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Protective effect of H-2545 on doxorubicin-induced acute cardiotoxicity
54iYDROXYDECANOATE AND DIAZOXIDE ARE NOT SPECIFIC MITOCHONDRIAL KATP CHANNEL EFFECTORS Manika Das, Kelvin Lim & Andrew P Halestrap. Dept Biochemistry, University of Bristol, Bristol BS8 ITD,
of UK
Many studies have Investigated the role of mltoKprp channels in ischaemic precondlttoning (IPC) by using diazoxide as a specific opener of this channel and 5hydroxydecanoate (5-HD) as a specific blocker it is generaiiy accepted that opening the mitokrp channel will cause an increase in matnx volume that previous studies in this laboratory have shown is able to activate respiration and oxidative phosphoryiation. Here we show that under physiological conditions, neither diazoxide (50 PM) nor 5HD (100 and 300 PM), alone or together, has any effect on matrix volume whether measured with light scattering or using radio-isotope techniques. Under the same condihons 1 nM valinomycin gives a lo-20% increase in volume that is readily detected by both techniques. We confirm reports that diazoxide acts to inhibit oxidation of succinate and 2oxogiutarate, suggesting an alternative mechanism for the protective action of diazoxide. perhaps involving free radical production by the respiratory chain. We also demonstrate that 5-HO is rapidly converted to 5-i-0CoA by fatty acyi CoA synthetase associated with the outer mitochondnai membrane. Thus 5-HD may exert its antagonistic effects through actions on cellular metabolism Supported
by The flritrsh
Hearl
FoundaOon
MONOCARBOXYLATE TRANSPORTER ISOFORMS IN THE HEART. Andrew J. Davies and Andrew P. Halestrap. Dept. of Biochemistry, University of Bristol, Bristol BS8 1TD UK. The transport of lactic acid and other monocarboxlates across the sarcolemma of heart cells IS catalysed by protonhnked monocarboxylate transporters (MCTs) These proteins enable the oxidation of lactic acid and ketone bodies tn the normal heart and the efflux of lactic acid undec hypoxic conditions. Kinetic analysis of lactate and pyruvate transport into rat heart cells has suggested the presence of two MCT Isoforms. one with a high afflnlty for substrates that is strongly inhibited by stlibene disuiphonates. and another with lower substrate and inhlbllor affinity These charactenst!cs initially suggested the presence of MCT-1 and MCT-2 respec6vely, but here we demonstrate that only MCTI can be detected by Western and Northern blotting We discount the presence of alternatfvely spliced or Ctermlnai truncated forms of MCT-2 by using an array of antibodies to different regions of MCT2 as well as RT-PCR of different regions of the mRNA transcript. In red blood cells MCTl exhibits a slightly lower mobility than in heart cells, suggesting a modified form We present data to suggest that this may represent a cell specific phosphorylabon of MCTl, perhaps by casem klnase II for which a consensus site exists In the C-terminus We suggest that the presence of phosphorylated and nonphosphorylated forms of MCTI may account for the klnetlcally distinct MCT Isoforms, rather than another member of the MCT family. This phosphorylabon may affect the association of MCTl with Its ancillary protein CD147 or a CO147 homologue such as GP70 Suoported
PROTECTM? EFFECT OF H-2545 ON DOXORUBICININDUCED ACUTJI CARDIOTOXICMY Peter Em&‘, Robar Halmoa’, Ambrus ‘foth’,‘. Krisltina Kovacs’. 7nltan Huente’, Kalman Hideg’. Kalman Toth’, Hala7s Sumcg~‘. University of Pees Medical School, Dept. of Blochemistq’. I” Dep. of Skdicine Inst of OrganidMedicmal Cbemist$, Pews, Hungw Doxorubicm is one of the most potent chemothaapeutic agents but ILS usabihty 1s retickd by its negatrvc effect on heart muscle Acute cardiotoxlcity of this rmtracyciine antibiohc ts mainly attributed to 1t.s free radical generating capability H-2545 LF an antioxidant compound, which have the abihty to accumulate at tlnz very nitc of free radical damage We mvestigatcd the effects of IS-2545 and itc melabohtr, H2954 on the etrcrgctic and haanodynamic parameters of doxorubicinperfused rut hearts. 1Ieart.s of adult CFY rata wue used for normolLnsve LangendortT pchsion High-enagy phcqhate levels were measured by 3’P NhJR spectroscope. L&l ventricukr dcveloycd prcmre, dpJ&. bavt rate and rmxlmal systoitc prcrsurc were monitonxi. Lipid peroxidahon was assed by the generation of thiolnubituric acid rmtive subslances. protein oxdation by the dimtrophmylhydrazine-method and DNA damage by sin&-strand DNA break formatmu. It-2545 and its metabolite pmmkxl the doxorubcm-induud creahne p4mphate and ATP depkhon BS well as lnorgaruc phosphate elevahon m a dose depcndmt namer (creatmc phosphate was 25% m doxorublcin(100 @l) and 85% m doxorublcin + H-2545~treated (20 *AIM) hearts atIer 60 mm) Iknrt functions were alvo preserved in II2545-trcatcd cases. II-2545 ah duced the extent of hpd peroxidation, pmtcin oxidation and ssDNA treak formahon. Our data support tk hypo&sis that tic acute cardiotox&y of doxombicin is induced by free radical formation and. in addition, suggest that Ii-2-2545 and its mctaboiite can cxcrt slgniticant protechon on doxorubicin-induced acute cardiomyopathy
by The Bntrsh
Heart
Foundation
DIFFEKENTLAL CY~OHNE EXPRESSION IN M~ocy’m mn NON-MYOCY~ES AFTER MYOC~RDLU INFARCTION .\lexander hrcn, Ilans C. Volz & Hems-Gerd Zn-nmer. I .udulg-lnsutute of Physlolrlgy, Lelpzlg, Germany
(&I-
‘I’he promflammatov cytokmes mterlcukm (Il.)-lp and II .A are mcreased after acute myocardial lnfarcnon (MI). Ltoreover, serum II.-6 level 1s elevated after LlI, but also associated wrth heart failure. In the present study, heart funcuon was monitored m a rat model of chronic XII, whereas the cytobc expresslon In the mfarctcd and nonmfarcted myocardrum as well as m hearts of sham-operated controls was measured by nbonuclease-protecuon assay. To ldenufv the cells contributing to the increased c):tokme expression, we further analyzed myocytes and nonmyocytcs isolated in the acute phase as well as m stages of congesuve heart failure (CHt;) after MI. There was a strong lnducuon tn cytokme expresslon m the myocytes of the Infarct area 6 h after MI. In the non-infarcted mvocardlum, cytolunc cxpressmn Increased only slightly m the nonmvocytes after 6 h. Thus .was nor different from shamoperated controls and may, therefore, be induced by stress and catrcholammcs. In CHF, however, cvtokme expresslon level m myocytes from the non-mfarctcd myocardlum was normal. It Increased slightly but significantly In the nonmyocytes 1 and 8 weeks after MI. In conclusion, we suggest that pro-Inflammatory cytohes, produced by the lschcmlc myoqtcs ma! hc mvolvcd rn the mtmnon of wound healing of the necronc area, whereas the effect of proInflammatory cytoluncs m CHF, if any, 1s not crucial.
of UK
Many studies have Investigated the role of mltoKprp channels in ischaemic precondlttoning (IPC) by using diazoxide as a specific opener of this channel and 5hydroxydecanoate (5-HD) as a specific blocker it is generaiiy accepted that opening the mitokrp channel will cause an increase in matnx volume that previous studies in this laboratory have shown is able to activate respiration and oxidative phosphoryiation. Here we show that under physiological conditions, neither diazoxide (50 PM) nor 5HD (100 and 300 PM), alone or together, has any effect on matrix volume whether measured with light scattering or using radio-isotope techniques. Under the same condihons 1 nM valinomycin gives a lo-20% increase in volume that is readily detected by both techniques. We confirm reports that diazoxide acts to inhibit oxidation of succinate and 2oxogiutarate, suggesting an alternative mechanism for the protective action of diazoxide. perhaps involving free radical production by the respiratory chain. We also demonstrate that 5-HO is rapidly converted to 5-i-0CoA by fatty acyi CoA synthetase associated with the outer mitochondnai membrane. Thus 5-HD may exert its antagonistic effects through actions on cellular metabolism Supported
by The flritrsh
Hearl
FoundaOon
MONOCARBOXYLATE TRANSPORTER ISOFORMS IN THE HEART. Andrew J. Davies and Andrew P. Halestrap. Dept. of Biochemistry, University of Bristol, Bristol BS8 1TD UK. The transport of lactic acid and other monocarboxlates across the sarcolemma of heart cells IS catalysed by protonhnked monocarboxylate transporters (MCTs) These proteins enable the oxidation of lactic acid and ketone bodies tn the normal heart and the efflux of lactic acid undec hypoxic conditions. Kinetic analysis of lactate and pyruvate transport into rat heart cells has suggested the presence of two MCT Isoforms. one with a high afflnlty for substrates that is strongly inhibited by stlibene disuiphonates. and another with lower substrate and inhlbllor affinity These charactenst!cs initially suggested the presence of MCT-1 and MCT-2 respec6vely, but here we demonstrate that only MCTI can be detected by Western and Northern blotting We discount the presence of alternatfvely spliced or Ctermlnai truncated forms of MCT-2 by using an array of antibodies to different regions of MCT2 as well as RT-PCR of different regions of the mRNA transcript. In red blood cells MCTl exhibits a slightly lower mobility than in heart cells, suggesting a modified form We present data to suggest that this may represent a cell specific phosphorylabon of MCTl, perhaps by casem klnase II for which a consensus site exists In the C-terminus We suggest that the presence of phosphorylated and nonphosphorylated forms of MCTI may account for the klnetlcally distinct MCT Isoforms, rather than another member of the MCT family. This phosphorylabon may affect the association of MCTl with Its ancillary protein CD147 or a CO147 homologue such as GP70 Suoported
PROTECTM? EFFECT OF H-2545 ON DOXORUBICININDUCED ACUTJI CARDIOTOXICMY Peter Em&‘, Robar Halmoa’, Ambrus ‘foth’,‘. Krisltina Kovacs’. 7nltan Huente’, Kalman Hideg’. Kalman Toth’, Hala7s Sumcg~‘. University of Pees Medical School, Dept. of Blochemistq’. I” Dep. of Skdicine Inst of OrganidMedicmal Cbemist$, Pews, Hungw Doxorubicm is one of the most potent chemothaapeutic agents but ILS usabihty 1s retickd by its negatrvc effect on heart muscle Acute cardiotoxlcity of this rmtracyciine antibiohc ts mainly attributed to 1t.s free radical generating capability H-2545 LF an antioxidant compound, which have the abihty to accumulate at tlnz very nitc of free radical damage We mvestigatcd the effects of IS-2545 and itc melabohtr, H2954 on the etrcrgctic and haanodynamic parameters of doxorubicinperfused rut hearts. 1Ieart.s of adult CFY rata wue used for normolLnsve LangendortT pchsion High-enagy phcqhate levels were measured by 3’P NhJR spectroscope. L&l ventricukr dcveloycd prcmre, dpJ&. bavt rate and rmxlmal systoitc prcrsurc were monitonxi. Lipid peroxidahon was assed by the generation of thiolnubituric acid rmtive subslances. protein oxdation by the dimtrophmylhydrazine-method and DNA damage by sin&-strand DNA break formatmu. It-2545 and its metabolite pmmkxl the doxorubcm-induud creahne p4mphate and ATP depkhon BS well as lnorgaruc phosphate elevahon m a dose depcndmt namer (creatmc phosphate was 25% m doxorublcin(100 @l) and 85% m doxorublcin + H-2545~treated (20 *AIM) hearts atIer 60 mm) Iknrt functions were alvo preserved in II2545-trcatcd cases. II-2545 ah duced the extent of hpd peroxidation, pmtcin oxidation and ssDNA treak formahon. Our data support tk hypo&sis that tic acute cardiotox&y of doxombicin is induced by free radical formation and. in addition, suggest that Ii-2-2545 and its mctaboiite can cxcrt slgniticant protechon on doxorubicin-induced acute cardiomyopathy
by The Bntrsh
Heart
Foundation
DIFFEKENTLAL CY~OHNE EXPRESSION IN M~ocy’m mn NON-MYOCY~ES AFTER MYOC~RDLU INFARCTION .\lexander hrcn, Ilans C. Volz & Hems-Gerd Zn-nmer. I .udulg-lnsutute of Physlolrlgy, Lelpzlg, Germany
(&I-
‘I’he promflammatov cytokmes mterlcukm (Il.)-lp and II .A are mcreased after acute myocardial lnfarcnon (MI). Ltoreover, serum II.-6 level 1s elevated after LlI, but also associated wrth heart failure. In the present study, heart funcuon was monitored m a rat model of chronic XII, whereas the cytobc expresslon In the mfarctcd and nonmfarcted myocardrum as well as m hearts of sham-operated controls was measured by nbonuclease-protecuon assay. To ldenufv the cells contributing to the increased c):tokme expression, we further analyzed myocytes and nonmyocytcs isolated in the acute phase as well as m stages of congesuve heart failure (CHt;) after MI. There was a strong lnducuon tn cytokme expresslon m the myocytes of the Infarct area 6 h after MI. In the non-infarcted mvocardlum, cytolunc cxpressmn Increased only slightly m the nonmvocytes after 6 h. Thus .was nor different from shamoperated controls and may, therefore, be induced by stress and catrcholammcs. In CHF, however, cvtokme expresslon level m myocytes from the non-mfarctcd myocardlum was normal. It Increased slightly but significantly In the nonmyocytes 1 and 8 weeks after MI. In conclusion, we suggest that pro-Inflammatory cytohes, produced by the lschcmlc myoqtcs ma! hc mvolvcd rn the mtmnon of wound healing of the necronc area, whereas the effect of proInflammatory cytoluncs m CHF, if any, 1s not crucial.