differences between the sensitivity of various regions to L-NAME after in vivo but not in vitro administration of the enzyme inhibitor. These findings indicate that intracerebroventricular injection of L-NAME is a useful tool for inhibiting brain nitric oxide synthase activities in vivo. The differences between the sensitivity of different brain regions to L-NAME as well as the relative fast recovery of nitric oxide synthase activities must be taken into account when L-NAME is administered intracerebroventricularly to rats.     

Nitric oxide synthase in human salivary glands

The distribution of the three nitric oxide synthase (NOS) isoforms was determined immunohistochemically in the human minor and major salivary glands with comparison to that of rat salivary glands. In contrast to rat glands, which contained a dense plexus of neuronal NOS-immunoreactive nerve fibers, only a minority of the nerve fibers in human glands showed neuronal NOS immunoreactivity. Human labial and submandibular glands contained sparse NOS-immunoreactive fibers, while only occasional nerve fibers in the parotid or sublingual glands were stained. Furthermore, in contrast to the animal glands, most duct epithelial cells in all human salivary glands were immunoreactive for neuronal NOS. No specific immunoreactivity for inducible or endothelial NOS were observed in the nerve fibers or duct epithelium. We provide evidence to suggest that the role of nitric oxide in the regulation of salivary gland function is different in human as compared to experimental animals. Nitricergic innervation in human tissue is very sparse and thus nitric oxide is probably of minor importance as a neural regulator of salivary glands. Instead, NOS localized in duct epithelial cells suggests that nitric oxide might directly regulate saliva secretion and it is a putative source of nitrates previously reportedly secreted into the saliva.     

Electron microscopic localization of 5′-nucleotidase in rat salivary glands

Summary The localization of 5-nucleotidase in rat parotid and submandibular glands was investigated at the electron microscope level by an immunohistochemical technique using a highly specific antibody, and the results were compared with those obtained using the newley developed cerium method for enzyme histochemistry. Both methods demonstrated that 5-nucleotidase is located on the external surface of the luminal plasma membranes of acinar cells as well as on intercalated and striated ductal cells. In the basolateral membranes of these cells, the portions adjacent to myoepithelial cells exhibited intense reaction products, but the other areas of plasma membranes contained only trace amounts of the reaction products. Both cerium-based enzyme histochemistry and immunohistochemistry showed that myoepithelial cells retain the enzyme on their plasma membranes. Neither method produced reaction products in the intracytoplasmic structure of constitutive cells of the salivary glands. We discuss the usefulness of the cerium-ion method for the demonstration of 5-nucleotidase activity and compare it with the traditional lead-ion method.     

Nitric oxide synthase isoforms I, III and protein kinase-Ctheta in skeletal muscle fibres of normal and streptozotocin-induced diabetic rats with and without Ginkgo biloba extract treatment

The expression of nitric oxide synthase (NOS) isoforms I, III and protein kinase-C (PKC) in rat vastus lateralis muscle was demonstrated immunohistochemically and then correlated to the physiological metabolic fibre types: SO (slow-oxidative), FOGI, FOGII (fast-oxidative glycolytic; I more glycolytic, II more oxidative), and FG (fast-glycolytic). NOS expression in muscles from different experimental groups (normal and diabetic rats, with and without Ginkgo biloba extract treatment) was assayed by Western blotting. Generally, NOS I and PKC were co-expressed in fibres with predominantly oxidative metabolism (SO, FOGII). This suggests an interplay of PKC and NOS I in nitric oxide production by oxidative fibres. NOS III was more highly expressed in fibres with predominantly glycolytic metabolism (FOGI, FG). A somewhat lower NOS I immunoreactivity was also found in NOS III positive fibres suggesting that NOS III and NOS I are co-expressed in these fibres. Western blotting revealed that NOS I as well as NOS III expression in the vastus lateralis muscle was down-regulated in diabetes and increased after Ginkgo biloba extract treatment. These effects may be associated with a diminished glucose uptake by myocytes of diabetic muscles and with an improved muscle function after Ginkgo biloba treatment.     

Evidence for coexistence of ATP and nitric oxide in non-adrenergic,non-cholinergic (NANC) inhibitory neurones in the rat ileum,colon and anococcygeus muscle

The possible coexistence of the two non-adrenergic, non-cholinergic (NANC) inhibitory neurotransmitters, adenosine 5-triphosphate and nitric oxide in the myenteric plexus was investigated using whole-mount preparations of rat ileum, proximal colon and anococcygeus muscle. The presence of adenosine 5-triphosphate in neurones was examined using the quinacrine fluorescence technique. After localizing and taking photographs of quinacrine-fluorescent neurones and nerve fibres, the same tissues were then fixed and processed for NADPH-diaphorase activity, a marker for nitric oxide-containing neurones. We have demonstrated for the first time that almost all quinacrine-fluorescent myenteric neurones in the proximal colon are also NADPH-diaphorase reactive, while only a subpopulation of quinacrine-fluorescent neurones in ileum and anococcygeus muscle were also NADPH-diaphorase reactive.     

Cytochemical studies on the localization of 5′-nucleotidase in the acinar cells of the rat salivary glands

Summary The cytochemical localization of 5-nucleotidase (5-AMPase), and its validity, were investigated in parotid and submandibular acinar cells of a rat. Biochemical determinations showed that adequate treatment with glutaraldehyde could minimize the loss of enzymatic activity, and that 5-AMPase and non-specific alkaline phosphatase (-GPase) possessed different pH optima.The cytochemical distribution of the reaction products from the 5-AMPase activity was distinct from those of -GPase. 5-AMPase activity was localized on the surface membranes of acinar, ductal and myoepithelial cells of both salivary glands. -GPase activity was evenly distributed on the entire plasma membranes of myoepithelial cells and on the basal plasmalemma of acinar cells. The reaction products, which appeared on the luminal and lateral plasma membranes of the acinar cells, were presumed to reflect the presence of 5-AMPase, while those on the myoepithelial surface and basal plasma membranes of the acinar cells demonstrated both 5-AMPase and -GPase.The results indicate that 5-AMPase activity can be utilized as a reliable marker enzyme of plasma membranes in the salivary acinar cells.     

Superoxide, nitric oxide, peroxynitrite and cytokine combinations all cause functional impairment and morphological changes in rat islets of Langerhans and insulin secreting cell lines, but dictate cell death by different mechanisms

We have shown that nitric oxide treatment for 30–90 min causes inhibition of insulin secretion, DNA damage and disturbs sub-cellular organization in rat and human islets of Langerhans and HIT-T15 cells. Here rat islets and beta-cell lines were treated with various free radical generating systems S-nitrosoglutathione (nitric oxide), xanthine oxidase plus hypoxanthine (reactive oxygen species), 3-morpholinosydnonimine (nitric oxide, super-oxide, peroxynitrite, hydrogen peroxide) and peroxynitrite and their effects over 4 h to 3 days compared with those of the cytokine combination interleukin-1, tumour necrosis factor- and interferon-. End points examined were de novo protein synthesis, cellular reducing capacity, morphological changes and apoptosis by acridine orange cytochemistry, DNA gel electrophoresis and electron microscopy. Treatment (24–72 h) with nitric oxide, superoxide, peroxynitrite or combined cytokines differentially decreased redox function and inhibited protein synthesis in rat islets of Langerhans and in insulin-containing cell lines; cytokine effects were arginine and nitric oxide dependent. Peroxynitrite gave rare apoptosis in HIT-T15 cells and superoxide gave none in any cell type, but caused the most beta cell-specific damage in islets. S-nitroso-glutathione was the most effective agent at causing DNA laddering or chromatin margination characteristic of apoptotic cell death in insulin-containing cells. Cytokine-induced apoptosis was observed specifically in islet beta cells, combined cytokine effects on islet function and death most resembled those of the mixed radical donor SIN-1.     

The Effects of Caffeine on <Emphasis Type="SmallCaps">l</Emphasis>-Arginine Metabolism in the Brain of Rats

In our study, the short-term effects of caffeine on L- arginine metabolism in the brains of rats were investigated. Caffeine was given orally at two different doses: 30 mg/kg and 100 mg/kg (a high non-toxic dose). Brain tissue arginase activity in rats from the caffeine-treated groups decreased significantly compared with the control group. Malondialdehyde (MDA) levels in the brain tissue and serum of animals in the caffeine groups also decreased significantly. Brain tissue and serum nitric oxide (NO) levels increased significantly after caffeine administration. Tumor necrosis factor-α (TNF-α) levels were also investigated in rat serum, but there was no statistically significant difference between the TNF-α levels of the caffeine-treated rats groups and the control rats. Our study indicates that brain arginase activity decreases after caffeine administration at doses of 30 mg/kg and 100 mg/kg. As a result, we can say that arginine induces production of NO in the organism.     

Guanosine stimulates neurite outgrowth in PC12 cells via activation of heme oxygenase and cyclic GMP

Undifferentiated rat pheochromocytoma (PC12) cells extend neurites when cultured in the presence of nerve growth factor (NGF). Extracellular guanosine synergistically enhances NGF-dependent neurite outgrowth. We investigated the mechanism by which guanosine enhances NGF-dependent neurite outgrowth. Guanosine administration to PC12 cells significantly increased guanosine 3,5-cyclic monophosphate (cGMP) within the first 24 h whereas addition of soluble guanylate cyclase (sGC) inhibitors abolished guanosine-induced enhancement of NGF-dependent neurite outgrowth. sGC may be activated either by nitric oxide (NO) or by carbon monoxide (CO). -Nitro-l-arginine methyl ester (l-NAME), a non-isozyme selective inhibitor of nitric oxide synthase (NOS), had no effect on neurite outgrowth induced by guanosine. Neither nNOS (the constitutive isoform), nor iNOS (the inducible isoform) were expressed in undifferentiated PC12 cells, or under these treatment conditions. These data imply that NO does not mediate the neuritogenic effect of guanosine. Zinc protoporphyrin-IX, an inhibitor of heme oxygenase (HO), reduced guanosine-dependent neurite outgrowth but did not attenuate the effect of NGF. The addition of guanosine plus NGF significantly increased the expression of HO-1, the inducible isozyme of HO, after 12 h. These data demonstrate that guanosine enhances NGF-dependent neurite outgrowth by first activating the constitutive isozyme HO-2, and then by inducing the expression of HO-1, the enzymes responsible for CO synthesis, thus stimulating sGC and increasing intracellular cGMP.     

Do vasoactive intestinal peptide (VIP)-and nitric oxide synthase-immunoreactive terminals synapse exclusively with VIP cell bodies in the submucous plexus of the guinea-pig ileum?

In the submucous plexus of the guinea-pig ileum, previous light-microscopic studies have revealed that vasoactive intestinal peptide (VIP)-immunoreactive and nitric oxide synthase (NOS)-immunoreactive terminals are found predominantly in association with VIP-immunoreactive nerve cell bodies. In this study, double-label immunohistochemistry at the light-microscopic level demonstrated co-localization of NOS-immunoreactivity and VIP-immunoreactivity in axon terminals in submucous ganglia. About 90% of nerve fibres with NOS-immunoreactivity or VIP-immunoreactivity were immunoreactive for both antigens; only about 10% of labelled varicosities contained only NOS-immunoreactivity or VIP-immunoreactivity. The VIP/NOS varicosities were more often seen in the central parts of the ganglia, close to the VIP-immunoreactive cell bodies. Ultrastructural immunocytochemistry with antibodies to VIP was used to determine if NOS/VIP terminals synapse exclusively with VIP-immunoreactive nerve cell bodies. We examined the targets of VIP-immunoreactive boutons in two submucous ganglia from different animals. Serial ultrathin sections were taken through the ganglia after they had been processed for VIP immunocytochemistry. For each cell body, the number of VIP inputs (synapses and close contacts) was determined. The number of VIP-immunoreactive synapses received by the cell bodies of submucous neurons varied from 0–4 and the number of VIP-immunoreactive close contacts varied from 3–10. There was no significant difference between VIP-immunoreactive nerve cell bodies and non-VIP nerve cell bodies in the number of VIP-immunoreactive synapses and close contacts they received. Thus, the implication from light microscopy that NOS/VIP terminals end predominantly on VIP nerve cells was not vindicated by electron microscopy.Abbreviations CCK Cholecystokinin - cGMP guanosine-3, 5-cyclic monophosphate - CGRP calcitonin gene-related peptide - ChAT choline acetyltransferase - DYN dynorphin - GAL galanin - GTP guanosine triphosphate - IR immunoreactive(ivity) - NO nitric oxide - NOS nitric oxide synthase - NMU neuromedin U - NPY neuropeptide Y - SOM somatostatin - SP substance P - VIP vasoactive intestinal peptide