Regulation of no synthesis by AMP-activated protein kinase

Synzymes of Superoxide Dismutase as Antiinflammatory Agents. D. Salvemini. MetaPhore Pharmaceuticals,1910 Innerbelt BusinessCenter Dr, St Louis, MO 63114, USA. Under normal circumstances,formation of superoxide anion (0;) is kept under tight control by endogenous superoxidedismutase (SOD) enzymes. However in acute and chronic inflammation, the production of 0,’ is increased at a rate that overwhelms the capacity of endogenous SOD enzyme defence system to remove them. The results of such imbalance results in 0,’ mediated damage. We have developed a series of low molecular weight synthetic mimetic (“synzyme”) of the native SOD enzvme (SODm). M40403 is a nrototvnic SODm posses&g catalytic ‘SOD activity approa&ng that of the native MnSOD enzyme while at the same time possessingoutstanding chemical and biological stability. Unlike other so-called SOD mimetics, M40403 selectivelyremoves superoxide without interfering with other relevant biological oxidants, such as nitric oxide, peroxynitrite, or hydrogen peroxide. This important property will allow us to develop a better understanding of the role of superoxide in health and disease by selectively removing superoxide in vivo. The pharmacological profile of M40403 in models of acute and chronic inflammation as well as reperfusion iniurv will be presented. The outcome of these-studies support the clinical annlication of this classof SODm in a varietv of diseasesd&en by superoxide or the product of it’s interactionwith nitric oxide, peroxynitrite.

NITRIC OXIDE, NO-DONORMEDIATED VASODILATATION AND RBDOX REGULATION OF VASCULAR SMOOTHiMUSCLE. $5 Tn gle*, F. Lovten, T.K. Jeffery, ‘A. Gambino, . . wganstall. Smooth Muscle Research Group & Dept. Phprky ,& Therapeutics, Canada; De t. P ystology or Pharma~ol,o,f,c%% Queensland, risbane,Australia. NO-donor drugs are used to mimic the effects of exogenous nitric oxide gas (NO) as well as the effects of endothelium-derived NO (EDNO). However, a differences in the NO, NO and NO-donor We compared ACh, nitroglycerin (GTN), sodium nitroprusside (SNP), spermine NONOate-mediated relaxation of precontracted endothelium-intact mouse aorta with that as (assumed to be NO free radical, (NO) generatedfrom Angeli‘s inhibitor, ODQ (lH-( 1,2,4-) oxadiazolo(4,3-a)-quinoxalon-l-one, dose-dependently inhibited responses to all agents, but with differing sensitivities to (ACh=GTN>SNP>NOODQ

res onses to NO. However, copper chelators and Fe*’ ana Fe’+ had no effect on relaxations to NO* or NO.. These data sug est that, in the mouse aor@ res onses mediatedby A 6 GTN, SNP, and spermine NOR Oate involve both NO’ and nitroxvl ions and show variable levelsof involvement of sGC&ctivation. (Supported by the Heart and StrokeFoundation of Alberta).

REGULATION OF NO SYNTHESISBY AMPACTIVATED PROTEIN KlNASE Bruce E. Kemp, Belinda J. Michell, ZhiPing Chen, David Stapleton and Sid MurthySt. Vincentis Institute for Medical Research,Fitzroy, Vie. 3065 Endothelial NOS is an important regulatory enzyme in the cardiovascularsystemwhose activity is controlled by protein phosphorylation. Multiple protein kinases including Akt/PKB, PKA and AMPK activateeNOS by phosphorylating Ser-1177 in response to various stimuli. Ischaemic stress activates the AMPK and eNOS in the heart. During both VEGF and bradykin signallingin endothelial cellsthere is a transient increase in Ser-1177phosphorylation coupled with a decreasein Thr-495 phosphorylation that reverses over 10 min. PKC signalling in endothelial cells inhibits eNOS activity by phosphorylating Thr-495 and dephosphorylating Ser-1177 whereas PKA signalling acts in reverseby increasing phosphorylation of Ser1177 and dephosphorylation of Thr-495 to activate eNOS. Both phosphatases PPl and PP2A are associated with eNOS PPl is responsible for deuhosnhorvlation of Thr-495 based on its soecificitv fo; this site in both eNOS and the corresponding syntheticphosphopeptide whereas PP2A is responsible for dephosphorylation of Ser-1177.Treatment of endothelialcellswith calyculin selectivelyblocked PKAmediated dephosphotylation of Thr-495 whereas okadaic acid s&c&ely blocked PKC-mediated denhosnhorvlation of Ser-1177. These results show that regulation of eNOS activity involves co-ordinated signalling through Ser-I 177 and Thr-495 by multiple protein kinasesand phosphatases.

REDOX AND METABOLISM IN THE VASCULAR WALL: AN OVERVIEW. Christopher D. Hardin, Dept. of Physiology, University of Missouri, Columbia, MO 65212 USA The metabolism of vascular smooth muscle appears highly organized with localized metabolism in support of co-localized function. VSM is a highly glycolytic tissue and glycolytic ATP production is regulated by, and can dramatically influence, cytoplasmic redox potential. In hyperglycemic states such as diabetes, VSM dysfunction has been proposed to be mediated by redox changes via increased polyol pathway flux and increasedde novo diacyglycerol synthesis resulting in eventualprotein kinase C activation. Using 13C-NMR, polyol pathway flux measured as fructose production from glucosecan be directly measured.However, polyol pathway flux is low compared to lactateproduction and hence the magnitude of shift in cytoplasmic redox potential is insignificant. To examine other possible sourcesof metabolic dvsfunction. we used VSM from a diabetic/dyslipidemic pig model and found plasma glucosedid not alter VSM oxidative substrateutilization while lipid profile correlated with alterations in the pattern of substrate utilization. In acute in vitro experiments, pahnitate administration mimicked the effects seen in the hyperlipidemic/diabetic pig model. Therefore, the metabolic dysfunction associatedwith diabetes/dyslipidemia may be due to mitochondrial damage from saturated fatty acids by either de novo ceramide synthesisor reactiveoxygen species.Support: AHA Established Investigator grant, NIH DK55039, MU Children’sMiracle Network. A157