l-citrulline prevents asymmetric dimethylarginine-mediated reductions in nitric oxide and nitrosative stress in primary human airway epithelial cells.

BACKGROUND: Asthma is associated with reduced systemic levels of l-arginine and increased asymmetric dimethylarginine (ADMA). This imbalance leads to nitric oxide synthase (NOS) uncoupling with reduced nitric oxide (NO) formation and greater oxidative and nitrosative stress. Whether this imbalance also occurs in bronchial epitheliumof asthmatics is unknown. OBJECTIVES: We used primary human bronchial epithelial cells (HBECs) from asthmatics and healthy controls to evaluate: (i) ADMA-mediated NOS uncoupling reduces epithelial production of NO and increases oxygen and nitrogen reactive species, and (ii) l-citrulline can reverse this mechanism by recoupling NOS, restoring NO production and reducing oxidative and nitrosative stress. RESULTS: In HBECsIL-13 and INFγ stimulated NOS2 and increased NOx levels. The addition of ADMA reduced NOx and increased H2 O2 levels (p<0.001). Treatment with l-citrulline (800, 1600 µm) rescued NOx when the l-arginine media concentration was 25 µm but failed to do so with higher concentrations (100 µm). Under reduced l-arginine media conditions, HBECs treated with l-citrulline increased the levels of argininosuccinate, an enzyme that metabolizes l-citrulline to l-arginine. l-citrulline prevented the ADMA-mediated increase in nitrotyrosine in HBECs in cells from asthmatics and controls. CONCLUSIONS AND CLINICAL RELEVANCE: Increasing ADMA reduces NO formation and increases oxidative and nitrosative stress in airway epithelial cells. l-citrulline supplementation restores NO formation, while preventing nitrosative stress. These results, suggest that l-citrulline supplementation may indeed be a powerful approach to restore airway NO production and may have a therapeutic potential in diseases in which there is a defective production of NO.

Oxidatively modified phosphatidylserines on the surface of apoptotic cells are essential phagocytic 'eat-me' signals: cleavage and inhibition of phagocytosis by Lp-PLA2.

Diversified anionic phospholipids, phosphatidylserines (PS), externalized to the surface of apoptotic cells are universal phagocytic signals. However, the role of major PS metabolites, such as peroxidized species of PS (PSox) and lyso-PS, in the clearance of apoptotic cells has not been rigorously evaluated. Here, we demonstrate that H2O2 was equally effective in inducing apoptosis and externalization of PS in naive HL60 cells and in cells enriched with oxidizable polyunsaturated species of PS (supplemented with linoleic acid (LA)). Despite this, the uptake of LA-supplemented cells by RAW264.7 and THP-1 macrophages was more than an order of magnitude more effective than that of naive cells. A similar stimulation of phagocytosis was observed with LA-enriched HL60 cells and Jurkat cells triggered to apoptosis with staurosporine. This was due to the presence of PSox on the surface of apoptotic LA-supplemented cells (but not of naive cells). This enhanced phagocytosis was dependent on activation of the intrinsic apoptotic pathway, as no stimulation of phagocytosis occurred in LA-enriched cells challenged with Fas antibody. Incubation of apoptotic cells with lipoprotein-associated phospholipase A2 (Lp-PLA2), a secreted enzyme with high specificity towards PSox, hydrolyzed peroxidized PS species in LA-supplemented cells resulting in the suppression of phagocytosis to the levels observed for naive cells. This suppression of phagocytosis by Lp-PLA2 was blocked by a selective inhibitor of Lp-PLA2, SB-435495. Screening of possible receptor candidates revealed the ability of several PS receptors and bridging proteins to recognize both PS and PSox, albeit with diverse selectivity. We conclude that PSox is an effective phagocytic 'eat-me' signal that participates in the engulfment of cells undergoing intrinsic apoptosis.

Trypsin/acrosin inhibitor activity of rat and guinea pig caltrin proteins. Structural and functional studies.

Dramatic inhibition of trypsin activity by rat caltrin and guinea pig caltrin I was spectrophotometrically demonstrated using the artificial substrate benzoylarginyl ethyl ester. Approximately 6% and 21% of residual proteolytic activity was recorded after preincubating the enzyme with 0.22 and 0.27 microM rat caltrin and guinea pig caltrin I, respectively. Reduction and carboxymethylation of the cysteine residues abolished the inhibitor activity of both caltrin proteins. Rat caltrin and guinea pig caltrin I show structural homology with secretory trypsin/acrosin inhibitor proteins isolated from boar and human seminal plasma and mouse seminal vesicle secretion and share a fragment of 13 amino acids of almost identical sequence (DPVCGTDGH/K/ITYG/AN), which is also present in the structure of Kazal-type trypsin inhibitor proteins from different mammalian tissues. Bovine, mouse, and guinea pig caltrin II, three caltrin proteins that have no structural homology with rat caltrin or guinea pig caltrin I, lack trypsin inhibitor activity. Rat caltrin, guinea pig caltrin I, and the mouse seminal vesicle trypsin inhibitor protein P12, which also inhibits Ca(2+) uptake into epididymal spermatozoa (mouse caltrin I), bound specifically to the sperm head, on the acrosomal region, as detected by indirect immunofluorescence. They also inhibited the acrosin activity in the gelatin film assay. Caltrin I may play an important role in the control of sperm functions such as Ca(2+) influx in the acrosome reaction and activation of acrosin and other serine-proteases at the proper site and proper time to ensure successful fertilization.

Purification, structure, and characterization of caltrin proteins from seminal vesicle of the rat and mouse.

Caltrins, small basic proteins that inhibit calcium uptake by epididymal spermatozoa, have been purified from seminal vesicle content of the mouse and rat. Mouse caltrin (M(r) 8,476) contains 75 amino acid residues, 14 basic, 5 acidic, and 7 cysteines while rat caltrin (M(r) 6,217) has 56 residues, 10 basic, 5 acidic, and 6 cysteines; their pI values are 10.2 and 9.3, respectively. The proteins did not react with Ellman's reagent unless the cystine residues were previously reduced. The primary structures were determined by sequencing fragments generated by trypsin, clostripain, and endoproteinase Lys-C digestion. The sequences were ordered to give the total structural formula. The two molecules have no sequence similarity and are different from those of the bull and guinea pig previously reported. Only rat caltrin has a sequence of 13 residues nearly identical to that in guinea pig caltrin I. Both rat and mouse caltrin react with antibodies against bovine and guinea pig caltrins. Reduction and alkylation of cysteine residues suppressed the immunologic response of mouse caltrin; however, modified rat caltrin retained partially its immunoreactivity with the antiserum against guinea pig caltrin I. The same treatment abolished the calcium transport inhibitory activity of mouse caltrin and greatly reduced that of rat caltrin. It is likely that rat and mouse caltrins have the same physiological function as proposed for bovine caltrin; namely, to regulate the development of the Ca(2+)-dependent processes that "capacitate" sperm for fertilization.