Ischemia increases intracellular calcium in lung capillary endothelium in situ

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CONTRIBUTION OF CHYDROXY-2-NONENAL LIPOFUSCIN FORMATION

TO

Pam& A. Srwda, KathL?en Conigan, Lin Tsd, Terry OberL$, Szweda. Case Western Reserm University, Clew&nd~NIH, Bethesdn3Uniwrsity of Wisconsin, Madison

and Luke I.

For a wide variety of organisms, aging is accompanied by the accumulation of fluorescent material within postmitotic cells. This material, termed lipofuscin, is localized in intracellular granules resembling lysosomal bodies. Lipofuscin appears to be composed, in part, of damaged, nondegradable forms of protein. We have shown that treatment of protein with the lipid peroxidation product I-hydroxy-Z-nonenal (HNE) in vitro leads to enzyme inactivation, protein crosslinktng, fluorescence with properties reminiscent of lipofuscin, and a loss of proteolytic susceptibility. Based on these observations, we hypothesize that modification of protein(s) by HNE plays a role in lipofusclnogenesls and aging. To test this hypothesis, we prepared antibody highly specific to a lysine-HNE fluorophore. This antibody was used to evaluate tissue from young and old Fisher-344 rats, utilizing histochemic&md lnununochemical techniques. Our results clearly demonstrate that the lysine-HNE fluorophore: 1) is present only ln tissue from old animals; 2) is localized primarily within lipofuscln granules; 3) occurs nearly exclusively on a specific protein component of lipofuscin. Thus, this study provides strong evidence that modification to protein by HNE contributes to lipofuscin formation.

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97*4 Isc+Gadoli nium(Gd)

(Isc) 123+4 Isc+ N-acetylcys

110*10 Isc+ Thapsigargin:

125+1* Isc+ Cromakal

130*4 Isc+Diphe nvleneiodi

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ROLE OF REACTIVE OXYGEN SPECIES IN ART ACTIVA’I’ION BY ANGIOTENSIN II IN VASCULAR SMOOTH MUSCLE w,U;hio-Fuka;R:WayneA+der. Gnmd tn(t Emory Unmmty,

I

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Drmsmn

h4a$&4kers,KafhyK. of Ca w&Ry, Atlanta,

GA

Reactiveoxygen species (ROS) are aucial componentsof receptor-mediatedsignal transductionin vascular smooth muscle cells (VSMW. AngiotensinII (Ang II), an important mediator of VSMC growth whose effects are dependenton the productionof ROS,also activates anti-apoptotic pathways through unknown mechanisms. Recently, the protein kinase Akt has been demonstrated to play a critical role in anti-apoptotic/cell survival signaling. Here we show that Ang II elicits a rapid and robust phosphorylationof Akt, with a peak at 5 min (W.4-fold increase). Exogenous I3202 (50-2W pM) also stimulates Akt phosphorylation WO.2-fold increase), suggesting that Akt is a redox-sensitivektnase. Ang II stimulationof Akt is mediated by phosphatidylinositol3kinase (PI3-K), because specific inhibitionof PI3-K by wortmannin or LY294002 completelyabolishes Akt phosphorylation. Furthermore, treatment of VSMCs with diphenyleneiodontum, an flavin-containingoxidase inhibitor,or overexpressionof catalase to blti Ang II-inducedintracellularH202 production, signtficantlyinhibitsAkt phosphorylationby Ang II. Becausewortmanninand LY294002 also abrogate HZOZ-induced Akt activation,PI3-K may be involved in coupling Ang II-inducedH202 formation to the Akt pathway. The redox-sensitivityof Akt may also be in part mediated by p38 mitogen-activatedprotein ktnase (p38MAPK), since SB203580 (10 pM), an inhibitorof the redox-sensitivep38MAPK pathway, but not PD98@59(30 @vi), a p42/44MAPK inhibitor, partially attenuates Ang II-induced Akt phosphorylation. Thus, our studies indicate that Akt is part of the remarkablespectrum of Ang II signalingpathways in VSMCs,and provide insights into a novel role for ROS in the regulationof anti-apoptotic/cell survival pathways.

OXYGEN

EPR OXIMETRY MAPPING (EPROM) OF CARTILAGE FORMED IN A HOLLOW FIBER BIOREACTOR S. Sendhil Velan’, Periannan Kuppusamy*, Erik Petersen’, Jay L. Zweid, Kenneth W. Fishbein’, Richard G. S. Spencer’ ‘NMR Unit, NIA, NIH, Baltimore, MD. ‘The EPR Center, Department of Medicine, Johns Hopkins University, Baltimore, MD We have developed an EPR-compatible hollow fiber bioreactor (HFBR) in which nearly cylindrically-symmetric neocartilage grows from inoculated chondrocytes. Oxygen, nutrient, and spin label are delivered via diffusion from a central porous fiber running the length of the bioreactor. We have studied diffusion and metabolism in neocartilage 17 days after initiation of growth, using electron Paramagnetic resonance oximetry mapping(EPROM). We employed ‘N-labeled perdeuterated tempone (PDT) which had a linewidth in the absence of oxygen, of 280 mG, and broadens to 1000 mG in 100% oxygen. Details regarding the time course of PDT detection during inflow into, and washout out of, in bioreactor without cells and also reduction of the PDT to the corresponding diamagnetic hydroxylamines by chondrocytespresent in the cartilage matrix was investigated. The Nitroxide distributed throughout the developing tissue, with local linewidth reflecting local oxygenation. The bioreactor was also used to study the rate of oxygen utilization radially across the cartilage. With the prominence of the central perfusing fiber in the resonance amplitude and total spin maps, but the disappearance of the fiber in oxygen maps, we conclude that the rate of oxygen diffusion in the cartilage/chondrocyte system is high. Oxygen utilization by chondrocytes in the cartilage matrix was studied by addition of cyanide to the perfusing medium. Cyanide inhibits oxidative phosphorylation and markedly reduce cellular oxygen utilization. Consistent with this, addition of cyanide increased oxygen levels measured by EPROM as compared to control tissue without cyanide.

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