Bacterial toxins activation of abbreviated urea cycle in porcine cerebral vascular smooth muscle cells.
Nitric oxide (NO) overproduction via induction of inducible nitric oxide synthase (iNOS) is implicated in vasodilatory shock in sepsis, leading to septic encephalopathy and accelerating cerebral ischemic injury. An abbreviated urea-cycle (l-citrulline-l-arginine-NO cycle) has been demonstrated in cerebral perivascular nitrergic nerves and endothelial cells but not in normal cerebral vascular smooth muscle cell (CVSMC). This cycle indicates that argininosuccinate synthase (ASS) catalyzes l-citrulline (l-cit) conversion to form argininosuccinate (AS), and subsequent AS cleavage by argininosuccinate lyase (ASL) forms l-arginine (l-arg), the substrate for NO synthesis. The possibility that ASS enzyme in this cycle was induced in the CVSMC in sepsis was examined. Blood-vessel myography technique was used for measuring porcine isolated basilar arterial tone. NO in cultured CVSMC and in condition mediums were estimated by diaminofluorescein (DAF)-induced fluorescence and Griess reaction, respectively. Immunohistochemical and immunoblotting analyses were used to examine iNOS and ASS induction. l-cit and l-arg, which did not relax endothelium-denuded normal basilar arteries precontracted by U-46619, induced significant vasorelaxation with increased NO production in these arteries and the CVSMCs following 6-hour exposure to 20μg/ml lipopolysaccharide (LPS) or lipoteichoic acid (LTA). Pre-treatment with pyrrolidine dithiocarbamate (PDTC) and salicylate (SAL) (NFκB inhibitors), aminoguanidine (AG, an iNOS inhibitor), and nitro-l-arg (NLA, a non-specific NOS inhibitor) blocked NO synthesis in the CVSMC and attenuated l-cit- and l-arg-induced relaxation of LPS- and LTA-treated arteries. Furthermore, immunohistochemical and immunoblotting studies demonstrated that expression of basal iNOS and ASS in the smooth muscle cell of arterial segments denuded of endothelium and the cultured CVSMCs was significantly increased following 6-hour incubation with LPS or LTA. This increased iNOS- and ASS-proteins expression in both preparations was inhibited by SAL, but was further increased by AG. These results indicate that LPS and LTA induce the l-cit-l-arg-NO cycle via induction of iNOS and ASS in the CVSMCs, accounting for massively increased NO-production and cerebral vasodilation in septic shock. Simultaneous inhibition of both pathways and NFκB-activation may be necessary to efficiently decrease or normalize NO production in the CVSMCs in this disease condition, and/or prevention and treatment of cerebral vessel-related brain dysfunctions. Our results further suggest to avoid using iNOS inhibitors alone which may cause upregulation of iNOS and ASS resulted from feedback-inhibition of iNOS activity. Accordingly, combined treatments with specific iNOS-activity inhibitor and inhibitor for iNOS genomic expression may provide a strategy in optimally managing brain sepsis and related encephalopathy associated with enhanced iNOS expression and NO overproduction.
Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, IL, USA.
Rajesh G
RG
Mishra
Department of Medical Research, Buddhist Tzu Chi General Hospital, Hualien, Taiwan; Institutes of Medical Sciences, Hualien, Taiwan; Pharmacology & Toxicology, Hualien, Taiwan; Tzu Chi Center of Vascular Medicine, College of Life Sciences, Hualien, Taiwan.
Tzu-Ling
TL
Tseng
Department of Medical Research, Buddhist Tzu Chi General Hospital, Hualien, Taiwan; Tzu Chi Center of Vascular Medicine, College of Life Sciences, Hualien, Taiwan; Tzu Chi University of Science and Technology, Hualien, Taiwan.
Mei-Fang
MF
Chen
Department of Medical Research, Buddhist Tzu Chi General Hospital, Hualien, Taiwan; Pharmacology & Toxicology, Hualien, Taiwan; Tzu Chi Center of Vascular Medicine, College of Life Sciences, Hualien, Taiwan.
Po-Yi
PY
Chen
Department of Medical Research, Buddhist Tzu Chi General Hospital, Hualien, Taiwan; Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, IL, USA; Institutes of Medical Sciences, Hualien, Taiwan; Pharmacology & Toxicology, Hualien, Taiwan; Department of Life Sciences, Hualien, Taiwan; Tzu Chi Center of Vascular Medicine, College of Life Sciences, Hualien, Taiwan. Electronic address: tlee@mail.tcu.edu.tw.
Tony J-F
TJ
Lee
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001124:Argininosuccinate Synthase / Q000378:metabolism; D001427:Bacterial Toxins / Q000494:pharmacology*; D002536:Cerebral Arteries / Q000166:cytology / Q000378:metabolism; D002956:Citrulline / Q000378:metabolism; D005260:Female; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D013552:Swine; D013682:Teichoic Acids / Q000494:pharmacology; D014508:Urea / Q000378:metabolism*; D014662:Vasoconstrictor Agents / Q000494:pharmacology
D001427:Bacterial Toxins; D008070:Lipopolysaccharides; D013682:Teichoic Acids; D014662:Vasoconstrictor Agents; D002956:Citrulline; D009569:Nitric Oxide; C009900:lipoteichoic acid; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D014508:Urea; D001120:Arginine; D052247:Nitric Oxide Synthase Type II; D001124:Argininosuccinate Synthase
Argininosuccinate synthase; Cerebral arterial smooth muscle; Encephalopathy; NFκB; Nitric oxide; Septic shock; iNOS