Free L-arginine Research Articles

Regulation of L-arginine transport and nitric oxide release in superfused porcine aortic endothelial cells.

1. We have investigated whether changes in extracellular ion composition and substrate deprivation modulate basal and/or bradykinin-stimulated L-arginine transport and release of nitric oxide (NO) and prostacyclin (PGI2) in porcine aortic endothelial cells cultured and superfused on microcarriers. 2. Saturable L-arginine transport (Km = 0.14 +/- 0.03 mM; Vmax = 2.08 +/- 0.54 nmol min-1 (5 x 10(6) cells)-1) was pH insensitive and unaffected following removal of extracellular Na+ or Ca2+. 3. Cationic arginine analogues, including L-lysine and L-ornithine, inhibited L-arginine transport, whilst 2-methylaminoisobutyric acid, beta-2-amino-bicyclo[2,2.1]-heptane-2-carboxylic acid, L-phenylalanine, 6-diazo-5-oxo-norleucine, L-glutamine, L-cysteine and L-glutamate were poor inhibitors. 4. Deprivation of L-arginine (30 min to 24 h) reduced intracellular free L-arginine levels from 0.87 +/- 0.07 to 0.40 +/- 0.05 mM (P < 0.05) and resulted in a 40% stimulation of L-arginine, L-lysine and L-ornithine transport. 5. L-arginine and NG-monomethyl-L-arginine (L-NMMA), but not N omega-nitro-L-arginine methyl ester (L-NAME), trans-stimulated efflux of L-[3H]arginine. 6. Depolarization of endothelial cells with 70 mM K+ reduced L-arginine influx and prevented the stimulation of transport by 100 nM bradykinin, but agonist-induced release of NO and PGI2 was still detectable. 7. Basal rates of L-arginine transport and NO release were unaffected during superfusion of cells with a nominally Ca(2+)-free solution. Bradykinin-stimulated L-arginine transport was insensitive to removal of Ca2+, whereas agonist-induced NO release was abolished. 8. Although bradykinin-stimulated NO release does not appear to be coupled directly to the transient increase in L-arginine transport, elevated rates of L-arginine influx via system y+ in response to agonist-induced membrane hyperpolarization or substrate deprivation provide a mechanism for enhanced L-arginine supply to sustain NO generation.

Tetrahydropyrimidine derivatives inhibit binding of a Tat-like, arginine-containing peptide, to HIV TAR RNA in vitro

The ability of a small molecule, 2-methyl,4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyrimidine (THP(A)), which accumulates intracellularly in various streptomyces, to inhibit the interaction of Tat peptide (R52) with TAR RNA is presented. Using gel-shift assay, we found that the inhibition constant K i of THP(A) is 50–100 nM, which is in the range of the binding constants of Tat peptide and protein. THP(A) is ∼ 10 6 times more tightly bound than the free l-arginine. The high binding affinity may be attributed to the special delocalized positive charge on the NCN group and the hydroxyl group at the 5 position of this molecule. A model for THP(A)-TAR interaction, analogous to the arginine guanidinum group-TAR interaction, is presented. The relatively high uptake of THP(A) by mammalian cells warrants in vivo Tat/TAR inhibition studies.

Mechanisms of Kupffer cell cytotoxicity in vitro against the syngeneic murine colon adenocarcinoma line MCA26

We have previously demonstrated that in vivo activation or inhibition of Kupffer cell (KC) cytotoxic function can reduce or enhance, respectively, the hepatic tumor burden in a syngeneic murine colon adenocarcinoma (MCA26) tumor model. In the current study, we have performed in vitro experiments to define the possible mechanisms of KC cytotoxicity against MCA26 cells. Addition of either anti-tumor necrosis factor (TNF) or anti-interleukin-1 alpha (IL-1 alpha) antisera reduced KC cytotoxicity in coculture against MCA26 targets in a dose-dependent fashion; addition of these sera together resulted in approximately additive inhibition, suggesting the existence of parallel pathways for these effector molecules. Nitric oxide as a mediator of cytotoxicity by KCs in coculture with MCA26 cells was evaluated by two approaches. Activated KCs produced detectable levels of nitric oxide; however, activated KC exerted cytotoxicity against MCA26 targets in the absence of exogenous free L-arginine. Thus, TNF and IL-1 play major roles in producing murine KC cytotoxicity against MCA26 colon cancer cells in vitro, whereas reactive nitric oxides do not.

Evidence for the presence of l-arginine in the glial components of the peripheral nervous system

l-Arginine is a precursor of nitric oxide that has been identified as an endogenous activator of soluble guanylate cyclase. We have recently reported the immunocytochemical localization of free l-arginine in glial cells in the central nervous system (CNS) using specific anti-arginine antibody. In the present study, we focused our attention on this particular amino acid in the peripheral nervous system (PNS). In the cochlea of the inner ear, arginine-like immunoreactivity was localized in satellite cells surrounding neurons of the spiral ganglion. In the dorsal root ganglia, satellite cells surrounding sensory neurons were found to be immunoreactive. In the superior cervical ganglion, l-arginine was concentrated in satellite cells around neuronal cells. In ganglia of the enteric plexus, supporting cells that covered neuronal cells were stained. These results show that free l-arginine in the PNS is concentrated in satellite and supporting cells, both of which correspond to glial cells in the CNS. Thus, those cells in ganglia of the PNS may support and/or control the neural activity by providing l-arginine to the neurons that they surround.

Predominant localization in glial cells of free l-arginine. Immunocytochemical evidence

Nitric oxide has been recently identified as an endogenous activator of the soluble guanylate cyclase in the brain as well as in vascular endothelial cells and macrophages. In the present study, we determined the localization of free arginine in the brain because nitric oxide was formed from the terminal guanido group of l-arginine. Anti-arginine antiserum was raised in guinea pigs by repeated injection of l-arginine covalently conjugated to guinea pig serum albumin via glutaraldehyde. Specicic anti-arginine antibody was purified from the antiserum by using an affinity gel coupled with l-arginine. Arginine-like immunoreactivity in the rat brain and spinal cord was found concentrated mainly in astrocytes including Bergmann glial cells in the cerebellum and processes of astrocytes around blood vessels. The present results suggest that glial cells, particularly astrocytes, are the main locus of l-arginine, a nitric oxide precursor, in the brain.

Stimulation of Growth and Polyamine Biosynthesis of the Ciliated ProtozoanTetrahymena thermophila.Regulation by L-Arginine

Tetrahymena thermophila cells grown in a synthetic nutrient medium for 9 h removed 97% of the free L-arginine but less than 50% of any of the other essential amino acids. The major portion of the arginine was degraded rapidly (76-92%) whereas 5-15% was conserved as intact and only 2.5-10% were incorporated into protein. However, if bovine serum albumin (BSA) was present in the medium as a macromolecular arginine source the incorporation of free arginine into protein was reduced to less than 1% but the degraded fraction was increased. Apparently, the uptake mode of arginine determines its fate: arginine taken up by phagocytosis is bound for protein biosynthesis, arginine taken up by membrane receptors is chanelled to degradation. Media without arginine did not support growth of Tetrahymena. Citrulline and ornithine, the precursors of arginine biosynthesis in yeast and vertebrates, were not able to substitute for arginine. Pronounced morphological changes, e.g. greatly reduced ribosome content, were observed in Tetrahymena cells after 24 h of arginine starvation in otherwise complete medium, but not in cells starved in water, salt solution, or buffer. Thus, arginine is an essential nutrient component for Tetrahymena and the rapid degradation of this compound involving the enzymes arginine deiminase (ADI) and citrulline hydrolase (CH) might be of regulatory importance for the unicellular, as it is the case with acetylcholine and catecholamines in mammalian organisms. Since the product of these enzymes, L-ornithine, is the substrate for the regulatory key enzyme of polyamine biosynthesis, ornithine decarboxylase (ODC), the effects of the presence of absence of arginine on the activities of each particular enzyme of the pathway were studied, including ODC and the enzyme ornithine-oxo-acid aminotransferase (O delta T), which is a competitor of ODC for the common substrate. The arginine-degradative pathway was stimulated by extracellular free but not by peptide-bound arginine and was modulated by extracellular protein which induced phagocytosis; O delta T was stimulated with a time lag. The stimulation of ODC was in a reciprocal relation to the arginine concentration and enhanced by phagocytosis and previous arginine starvation.(ABSTRACT TRUNCATED AT 400 WORDS)

Occurrence of Guanidino Compounds in Several Plants

The stored nitrogen in perennial plants plays an important role in their regrowth in spring. Free L-arginine is one of the major forms of stored nitrogen (6, 9) .. LArginine is metabolized via the urea cycle in animal and plant tissues. In animal tissues, L-arginine is also metabolized via r-guanidinobutyramide, r-guanidinobutyric acid, and r-aminobutyric acid to the TCA cycle. In plant tissues, few attempts have been made to study arginine-metabolic routes other than the urea cycle. In white spruce tissues (3), L-arginine is metabolized via r-guanidinobutyric acid. Also in citrus tissues (4, 5), the operation of the same metabolic route has been reported. In these experiments, it was observed that various guanidino compounds reacted with the Sakaguchi reagent. To obtain more information about the guanidino compounds involved in the arginine metabolism, the analytical system was evaluated and the presence of these compounds in several plant species was examined.

Identification and properties of peptidylarginine deiminase from rabbit skeletal muscle.

Peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, was identified and partially purified from rabbit skeletal muscle. The specific activity of the crude extract from the muscle was aboutt 120 times that of the extract from newborn rat epidermis. The enzyme was purified abou 250-fold over the initial extract of rabbit skeletal muscle in a yield of about 53%. The enzyme requires Ca2+ as an essential cofactor and the activity was not inhibited by monoiodoacetate or PCMB. The molecular weight of the enzyme was found to be about 115,000 by gel chromatography on Bio-Gel P-300. The enzyme catalyzed the deimination of arginyl residue when both the alpha-amino and alpha-carboxyl groups were substituted, and showed low activity when substrates had either a free alpha-amino or a free alpha-carboxyl group. The action of the enzyme on free L-arginine was negligible. Proteins such as protamine, histone, and ribonuclease A were better substrates than the other small synthetic peptides tested.

Properties of peptidylarginine deiminase from the epidermis of newborn rats.

An enzyme which catalyzes the coversion of arginyl residues to citrullyl residues in protein was obtained from the extract of the epidermis of newborn rats. The enzyme required Ca2+ for its activity. The enzyme activity was enhanced in the presence of DTT. The maximum activity was observed at pH 7.5 at 50 degrees C in the presence of 10 mm CaCl2 and 2 mM DTT. The activity was inhibited strongly by treatment of the enzyme with monoiodoacetate or PCMB, which suggests that the epidermal enzyme is an SH-enzyme. The molecular weight of the enzyme was calculated by gel filtration to be about 48,000. It was essential for the alpha-amino or alpha-carboxyl group of the L-arginine substrate to be involved in a peptide linkage. The enzyme showed marked activities towards N-substituted L-arginine derivatives such as BZ-L-Arg, BZ-L-Arg-NH2, and BZ-Gly-L-Arg, But the action of the enzyme on free L-arginine was negligible. The enzyme activity was affected by the nature of the residue neighboring the arginyl residue in proteins. The authors propose the name "peptidylarginine deiminase" for this enzyme. A considerable specificity of the enzyme for proteins from the epidermal cells in terminal differentiation was observed. The results suggest that citrullyl residues in membranous protein of horny cells of the epidermis of newborn rat are formed by the action of epidermal peptidylarginine deiminase.