Kegg Pathway: Arginine and proline metabolism

Am J Physiol Lung Cell Mol Physiol. 2009 Oct 2;
Chen B, Calvert AE, Cui H, Nelin LD

Vascular remodeling and smooth muscle cell proliferation are hallmark pathogenic features of pulmonary artery hypertension (PAH). Alterations in the metabolism of L-Arginine via arginase and nitric oxide synthase play a critical role in the endothelial dysfunction seen in PAH. L-Arginine metabolism by arginase produces L-ornithine and urea. L-ornithine is a precursor for polyamine and proline synthesis, ultimately leading to an increase in cellular proliferation. Given the integral role of the smooth muscle layer in the pathogenesis of hypoxia-induced PAH, we hypothesized that hypoxia would increase cellular proliferation via arginase induction in human pulmonary artery smooth muscle cells (hPASMC). We found that arginase II mRNA and protein expression were significantly increased in cultured hPASMC exposed to 1% O2 for 24 and 48 hours, which coincided with an increase in arginase activity at 48 hours. There were no hypoxia-induced changes in levels of arginase I mRNA or protein in cultured hPASMC. Exposure to hypoxia resulted in more than one and a half times as many viable cells after 120 hours than normoxic exposure. The addition of the arginase inhibitor, S-(2-boronoethyl)-L-cysteine, completely prevented both the hypoxia-induced increase in arginase activity and proliferation in hPASMC. Furthermore, transfection of siRNA targeting arginase II in hPASMC resulted in knock down of arginase II protein levels and complete prevention of the hypoxia-induced cellular proliferation. These data support our hypothesis that hypoxia increases proliferation of hPASMC through the induction of arginase II. Key words: pulmonary hypertension, L-Arginine, vascular remodeling.

Anal Chim Acta. 2009 Sep 14; 650(1): 3-9
Chen J, Wang W, Lv S, Yin P, Zhao X, Lu X, Zhang F, Xu G

In this study, urinary metabolites from liver cancer patients and healthy volunteers were studied by a metabonomic method based on ultra performance liquid chromatography coupled to mass spectrometry. Both hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) were used to separate the urinary metabolites. Principle component analysis (PCA) and partial least squares to latent structure-discriminant analysis (PLS-DA) models were built to separate the healthy volunteers from the liver cancer patients and to find compounds that are expressed in significantly different amounts between the two populations. 21 metabolite ions were considered as potential biomarkers according to the Variable importance in the Project (VIP) value and S-plot. Compared with RPLC, a more sensitive and stable response can be recorded in HILIC mode due to the high content of organic solvent used. Moreover, the liver cancer group and the healthy volunteers can be better separated based on the data from the HILIC separation, which indicates that HILIC is suitable for urinary metabonomic analysis. In HILIC mode, several polar compounds related to Arginine and proline metabolism, alanine and aspartate metabolism, lysine degradation, nicotinate and nicotinamide metabolism were found to be significantly changed in the concentrations of the two different populations: healthy and cancer. In contrast, in RPLC mode, these changed compounds are related to fatty acids oxidation.

metabolism. 2009 Aug 24;
Tomlinson C, Rafii M, Ball RO, Pencharz P

d-Amino acids (d-AAs) in stable isotope tracers may result in erroneous estimates of enrichment, particularly if urine is used as a surrogate for plasma enrichment. Previous studies suggest that a d-AA content of less than 0.2% will not result in significant error in studies with adult humans. To describe the effects of d-AA content of less than 0.2%, in 3 different AA tracers, on isotope enrichment in urine and plasma, Arginine, proline, and phenylalanine (Phe) tracers were given enterally to human neonates. Enrichment was measured in urine and plasma using chiral chromatography and tandem mass spectrometry. The Phe tracer was also given parenterally to human neonates and enterally to children and adults to further characterize the d-AA effect. All isotopes had a confirmed d-AA content of less than 0.2%. Labeled d-Arginine resulted in an overestimate for enrichment of 20% in plasma and 87% in urine. A smaller effect was seen for d-Phe, which resulted in a 5% overestimate for plasma and 40% in urine. d-proline had no significant effect. Using the same Phe tracer, a developmental effect was found, with a reduction in the overestimate in children compared with infants and no effect on enrichment in adults. Investigators using commercially produced, stable isotope-labeled AAs need to be aware that there is no safe level of d contamination; a d-AA content less than 0.2% may result in significant overestimate for enrichment, even in plasma, for infants and children. This source of error can be avoided by the use of chiral chromatography.

Curr Microbiol. 2009 Oct; 59(4): 483-8
Lee SY, Kim YH, Min J

pEMBTL-SY1, which can over produce the ArgR protein in Corynebacterium glutamicum, was constructed. The DNA-binding affinity of ArgR was analyzed using a Chromatin Immunoprecipitation (ChIP) assay. The level of ArgR protein expression in the plasmid-carrying C. glutamicum (pEMBTL-SY1) was higher than that in the wild-type strain. On the other hand, there was no increase in the DNA-binding affinity of ArgR on the upstream of argB and the level of ornithine production. The DNA-binding affinity of ArgR on the arg operon and the level of ornithine production in the presence of three metabolites, ornithine, Arginine, and proline, were examined as feedback controlling effectors in the Arginine biosynthesis pathway in C. glutamicum. The ChIP assay showed that the supplemented metabolites altered the ArgR-binding affinity on the upstream of argB, which is consistent with the change in ornithine production. This suggests that the regulation of ornithine biosynthesis by the transcriptional regulator, ArgR, depends on the DNA-binding affinity of the arg operon, which is regulated by the feedback controlling effectors, rather than on the level of ArgR protein expression.

J Proteome Res. 2009 Oct; 8(10): 4844-50
Qiu Y, Cai G, Su M, Chen T, Zheng X, Xu Y, Ni Y, Zhao A, Xu LX, Cai S, Jia W

Colorectal carcinogenesis involves the overexpression of many immediate-early response genes associated with growth and inflammation, which significantly alters downstream protein synthesis and small-molecule metabolite production. We have performed a serum metabolic analysis to test the hypothesis that the distinct metabolite profiles of malignant tumors are reflected in biofluids. In this study, we have analyzed the serum metabolites from 64 colorectal cancer (CRC) patients and 65 healthy controls using gas chromatography time-of-flight mass spectrometry (GC-TOFMS) and Acquity ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (Acquity UPLC-QTOFMS). Orthogonal partial least-squares discriminate analysis (OPLS-DA) models generated from GC-TOFMS and UPLC-QTOFMS metabolic profile data showed robust discrimination from CRC patients and healthy controls. A total of 33 differential metabolites were identified using these two analytical platforms, five of which were detected in both instruments. These metabolites potentially reveal perturbation of glycolysis, Arginine and proline metabolism, fatty acid metabolism and oleamide metabolism, associated with CRC morbidity. These results suggest that serum metabolic profiling has great potential in detecting CRC and helping to understand its underlying mechanisms.

J Hypertens. 2009 Aug; 27 Suppl 6: S11-6
Paulis L, Pechanova O, Zicha J, Krajcirovicova K, Barta A, Pelouch V, Adamcova M, Simko F

OBJECTIVE: Melatonin was shown to reduce blood pressure, enhance nitric oxide availability and scavenge free radicals. There is, however, a shortage of data with respect to the effect of melatonin on pathological left ventricular remodelling associated with haemodynamic overload. DESIGN: We investigated whether melatonin was able to prevent left ventricular hypertrophy (LVH) and fibrosis associated with N(G)-nitro-L-Arginine-methyl ester (L-NAME)-induced hypertension. METHODS: Four groups of male Wistar rats were investigated: control, L-NAME (50 mg/kg per day), melatonin (10 mg/kg per day) and L-NAME plus melatonin. Blood pressure was measured non-invasively each week. After 5 weeks of treatment the animals were killed and nitric oxide synthase (NOS) activity, endothelial and inducible NOS expression, the level of collagenous proteins, hydroxyproline and conjugated dienes in the left ventricle were determined. RESULTS: The administration of L-NAME inhibited NOS activity, increased conjugated dienes concentration, elevated blood pressure and induced LVH and fibrosis (indicated by increased collagenous proteins and hydroxyproline levels). The addition of melatonin to L-NAME treatment failed to prevent the attenuation of NOS activity and the development of LVH and prevented hypertension only partly. The administration of melatonin, however, completely prevented the increase in conjugated dienes concentration and the development of left ventricular fibrosis. NOS expression was not different among experimental groups. CONCLUSION: Melatonin prevented the development of left ventricular fibrosis and the increase in oxidative load in rats with L-NAME-induced hypertension. The antifibrotic effect of melatonin seems to be independent of its effects on NOS activity and might be linked to its antioxidant properties.

Amino Acids. 2009 Aug 1;
Mohapatra S, Minocha R, Long S, Minocha SC

The polyamine metabolic pathway is intricately connected to metabolism of several amino acids. While ornithine and Arginine are direct precursors of putrescine, they themselves are synthesized from glutamate in multiple steps involving several enzymes. Additionally, glutamate is an amino group donor for several other amino acids and acts as a substrate for biosynthesis of proline and gamma-aminobutyric acid, metabolites that play important roles in plant development and stress response. Suspension cultures of poplar (Populus nigra x maximowiczii), transformed with a constitutively expressing mouse ornithine decarboxylase gene, were used to study the effect of up-regulation of putrescine biosynthesis (and concomitantly its enhanced catabolism) on cellular contents of various protein and non-protein amino acids. It was observed that up-regulation of putrescine metabolism affected the steady state concentrations of most amino acids in the cells. While there was a decrease in the cellular contents of glutamine, glutamate, ornithine, Arginine, histidine, serine, glycine, cysteine, phenylalanine, tryptophan, aspartate, lysine, leucine and methionine, an increase was seen in the contents of alanine, threonine, valine, isoleucine and gamma-aminobutyric acid. An overall increase in percent cellular nitrogen and carbon content was also observed in high putrescine metabolizing cells compared to control cells. It is concluded that genetic manipulation of putrescine biosynthesis affecting ornithine consumption caused a major change in the entire ornithine biosynthetic pathway and had pleiotropic effects on other amino acids and total cellular carbon and nitrogen, as well. We suggest that ornithine plays a key role in regulating this pathway.

J Vet Intern Med. 2009 May-Jun; 23(3): 559-63
Chan DL, Rozanski EA, Freeman LM

BACKGROUND: Alterations in circulating amino acids have been documented in animal models and in critically ill people but have not been evaluated in dogs with spontaneously occurring disease. HYPOTHESIS/OBJECTIVES: To compare amino acid concentrations in critically ill dogs and healthy controls and to investigate potential relationships among amino acids, markers of inflammation, illness severity, and clinical outcome. ANIMALS: Forty-eight critically ill dogs and 24 healthy control dogs. METHODS: Plasma was analyzed for amino acids and C-reactive protein (CRP) was measured in serum. The Fischer ratio (the molar ratio of branched chain amino acids [BCAA] to aromatic amino acids [AAA]) and survival prediction index (SPI2) were calculated. RESULTS: Median CRP concentrations were significantly higher in the critically ill dogs compared with controls (P < .001). Critically ill dogs had significantly lower concentrations of alanine (P= .001), Arginine (P < .001), citrulline (P < .001), glycine (P < .001), methionine (P < .001), proline (P < .001), and serine (P= .001) but significantly higher concentrations of lysine (P= .02) and phenylalanine (P < .001; Table 1). This pattern resulted in a significantly lower Fischer ratio (P= .001) in the critically ill group. Median SPI2 score was significantly higher in dogs that survived (P= .03). Concentrations of Arginine (P= .02), isoleucine (P= .01), leucine (P= .04), serine (P= .04), valine (P= .04), total BCAA (P= .03), and the Fischer ratio (P= .03) were significantly higher in survivors compared with nonsurvivors. CONCLUSIONS AND CLINICAL IMPORTANCE: Critically ill dogs have altered amino acid profiles and additional research to investigate potential benefits of amino acid supplementation is warranted.

J Exp Zool A Ecol Genet Physiol. 2009 Nov 1; 311(9): 719-26
Izeirovski S, Moffett SB, Moffett DF, Onken H

Isolated anterior midguts of larval Aedes aegypti were bathed in aerated mosquito saline containing serotonin (0.2 micromol L(-1)) and perfused with NaCl (100 mmol L(-1)). The lumen negative transepithelial voltage (V(te)) was measured and luminal alkalinization was determined through the color change of luminal m-cresol purple from yellow to purple after luminal perfusion stops. Addition of 10 mmol L(-1) amino acids (Arginine, glutamine, histidine or proline) or dicarboxylic acids (malate or succinate) to the luminal perfusate resulted in more negative V(te) values, whereas addition of glucose was without effect. In the presence of TRIS chloride as luminal perfusate, addition of nutrients did not change V(te). These results are consistent with Na(+)-dependent absorption of amino acids and dicarboxylic acids. Effects of serotonin withdrawal indicated that nutrient absorption is stimulated by this hormone. Strong luminal alkalinization was observed with mosquito saline containing serotonin on the hemolymph-side and 100 mmol L(-1) NaCl in the lumen, indicating that alkalinization does not depend on luminal nutrients. Omission of glucose or dicarboxylic acids from the hemolymph-side solution had no effect on luminal alkalinization, whereas omission of amino acids significantly decelerated it. Re-addition of amino acids restored alkalinization, suggesting the involvement of amino acid metabolism in luminal alkalinization.

Am J Clin Nutr. 2009 Sep; 90(3): 857S-861S
Brosnan ME, Brosnan JT

Glutamate plays a central role in hepatic amino acid metabolism, both because of its role in the transdeamination of most amino acids and because the catabolism of Arginine, ornithine, proline, histidine, and glutamine gives rise to glutamate. It is now appreciated that different hepatic functions are restricted to hepatocyte subpopulations within different acinar zones. This is also a feature of glutamate metabolism. Glutamine catabolism and synthesis are physically separated by zonation, with glutamine synthetase restricted to a narrow band of hepatocytes in zone 3 of the hepatic acinus, whereas glutaminase occurs in zone 1. Arginine and ornithine metabolism is also restricted to particular hepatocyte subpopulations. Ornithine aminotransferase, the regulated enzyme of Arginine and ornithine catabolism, is restricted to the same zone 3 cells as glutamine synthetase, whereas the urea cycle is found in the remaining hepatocytes. This separation facilitates the independent regulation of these 2 different metabolic processes. We know the acinar localization of only a small fraction of the approximately 15,000 genes expressed in the liver. Knowledge of the acinar localization of metabolic processes is essential for an appreciation of their relation to other hepatic functions and their regulation.

FEBS Lett. 2009 Aug 6; 583(15): 2565-72
Wootla B, Mahendra A, Dimitrov JD, Friboulet A, Borel-Derlon A, Rao DN, Uda T, Borg JY, Bayry J, Kaveri SV, Lacroix-Desmazes S

Anti-factor VIII (FVIII) inhibitory IgG may arise as alloantibodies to therapeutic FVIII in patients with congenital hemophilia A, or as autoantibodies to endogenous FVIII in individuals with acquired hemophilia. We have described FVIII-hydrolyzing IgG both in hemophilia A patients with anti-FVIII IgG and in acquired hemophilia patients. Here, we compared the properties of proteolytic auto- and allo-antibodies. Rates of FVIII hydrolysis differed significantly between the two groups of antibodies. proline-phenylalanine-Arginine-methylcoumarinamide was a surrogate substrate for FVIII-hydrolyzing autoantibodies. Our data suggest that populations of proteolytic anti-FVIII IgG in acquired hemophilia patients are different from that of inhibitor-positive hemophilia A patients.

Rapid Commun Mass Spectrom. 2009 Aug; 23(15): 2307-15
Guedes S, Vitorino R, Domingues R, Amado F, Domingues P

Albumin is an important plasma antioxidant protein, contributing to protecting mechanisms of cellular and regulatory long-lived proteins. The metal-catalyzed oxidation (MCO) of proteins plays an important role during oxidative stress. In this study, we examine the oxidative modification of albumin using an MCO in vitro system. Mass spectrometry, combined with off-line nano-liquid chromatography, was used to identify modifications in amino acid residues. We have found 106 different residues oxidatively damaged, being the main oxidized residues lysines, cysteines, Arginines, prolines, histidines and tyrosines. Besides protein hydroxyl derivatives and oxygen additions, we detected other modifications such as deamidations, carbamylations and specific amino acid oxidative modifications. The oxidative damage preferentially affects particular subdomains of the protein at different time-points. Results suggest the oxidative damage occurs first in exposed regions near cysteine disulfide bridges with residues like methionine, tryptophan, lysine, Arginine, tyrosine and proline appearing as oxidatively modified. The damage extended afterwards with further oxidation of cysteine residues involved in disulfide bridges and other residues like histidine, phenylalanine and aspartic acid. The time-course evaluation also shows the number of oxidized residues does not increase linearly, suggesting that oxidative unfolding of albumin occurs through a step-ladder mechanism.

DNA Cell Biol. 2009 Sep; 28(9): 443-9
Sobti RC, Kaur P, Kaur S, Janmeja AK, Jindal SK, Kishan J, Raimondi S

The interaction of genetic and environmental factors can determine individual susceptibility to various cancers. We studied the influence of NAT2 and codon 72 p53 polymorphisms on 151 patients with lung cancer and an equal number of matched population controls. Polymorphisms of NAT2 and p53 were determined by PCR-RFLP techniques. The results were analyzed using logistic regression analysis. A statistically significant relationship between NAT2*5 and NAT2*6 alleles and lung cancer risk was observed. In addition, the population with slow acetylator alleles for NAT2*5 and NAT2*6 had a significantly higher risk of lung cancer compared with rapid acetylator alleles both in smokers and nonsmokers. The combined genotype of heterozygous Arginine (Arg)/proline (Pro), Pro/Pro, and slow acetylator alleles of NAT2*5 and NAT2*6 showed higher, although not significant, risk of lung cancer compared with Arg/Arg and rapid acetylator alleles of NAT2*5 and NAT2*6. In conclusion, these findings suggest that the influence of NAT2 genotype, alone or in combination with p53 genotype, may confer increased susceptibility to lung cancer.

J Appl Physiol. 2009 Sep; 107(3): 880-6
Couppé C, Hansen P, Kongsgaard M, Kovanen V, Suetta C, Aagaard P, Kjaer M, Magnusson SP

Age-related loss in muscle mass and strength impairs daily life function in the elderly. However, it remains unknown whether tendon properties also deteriorate with age. Cross-linking of collagen molecules provides structural integrity to the tendon fibrils and has been shown to change with age in animals but has never been examined in humans in vivo. In this study, we examined the mechanical properties and pyridinoline and pentosidine cross-link and collagen concentrations of the patellar tendon in vivo in old (OM) and young men (YM). Seven OM (67 +/- 3 years, 86 +/- 10 kg) and 10 YM (27 +/- 2 years, 81 +/- 8 kg) with a similar physical activity level (OM 5 +/- 6 h/wk, YM 5 +/- 2 h/wk) were examined. MRI was used to assess whole tendon dimensions. Tendon mechanical properties were assessed with the use of simultaneous force and ultrasonographic measurements during ramped isometric contractions. Percutaneous tendon biopsies were taken and analyzed for hydroxylysyl pyridinoline (HP), lysyl pyridinoline (LP), pentosidine, and collagen concentrations. We found no significant differences in the dimensions or mechanical properties of the tendon between OM and YM. Collagen concentrations were lower in OM than in YM (0.49 +/- 0.27 vs. 0.73 +/- 0.14 mg/mg dry wt; P < 0.05). HP concentrations were higher in OM than in YM (898 +/- 172 vs. 645 +/- 183 mmol/mol; P < 0.05). LP concentrations were higher in OM than in YM (49 +/- 38 vs. 16 +/- 8 mmol/mol; P < 0.01), and pentosidine concentrations were higher in OM than in YM (73 +/- 13 vs. 11 +/- 2 mmol/mol; P < 0.01). These cross-sectional data raise the possibility that age may not appreciably influence the dimensions or mechanical properties of the human patellar tendon in vivo. Collagen concentration was reduced, whereas both enzymatic and nonenzymatic cross-linking of concentration was elevated in OM vs. in YM, which may be a mechanism to maintain the mechanical properties of tendon with aging.

Anal Biochem. 2009 Oct 1; 393(1): 80-7
Tenorio-Laranga J, Valero ML, Männistö PT, Sánchez del Pino M, García-Horsman JA

In vitro, prolyl oligopeptidase (POP) cleaves proline-containing bioactive peptides such as substance P, gonadotropin-releasing hormone, thyrotropin-releasing hormone, Arginine-vasopressin, and neurotensin. Based on specific in vivo inhibition, POP has been suggested to be involved in cognitive and psychiatric processes but the identity of its physiological substrates has remained inconclusive. We have combined (a) sample snap-freezing and boiling buffer extraction, to limit protein degradation and reduce sample complexity; (b) pH two-dimensional liquid reverse-phase chromatography to enhance resolution; and (c) iTRAQ isobaric labeling to identify the rat brain peptides whose levels were differentially changed due to in vivo POP inhibition. In the hypothalamus, all the substrates found were part of precursors of secreted peptides such as copeptin, PACAP-related peptide, somatostatin, and proSAAS derived peptides, while in the cerebellum the peptides were derived from carcinoma-amplified sequence 1 homolog and calmodulin. In the striatum, somatostatin precursor derived peptide, fragments from E3-SUMO protein ligase RanBP2, and the subunit 5A of cytochrome c oxidase were increased. When analyzing the peptides that were significantly reduced by POP inhibition we found fragments from large protein complexes but, exclusively in the cerebellum, bioactive peptides such as cerebellin and fibrinopeptides A and B were detected.

Proteomics. 2009 Jun; 9(11): 2910-28
Moreno R, Martínez-Gomariz M, Yuste L, Gil C, Rojo F

The Crc protein is a global translational regulator involved in catabolite repression of catabolic pathways for several non-preferred carbon sources in Pseudomonads when other preferred substrates are present. Using proteomic and transcriptomic approaches, we have analyzed the influence of Crc in cells growing in a complete medium, where amino acids are the main carbon source. Inactivation of the crc gene modified the expression of at least 134 genes. Most of them were involved in the transport and assimilation of amino acids or sugars. This allowed envisioning which amino acids are preferentially used. Crc did not inhibit the pathways for proline, alanine, glutamate, glutamine and histidine. These amino acids are good carbon sources for P. putida. In the case of Arginine, lysine, aspartate and asparagine, which can be assimilated through several pathways, Crc favored one particular route, inhibiting other alternatives. Finally, Crc-inhibited genes needed to assimilate valine, isoleucine, leucine, tyrosine, phenylalanine, threonine, glycine and serine, amino acids that provide a less efficient growth. Crc has therefore a key role in coordinating metabolism, controlling the sequential assimilation of amino acids when cells grow in a complete medium. Inactivation of crc reduced growth rate, suggesting that Crc optimizes metabolism.

Br J Pharmacol. 2009 Jul; 157(6): 922-30
Morris SM

As Arginine can serve as precursor to a wide range of compounds, including nitric oxide, creatine, urea, polyamines, proline, glutamate and agmatine, there is considerable interest in elucidating mechanisms underlying regulation of its metabolism. It is now becoming apparent that the two isoforms of arginase in mammals play key roles in regulation of most aspects of Arginine metabolism in health and disease. In particular, work over the past several years has focused on the roles and regulation of the arginases in vascular disease, pulmonary disease, infectious disease, immune cell function and cancer. As most of these topics have been considered in recent review articles, this review will focus more closely on results of recent studies on expression of the arginases in endothelial and vascular smooth muscle cells, post-translational modulation of arginase activity and applications of arginase inhibitors in vivo.

Cell Tissue Res. 2009 Aug; 337(2): 235-42
Tomiosso TC, Nakagaki WR, Gomes L, Hyslop S, Pimentel ER

The Achilles tendon can support high tension forces and may experience lesions. The damaged tissue does not regenerate completely, with the organization and mechanical properties of the repaired tendon being inferior to those of a healthy tendon. Nitric oxide (NO) plays an important role in wound repair. We have examined the structural reorganization and repair in Achilles tendon after injury in rats treated with the NO synthase inhibitor L-NAME. The right Achilles tendon of male Wistar rats was partially transected. One group of rats was treated with L-NAME (~300 mg/kg per day, given in drinking water) for 4 days prior to tendon sectioning and throughout the post-operative period. Control rats received water without L-NAME. The tendons were excised at 7, 14, and 21 days post-injury and used to quantify hydroxyproline and for mechanical tests. Tendons were also processed for histomorphological analysis by polarized light microscopy, which showed that the collagen fibers were disorganized by day 7 in non-treated and L-NAME-treated rats. In non-treated rats, the organization of the extracellular matrix was more homogeneous by days 14 and 21 compared with day 7, although this homogeneity was less than that in normal tendon. In contrast, in injured tendons from L-NAME-treated rats, the collagen fibers were still disorganized on day 21. Tendons from treated rats had more hydroxyproline but lower mechanical properties compared with those from non-treated rats. Thus, NO modulates tendon healing, with a reduction in NO biosynthesis delaying reorganization of the extracellular matrix, especially collagen.

J Biol Chem. 2009 Jul 24; 284(30): 20022-33
Guerreiro JR, Lameu C, Oliveira EF, Klitzke CF, Melo RL, Linares E, Augusto O, Fox JW, Lebrun I, Serrano SM, Camargo AC

Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-de pend ent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of Arginine in human embryonic kidney cells and (ii) increase of Arginine plasma concentration in SHR. Moreover, alpha-methyl-dl-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases.

J Med Chem. 2009 Jun 25; 52(12): 3627-35
Qu H, Cai M, Mayorov AV, Grieco P, Zingsheim M, Trivedi D, Hruby VJ

A new series of melanotropin analogues with His or Arg residues in the core pharmacophores of MTII, SHU9119, and Ac-NDP-gamma-MSH-NH(2) replaced by Pro or trans-/cis-4-guanidinyl-Pro derivatives were designed and synthesized to introduce selectivity toward the human melanocortin 4 receptor (hMC4R). Analogues 1, 2, 3, 6, 7, 8 were found to be hMC4R selective. Second messenger studies have demonstrated that analogues 1 and 2 are insurmountable inhibitors of MTII agonist activity at the hMC4R. Molecular modeling studies suggest that the hMC4R selectivity is due to a beta-turn shift induced by the Pro ring that makes the global minimum structures of these analogues resemble the NMR solution structure of the hASIP melanocortin receptor binding motif. Substitution of His in MTII also provided functional selectivity for the hMC3R or the hMC4R. These findings are important for a better understanding of the selectivity mechanism at the hMC3R/hMC4R and the development of therapeutic ligands selectively targeting the hMC4R.