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Purpose: Diazoxide (DZX) is frequently used for induction of effective pharmacological precondi- tioning (P-PC) of the myocardium in the animal models. This compound was originally classified as an opener of the mitochondrial (MIT) ATP-dependent potassium channels (KATP). But, afterwards it was demonstrated that DZX also i) acts as an inhibitor of the Krebs cycle at the level of succinate dehydrogenase, ii) supports free radicals generation and iii) acts more as a PKC- e activator than as theactivatorofKATP channels,whichisitssecondaryeffect only.Taking intoconsideration allthese effects of DZX, no satisfactory explanation for the molecular mechanism of its cardioprotective action has been introduced up to now. In the independent studies it was demonstrated that the acutestreptozotocin-diabetes (DIA)alsoconferscardioprotectiveeffect andthatthefindingof con- siderably stimulated heart MIT Mg2 + -ATPase is intimately involved in this protection. Aim of the present study was to elucidate the role of the MIT Mg2 + -ATPase and membrane fluidity in the mechanismofDZXactionand,inthisway,tobringthepreconditioningwithDZXclosertopractical applicability. Methods: Experimentswereperformedon9-11weekoldmaleWistarratsdevidedintohealthyand DIAgroups.AcuteDIA(8days)wasinducedbyasingledoseofstreptozotocin(65mg.kg-1,i.p.).HeartmitochondriaisolatedbydifferentialcentrifugationwereexposedtoDZX(0-7 m mol.l-1). MIT ATP synthase activity, detected in the reaction that proceeded in the opposite direction i.e., as Mg2 + ATPase was assessed by measuring of Pi liberated from ATP splitting. Fluidity of the lipid bilayer of the MIT membrane was estimated by measuring fluorescence anisotropy with the probe 1,6-diphenyl-1,3,5-hexatriene. Results: Startingwiththeconcentrationof3.5 m mol.l-1DZXinducedasignificant(p , 0.05)increase inMg2 + -ATPaseactivityinMITfromhealthyhearts.AsimilartrendwasobservedalsoinDIAhearts, butitreachednostatisticalsignificanceevenat7 m mol.l-1.Thestimulationoftheenzymeactivitywas notaccompaniedwithanyconsiderablechangesinMITmembranefluidity. Conclusions: ConcentrationsofDZXequaltoorabove5 m mol.l-1stimulate(p , 0.05)theactivityof MITMg2 + -ATPasebyadirectinteractionwiththeenzymemoleculeasitwasrevealedbytheenzyme kineticsanalysis.StimulationofMITMg2 + -ATPaseplaysanessentialbutnotexclusiveroleincardio-protectiveeffectprovidedbybothDZXandDIA.Grants:VEGA2/0101/12,2/0094/12,APVV0102-11,KEGA003UK-4/2012.