High Concentrations Of L-arginine Research Articles

The Effects of L-Arginine and Nitric Oxide Donors on the Induction of Mitochondrial Permeability Transition Pore by Calcium Ions and Palmitoylcarnitine. Possible Involvement of Mitochondrial NO/cGMP/PKG Signaling System

This study was focused on the mechanisms of the protective effects of L-arginine and the nitric oxide donor SNAP under the induction of cyclosporine A-dependent mitochondrial permeability transition pore (MPTP) by excess of Ca2+ or D,L-palmitoylcarnitine (PC). The possible involvement of a signaling system that included mitochondrial enzymes NO-synthase (mtNOS), guanylate cyclase (GC), and cGMP-dependent protein kinase G (PKG) (mtNOS/GC/PKG-SS) in the regulation of MPTP was investigated. The experiments were performed on isolated liver mitochondria using inhibitory analysis. The results of the study have shown that L-arginine and SNAP caused a dose-dependent effects on the opening of MPTP. L‑Arginine and SNAP at low concentrations (5–100 μM) activated protective mechanisms, providing an increase in the mitochondrial calcium buffer capacity (CRC) and the critical (threshold) concentration of palmitoylcarnitine (PC*) required for MPTP induction. At higher concentrations, L-arginine (more than 500 μM) and SNAP (more than 100 μM) caused the opposite effect, that is, they suppressed respiration and promoted pore opening. Inhibitors of NOS, GC, and PKG eliminated the protective effects observed at low concentrations of L‑arginine and SNAP. The experiments conducted with a specific inhibitor of inducible NOS (W1400) showed that this calcium-independent enzyme did not participate in the regulation of MPTP in our experiments. Based on the results obtained, it can be assumed that the calcium-dependent mitochondrial signaling system mtNOS/GC/PKG-SS is involved in the regulation of MPTP, providing an increase in CRC and PC*. At high concentrations of L-arginine or SNAP, excess of NO overcame the protection provided by mtPKG and promoted pore opening.

Drugs linked to plasma homoarginine in chronic kidney disease patients-a cross-sectional analysis of the German Chronic Kidney Disease cohort.

Elevated plasma concentrations of symmetric and asymmetric dimethylarginine (SDMA and ADMA, respectively) and a lower plasma concentration of the structurally related homoarginine are commonly observed in patients with chronic kidney disease (CKD) and independently predict total mortality as well as progression of renal disease. We aimed to identify drugs that may alter this adverse metabolite pattern in a favourable fashion. Plasma ADMA, SDMA, homoarginine and l-arginine were determined by liquid chromatography-tandem mass spectrometry in 4756 CKD patients ages 18-74 years with an estimated glomerular filtration rate (eGFR) of 30-60 mL/min/1.73 m2 or an eGFR >60 mL/min/1.73 m2 and overt proteinuria who were enrolled in the German Chronic Kidney Disease (GCKD) study. Associations between laboratory, clinical and medication data were assessed. Intake of several commonly used drugs was independently associated with plasma concentrations of homoarginine and/or related metabolites. Among these, the peroxisome proliferator-activated receptor alpha (PPAR-α) agonist fenofibrate was associated with the most profound differences in ADMA, SDMA and homoarginine plasma concentrations: 66 patients taking fenofibrate had a multivariable adjusted odds ratio (OR) of 5.83 [95% confidence interval (CI) 2.82-12.03, P < 0.001] to have a plasma homoarginine concentration above the median. The median homoarginine plasma concentration in patients taking fenofibrate was 2.30 µmol/L versus 1.55 in patients not taking the drug (P < 0.001). In addition, fibrates were significantly associated with lower plasma SDMA and higher l-arginine concentrations. In contrast, glucocorticoids were associated with lower plasma homoarginine, with adjusted ORs of 0.52 (95% CI 0.40-0.67, P < 0.001) and 0.53 (95% CI 0.31-0.90, P = 0.018) for prednisolone and methylprednisolone, respectively. In a large cohort of CKD patients, intake of fenofibrate and glucocorticoids were independently associated with higher and lower plasma homoarginine concentrations, respectively. Effects on plasma homoarginine and methylarginines warrant further investigation as potential mechanisms mediating beneficial or adverse drug effects.

Arginine and Citrulline Catabolic Pathways Encoded by the arc Gene Cluster of Lactobacillus brevis ATCC 367.

High concentrations of l-arginine or l-citrulline in the growth medium provided the wine bacterium Lactobacillus brevis with a significant growth advantage. The arginine deiminase pathway (ADI) arc gene cluster of Lactobacillus brevis contains three genes-arcD, arcE1, and arcE2-encoding putative l-arginine/l-ornithine exchangers. Uptake experiments with Lactococcus lactis cells expressing the genes showed that all three transported l-ornithine with affinities in the micromolar range. Similarly, ArcD and ArcE2 transported l-arginine, while ArcE1 transported l-citrulline, an intermediate of the ADI pathway. Chase experiments showed very efficient exchange of l-arginine and l-ornithine by ArcD and ArcE2 and of l-citrulline and l-ornithine by ArcE1. Low affinities (millimolar range) combined with low translocation rates were found for ArcD and ArcE2 with l-citrulline and for ArcE1 with l-arginine. Resting cells of Lactobacillus brevis grown in the presence of l-arginine and l-citrulline rapidly consumed l-arginine and l-citrulline, respectively, while producing ammonia and l-ornithine. About 10% of l-arginine degraded was excreted by the cells as l-citrulline. Degradation of l-arginine and l-citrulline was not subject to carbon catabolite repression by glucose in the medium. At a high medium pH, l-citrulline in the medium was required for induction of the l-citrulline degradation pathway. Pathways are proposed for the catabolic breakdown of l-arginine and l-citrulline that merge at the level of ornithine transcarbamylase in the ADI pathway. l-Arginine uptake is catalyzed by ArcD and/or ArcE2, l-citrulline by ArcE1. l-Citrulline excretion during l-arginine breakdown is proposed to be catalyzed by ArcD and/or ArcE2 through l-arginine/l-citrulline exchange.IMPORTANCELactobacillus brevis, a bacterium isolated from wine, as well as other food environments, expresses a catabolic pathway for the breakdown of l-citrulline in the medium that consists of the l-citrulline/l-ornithine exchanger ArcE1 and part of the catabolic arginine deiminase (ADI) pathway enzymes. The proposed pathways for l-arginine and l-citrulline breakdown provide a mechanism for l-citrulline accumulation in fermented food products that is the precursor of the carcinogen ethyl carbamate.

Freeze-Drying of l-Arginine/Sucrose-Based Protein Formulations, Part 2: Optimization of Formulation Design and Freeze-Drying Process Conditions for an l-Arginine Chloride-Based Protein Formulation System

We recently reported that the presence of chloride counter ions in freeze-dried l-arginine/sucrose formulations provided the greatest protein stability, but led to low collapse temperatures and glass transition temperatures of the freeze concentrates. The objectives of this study were to identify l-arginine chloride-based formulations and optimize freeze-drying process conditions to deliver a freeze-dried product with good physical quality attributes (including cake appearance, residual moisture, and reconstitution time). Additional properties were tested such as thermal properties, cake microstructure, and protein physical stability. Excipient concentrations were varied with and without a model protein (bovine serum albumin, BSA). Formulations were frozen with and without annealing or with and without controlled nucleation. Primary drying was conducted at high and low shelf temperature. Cakes with least defects and optimum physical attributes were achieved when protein to excipient ratios were high. Controlled nucleation led to elegant cakes for most systems at a low shelf temperature. Replacing BSA by a monoclonal antibody showed that protein (physical) stability was slightly improved under stress storage temperature (i.e., 40°C) in the presence of a low concentration of l-arginine in a sucrose-based formulation. At higher l-arginine concentrations, cake defects increased. Using optimized formulation design, addition of l-arginine chloride to a sucrose-based formulation provided elegant cakes and benefits for protein stability.

A blinded, randomized study of l-arginine in small clot embolized rabbits

ObjectiveTissue plasminogen activator (tPA) is administered to acute ischemic stroke victims in a vehicle formulation containing high concentrations of l-arginine (3.5g/100mg vial), a well-known nitric oxide synthase (NOS) substrate and precursor to nitric oxide (NO), as well as an enhancer of cerebral blood flow. MethodsWe studied the effects of tPA vehicle compared to tPA (3.3mg/kg) formulated in the same vehicle containing l-arginine, normal saline or normal saline containing l-arginine, on behavioral function following small clot embolic strokes in rabbits using clinical rating scores and quantal analysis curves as the primary end point. Treatments were administered intravenously (1ml/kg; 20% bolus/80% infused over 30min) starting 1h following the injection of small-sized blood clots into the brain vasculature and terminal behavior was measured 2days following embolization. Behavioral rating scores were used to calculate the effective stroke dose (P50 in mg) that produces neurological deficits in 50% of the rabbits. ResultsIn this study, tPA significantly (p=0.001) improved behavior compared to all other treatments including tPA vehicle, saline and saline-l-arginine, increasing the P50 by 141% over tPA vehicle. Saline-l-arginine was not significantly different from either saline or tPA vehicle (p>0.05). ConclusionThis study demonstrates that the l-arginine component of the tPA vehicle does not contribute to the reproducible clinical improvement observed following tPA administration in rabbits. Moreover, the administration of l-arginine was not an effective method to promote behavioral recovery following embolic strokes in the stringent rabbit small clot stroke model, nor did l-arginine exacerbate behavioral deficits or intracerebral hemorrhage in embolized rabbits.

Amino Acidic Control of Calcium Phosphate Precipitation by Using the Vapor Diffusion Method in Microdroplets

Calcium phosphate precipitation was carried out in the presence of l-aspartic acid (l-asp, iep = 2.77), l-alanine (l-ala, iep = 6.00), and l-arginine (l-arg, iep = 10.76) at different concentrations by using a vapor diffusion sitting drop method (VDSD) in microdroplets. Irrespective of the nature and the concentration of the amino acid used, the early stage in the precipitation consisted in the formation of a white viscous suspension composed of amorphous calcium phosphate (ACP) spherulites. After 1 week, different calcium phosphate phases were found depending on the amino acid nature and concentration. At higher concentrations of l-aspartic acid, brushite (dicalcium phosphate dihydrate, DCDP) platelets and a few needle-like carbonate-hydroxyapatite (HA) crystals were found. In the presence of higher concentrations of l-alanine, the precipitate was composed of both needle-like HA and octacalcium phosphate (OCP) platelets. Finally, at higher concentrations of l-arginine, we obtained carbonate-HA nanocrysta...

L-arginine via nitric oxide is an inhibitory feedback modulator ofAplysiafeeding

An increase in L-arginine hemolymph concentration acts as a postingestion signal inhibiting Aplysia feeding. At physiological concentrations (a 10-μM increase over background), the inhibitory effect of L-arginine is too weak to block feeding in hungry animals. However, a 10-μM increase in L-arginine concentration acts along with another inhibitory stimulus, the sustained presence of food odor, to inhibit feeding after a period of access to food. A physiological concentration of L-arginine also blocked the excitatory effect of a stimulus enhancing feeding, pheromones secreted by mating conspecifics. High concentrations of L-arginine (2.5 mM) alone also inhibited ad libitum feeding. L-arginine is the substrate from which nitric oxide synthase (NOS) produces nitric oxide (NO). Both an NO donor and a 10-μM increase in L-arginine inhibited biting in response to a weak food stimulus. Treatment with NOS inhibitors initiated food-finding and biting in the absence of food, indicating that food initiates feeding against a background of tonic nitrergic inhibition. Increased feeding in response to blocking NOS is accompanied by firing of the metacerebral (MCC) neuron, a monitor of food arousal. The excitatory effect on the MCC of blocking NOS is indirect. The data suggest that L-arginine acts by amplifying NO synthesis, which acts as a background stimulus inhibiting feeding. Background modulation of neural activity and behavior by NO may also be present in other systems, but such modulation may be difficult to identify because its effects are evident only in the context of additional stimuli modulating behavior.

Functional relationship between cationic amino acid transporters and β-defensins: Implications for dry skin diseases and the dry eye

The ocular surface, constantly exposed to environmental pathogens, is particularly vulnerable to infection. Hence an advanced immune defence system is essential to protect the eye from microbial attack. Antimicrobial peptides, such as β-defensins, are essential components of the innate immune system and are the first line of defence against invaders of the eye. High concentrations of l-arginine and l-lysine are necessary for the expression of β-defensins. These are supplied by epithelial cells in inflammatory processes. The limiting factor for initiation of β-defensin production is the transport of l-arginine and l-lysine into the cell. This transport is performed to 80% by only one transporter system in the human, the y +-transporter. This group of proteins exclusively transports the cationic amino acids l-arginine, l-lysine and l-ornithine and is also known under the term cationic amino acid transporter proteins (CAT-proteins). Various infections associated with l-arginine deficiency (for example psoriasis, keratoconjuctivitis sicca) are also associated with an increase in β-defensin production. For the first time, preliminary work has shown the expression of human CATs in ocular surface epithelia and tissues of the lacrimal apparatus indicating their relevance for diseases of the ocular surface. In this review, we summarize current knowledge on the human CATs that appear to be integrated in causal regulation cascades of β-defensins, thereby offering novel concepts for therapeutic perspectives.

The Novel Imidazopyridine 2-[2-(4-Methoxy-pyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine (BYK191023) Is a Highly Selective Inhibitor of the Inducible Nitric-Oxide Synthase

We have identified imidazopyridine derivatives as a novel class of NO synthase inhibitors with high selectivity for the inducible isoform. 2-[2-(4-Methoxy-pyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine (BYK191023) showed half-maximal inhibition of crudely purified human inducible (iNOS), neuronal (nNOS), and endothelial (eNOS) NO synthases at 86 nM, 17 microM, and 162 microM, respectively. Inhibition of inducible NO synthase was competitive with l-arginine, pointing to an interaction of BYK191023 with the catalytic center of the enzyme. In radioligand and surface plasmon resonance experiments, BYK191023 exhibited an affinity for iNOS, nNOS, and eNOS of 450 nM, 30 microM, and >500 microM, respectively. Inhibition of cellular nitrate/nitrite synthesis in RAW, rat mesangium, and human embryonic kidney 293 cells after iNOS induction showed 40- to 100-fold higher IC(50) values than at the isolated enzyme, in agreement with the much higher l-arginine concentrations in cell culture media and inside intact cells. BYK191023 did not show any toxicity in various rodent and human cell lines up to high micromolar concentrations. The inhibitory potency of BYK191023 was tested in isolated organ models of iNOS (lipopolysaccharide-treated and phenylephrine-precontracted rat aorta; IC(50) = 7 microM), eNOS (arecaidine propargyl ester-induced relaxation of phenylephrine-precontracted rat aorta; IC(50) > 100 microM), and nNOS (field-stimulated relaxation of phenylephrine-precontracted rabbit corpus cavernosum; IC(50) > 100 microM). These data confirm the high selectivity of BYK191023 for iNOS over eNOS and nNOS found at isolated enzymes. In summary, we have identified a new highly selective iNOS inhibitor structurally unrelated to known compounds and l-arginine. BYK191023 is a valuable tool for the investigation of iNOS-mediated effects in vitro and in vivo.

Involvement of nitric oxide during in vitro fertilization and early embryonic development in mice.

Nitric oxide (NO) has emerged as an important intracellular and intercellular messenger, controlling many physiological processes and participating in the fertilization process via the autocrine and paracrine mechanisms. This study investigated whether nitric oxide synthase (NOS) inhibitior (L-NAME) and L-arginine could regulate in vitro fertilization and early embryonic development in mice. Mouse epididymal spermatozoa, oocytes, and embryos were incubated in mediums of variable conditions with and without L-NAME or L-arginine (0.5, 1, 5 and 10 mM). Fertilization rate and early embryonic development were significantly inhibited by treating sperms or oocytes with L-NAME (93. 8% vs 66.3%, 92.1% vs 60.3%), but not with L-arginine. In contrast, fertilization rate and early embryonic development were conspicuously reduced when L-NAME or L-arginine was added to the culture media for embryos. Early embryonic development was inhibited by microinjection of L-NAME into the fertilized embryos in a dose-dependent manner, but only by high concentrations of L-arginine. These results suggest that a moderate amount of NO production is essential for fertilization and early embryo development in mice.

Effect of L-arginine on Na+,K+-ATPase activity in rat aorta endothelium.

The effect of L-arginine on the Na+,K+-ATPase activity in rat aorta endothelium was studied at its physiological concentrations in the range of 10(-6)-10(-3) M. The enzyme activity was 35.5% increased by low concentrations of L-arginine (<or=10(-5) M) and its activity was 32.3-37.1% decreased at the L-arginine concentrations of 10(-4)-10(-3) M. A similar inhibition (by 34.5-42.8%) was also found in the presence of a NO-donor nitroglycerol (10(-4)-10(-3) M). An optical isomer of L-arginine, D-arginine, at the concentrations of <or=10(-5) M also increased the enzyme activity by 37.1%, but its inhibiting effect was much less pronounced and was 15.7% at the D-arginine concentration of 10(-3) M. An inhibitor of NO-synthase, L-NAME (NG-nitroarginine, methyl ester), failed to inhibit Na+,K+-ATPase. However, the presence of L-NAME abolished the inhibition of Na+,K+-ATPase by high concentrations of L-arginine. Thus, the effect of L-arginine on the endothelial Na+-pump depended on its concentration, and it is suggested that the enzyme inhibition by high concentrations of L-arginine should be associated with activation of the endogenous synthesis of NO.

Albumin restores lysophosphatidylcholine-induced inhibition of vasodilation in rat aorta

Impairment of vasodilation by oxidized low-density lipoprotein has been attributed to lysophosphatidylcholine (LPC). Albumin avidly binds LPC. Therefore, hypoalbuminemia may directly impair vasodilation and thus contribute to increased risk of atherosclerosis in nephrotic syndrome. The addition of albumin reduces LPC in erythrocytes and endothelial cells. We hypothesized that the addition of albumin will salvage vasodilation in aortic rings previously exposed to LPC. LPC increases superoxide production and disturbs L-arginine availability. Therefore, we also decreased superoxide with a superoxide dismutase mimic, MnCl(2), and supplemented L-arginine in an attempt to restore vasodilation. Rat aorta rings, which had been incubated with various concentrations of LPC and human serum albumin (HSA), were mounted in organ chambers. Relaxation was studied with acetylcholine (0.01 to 100 micromol/L) after precontraction with phenylephrine (CON, 0.3 micromol/L; LPC, 0.03 micromol/L). In some studies MnCl(2) or L-arginine was added to the organ chamber. LPC had time- and dose-dependent inhibitory effects on acetylcholine-mediated vasodilation, but no effect on nitroprusside-mediated vasodilation. Preincubation with albumin (50 or 6 g/L) could protect vasodilation against very high levels of LPC. After preincubation with LPC, the addition of albumin to the incubation salvaged vasodilation. Albumin was more effective after short LPC incubation. MnCl(2) had no specific effect on the LPC-mediated disturbance in vasodilation. L-arginine completely salvaged vasodilation at low concentrations of LPC. However, even high concentrations of L-arginine (1 mmol/L) could not improve vasodilation at LPC levels at which vasodilation was restored by albumin. LPC affects several pathways that inhibit vasodilation, all of which are salvaged by addition of albumin.

Tetrahydrobiopterin-dependent Inhibition of Superoxide Generation from Neuronal Nitric Oxide Synthase

The binding of calcium/calmodulin stimulates electron transfer between the reductase and oxygenase domains of neuronal nitric oxide synthase (nNOS). Here, we demonstrate using electron spin resonance spin-trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide that pterin-free nNOS generates superoxide from the reductase and the oxygenase domain by a calcium/calmodulin-dependent mechanism. Tetrahydrobiopterin (BH(4)) diminishes the formation of superoxide by a mechanism that does not cause inhibition of NADPH consumption. In contrast, BH(4) analogs 7,8-dihydrobiopterin and sepiapterin do not affect superoxide yields. L-Arginine alone inhibits the generation of superoxide by nNOS but not by C331A-nNOS mutant that has a low affinity for L-arginine. A greater decrease in superoxide yields is observed when nNOS is preincubated with L-arginine. This effect is in accordance with the slow binding rates of L-arginine to NOS in the absence of BH(4). L-Arginine alone or in combination with BH(4) decreases the rates of NADPH consumption. The effect of L-arginine on superoxide yields, however, was less dramatic than that caused by BH(4) as much higher concentrations of L-arginine are necessary to attain the same inhibition. In combination, L-arginine and BH(4) inhibit the formation of superoxide generation and stimulate the formation of L-citrulline. We conclude that, in contrast to L-arginine, BH(4) does not inhibit the generation of superoxide by controlling electron transfer through the enzyme but by stimulating the formation of the heme-peroxo species.

Effect of arginine on basal and high potassium induced efflux of [ 3H] d-aspartate from rat striatal slices

There are conflicting reports in the literature regarding the effects of nitric oxide as well as the involvement of the cyclic GMP-pathway on the transmitter release. To study the influence of the availability of the nitric oxide precursor arginine on the glutamate transmission process, rat striatal slices, preloaded with the tritiated glutamate analogue d-aspartate were used. l-arginine stimulated in a concentration-dependent way (0.01–10.0 mM) the high potassium-induced efflux of [ 3H] d-aspartate. The basal release was increased only by 10 mM l-arginine. Neither the basal nor the depolarization induced efflux of [ 3H] d-aspartate were affected by d-arginine. The l-arginine effect was abolished by the nitric oxide synthase inhibitor l-arginine methyl ester and was not modified by cyclic GMP. Only at high concentrations of l-arginine (10 mM) an elevation of cyclic GMP level could be demonstrated. The results are discussed in terms of direct presynaptic action of nitric oxide on [ 3H] d-aspartate efflux and a possible modulation of glutamate release by the availability of arginine.

The C331A Mutant of Neuronal Nitric-Oxide Synthase Is Defective in Arginine Binding

It has been proposed that Cys99 of human endothelial nitric oxide synthase (eNOS) is responsible for tetrahydrobiopterin (BH4) binding. To examine this possibility rigorously, we expressed rat neuronal NOS (nNOS) in Escherichia coli, with the homologous Cys331 to Ala mutation, and characterized structural and functional attributes of the purified, mutated enzyme. C331A-nNOS, as isolated, was catalytically incompetent. Upon prolonged incubation with L-arginine (L-Arg), not only BH4 binding but also catalytic activity could be restored. In contrast to wild-type nNOS (WT-nNOS), which exhibits an absorbance maximum at 407 nm that shifts immediately upon L-arginine addition to a high spin form, the C331A-nNOS mutant, as isolated, exhibited an absorbance maximum at 420 nm. C331A-nNOS, as isolated, did not bind detectable levels of either [3H]Nomega-nitro-L-arginine or [3H]BH4, but [3H]BH4 binding was reinstated after extended incubation with excess L-arginine. On the other hand, C331A-nNOS and WT-NOS were identical with regard to imidazole binding affinity, CaM binding affinity, and rates of cytochrome c and 2, 6-dichlorophenolindophenol reduction. EPR spectroscopy revealed conversion of low to high spin heme after extended incubation with high concentrations of L-arginine (0.1-10 mM). The estimated Kd for L-arginine binding to C331A-nNOS was two orders of magnitude greater than WT-nNOS (>100 microM versus 2-3 microM). Here we propose that Cys331 plays an important role in stabilizing L-arginine binding to nNOS. Our findings suggest that the primary dysfunction in the C331A mutant of nNOS, as isolated, is disruption of the BH4-substrate binding interactions as broadcast from this mutated cysteine residue. Prolonged incubation with L-arginine appears to cause remodeling of the mutant protein to a form similar to that of WT-nNOS, allowing for normalized BH4 binding and nitric oxide synthetic activity.

Inducible Nitric-oxide Synthase Generates Superoxide from the Reductase Domain

In the absence of L-arginine, the heme center of the oxygenase domain of neuronal nitric-oxide synthase reduces molecular oxygen to superoxide (O-2). Our recent work has provided evidence that inducible NOS (iNOS) may also catalyze O-2 formation in macrophages. However, there has been a lack of direct evidence of superoxide generation from the purified iNOS, and it was previously hypothesized that significant O-2 production does not occur. Moreover, the mechanism and enzyme site responsible for O-2 generation is unknown. To determine whether iNOS produces O-2 and to identify the mechanism of this process, we performed electron paramagnetic resonance measurements on purified iNOS using the spin trap 5,5-dimethyl-1-pyrroline N-oxide. In the presence of NADPH, prominent O-2 adduct signals were detected from iNOS. These signals were totally abolished by superoxide dismutase but not affected by catalase. High concentrations of L-arginine decreased this O-2 formation, whereas its enantiomer D-arginine did not. Pre-incubation of iNOS with the flavoprotein inhibitor diphenyleneiodonium totally blocked these O-2 signals. Conversely, pretreatment of the enzyme with the heme blocker cyanide had no effect on O-2 generation. Furthermore, strong O-2 generation was directly detected from the isolated iNOS reductase domain. Together, these data demonstrate that iNOS does generate O-2, and this mainly occurs at the flavin-binding sites of the reductase domain.

L-arginine in low concentration improves rat intestinal water and sodium absorption from oral rehydration solutions.

The nitric oxide (NO) precursor L-arginine has been shown to produce variable effects on intestinal absorptive function, including ion transport. To determine whether there is an optimal concentration of L-arginine, promoting proabsorptive effects from oral rehydration solutions (ORS) with 90 or 60 mM sodium. In vivo perfusion of rat jejunum with determination of net water absorption, unidirectional fluid exchanges, sodium and calcium transport, and glucose absorption. L-Arginine (1 mM) added to the 90 mM sodium ORS increased intestinal absorption of both sodium and water. Higher concentrations of L-arginine (2 to 10 mM) lacked this stimulatory effect. At 20 mM, L-arginine decreased sodium absorption below baseline. With a 60 mM sodium ORS, 2 mM L-arginine had a maximal fluid and electrolyte proabsorptive effect. At 20 mM L-arginine, net water absorption was indistinguishable from that obtained in the absence of L-arginine, and lower than with 2 mM L-arginine. Sodium absorption remained raised above baseline in perfusions with 10 and 20 mM L-arginine. Morphologically, villi from perfusions with increased absorption showed a large expansion of intercellular and lamina propria intercellular spaces. Low concentrations of L-arginine seem to stimulate water and electrolyte absorption by the small intestine. This effect is consistent with NO induced vasodilation, may be vaso-constrictive and thereby reverse fluid and electrolyte transport.

CELL DEATH, SURVIVAL AND PROLIFERATION IN TETRAHYMENA THERMOPHILA. EFFECTS OF INSULIN, SODIUM NITROPRUSSIDE, 8-BROMO CYCLIC GMP, N G-METHYL-L-ARGININE AND METHYLENE BLUE

Cells of the ciliate Tetrahymena thermophilaproduce compounds that act as autocrine (paracrine) survival and/or growth factors. 8-Bromo cyclic GMP, sodium nitroprusside, hemin, protoporphyrin IX, human recombinant and bovine insulin were tested for their ability to substitute for the cell-produced factors and stimulate cell survival and proliferation. The cells were inoculated into conical flasks in a nutritionally complete, chemically defined medium at known cell densities from 5 to 5000 cells/ml. In unsupplemented medium cells at 5 to 500 cells/ml (‘low initial cell density cultures’) died within 8 h, whereas cells at 1000 and 5000 cells/ml (‘high initial cell density cultures’) proliferated with lag phases lasting for up to 4 h. In the presence of insulin compounds, hemin, protoporphyrin IX, or 8-bromo cyclic GMP, cells also proliferated at all low initial cell densities. Sodium nitroprusside was effective over two separate concentration ranges: at the nanomolar levels as well at low pico- to femtomolar levels. At initial population densities of up to 50 cells/ml the cells at both concentrations of sodium nitroprusside survived about 4-fold longer than the controls. At 500 initial cells/ml, cells at thehigh concentrations of sodium nitroprusside survived about 4-fold longer than those of the control cultures; they proliferated in the low concentrations of sodium nitroprusside. Concentrations of hemin, too low to have any effects on their own, had synergistic effects with sodium nitroprusside. N G-methyl-L-arginine inhibited proliferation at high initial cell densities. This inhibitory action was reduced by high concentrations of L-arginine, protoporphyrin IX, sodium nitroprusside, or 8-bromo cGMP, but not by insulin. Methylene blue inhibited cell proliferation at high initial cell densities. This inhibition was circumvented by addition of 8-bromo cGMP. The findings that insulin-related material may be released from Tetrahymenaand that insulin and sodium nitroprusside increase intracellular cGMP in these cells are discussed in relation to the presented results. Together these observations suggest that cGMP is responsible for supporting cell survival in Tetrahymenaand switching the cells into their proliferative mode, and that cell-produced signal molecules and insulin stimulate an NO-dependent guanylate cyclase into producing cGMP.

Hepatocyte nitric oxide biosynthesis inhibits glucose output and competes with urea synthesis for L-arginine.

Inflammatory stimulation of the liver is known to induce nitric oxide (NO) biosynthesis. NO can interfere with the activity of a number of enzymes important to cellular metabolism. This study was carried out to investigate the influence of NO on rat hepatocyte glucose output and urea production. Induction of NO synthesis by incubation with a combination of cytokines and lipopolysaccharide led to a 48.8 +/- 2.4% inhibition of glucose output and to a 45.0 +/- 6.4% suppression of urea production. Inhibition of NO synthesis with NG-monomethyl-L-arginine was able to totally prevent these effects. High concentrations of L-arginine overcame the inhibition of urea production caused by endogenous NO synthesis. Exposure of HC to NO donors resulted in a concentration-dependent inhibition of glucose output, without having any effect on urea production. Hepatocellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity was also found to be inhibited by endogenously produced NO (33.5 +/- 5.2%), as well as by exogenously applied NO. However, an exact correlation between GAPDH activity and glucose output could not be established. These data indicate that NO biosynthesis may contribute to the development of hepatic dysfunction in chronic sepsis.

Dysfunction of nitric oxide in the spastic basilar arteries after subarachnoid hemorrhage

The function of nitric oxide in spastic cerebral arteries after subarachnoid hemorrhage (SAH) was angiographically investigated in dogs. On days 4 and 7, after two intracisternal injections of autologous blood, higher concentrations of l-arginine than those of endogenous l-arginine in the cerebrospinal fluid produced a transient vasodilation of the spastic basilar artery, whereas N G - monomethyl- l-arginine ( l-NMMA) produced no significant vasoconstriction. The vasodilator effect of l-arginine after SAH was stronger on day 4 than day 7, but less than that in intact dogs. Vasopressin, which is known to activate the endothelial l-arginine pathway, could induce a vasodilation only after the treatment with l-arginine. Intracisternal injection of superoxide dismutase (SOD), which caused no effect by itself, enhanced the duration of the vasodilator effect of l-arginine on the basilar artery on day 4 and both the magnitude and duration of that effect on day 7. Thus, the basal release of nitric oxide was impaired after SAH, but the ability to synthesize nitric oxide in the vascular wall was not abolished. Enhancement of l-arginine's effect by SOD suggested that the inactivation of nitric oxide by superoxide anion contributed to the development of vasospasm.