Purpose: Diazoxide (DZX) is frequently used for induction of effective pharmacological preconditioning (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-ε activator than as the activator of KATP channels, which is its secondary effect only. Taking into consideration all these 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 acute streptozotocin-diabetes (DIA) also confers cardioprotective effect and that the finding of considerably 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 mechanism of DZX action and, in this way, to bring the preconditioning with DZX closer to practical applicability.
Methods: Experiments were performed on 9-11 week old male Wistar rats devided into healthy and DIA groups. Acute DIA (8 days) was induced by a single dose of streptozotocin (65 mg.kg-1, i.p.). Heart mitochondria isolated by differential centrifugation were exposed to DZX (0-7 μmol.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: Starting with the concentration of 3.5 μmol.l-1 DZX induced a significant (p<0.05) increase in Mg2+-ATPase activity in MIT from healthy hearts. A similar trend was observed also in DIA hearts, but it reached no statistical significance even at 7 μmol.l-1. The stimulation of the enzyme activity was not accompanied with any considerable changes in MIT membrane fluidity.
Conclusions: Concentrations of DZX equal to or above 5 μmol.l-1 stimulate (p<0.05) the activity of MIT Mg2+-ATPase by a direct interaction with the enzyme molecule as it was revealed by the enzyme kinetics analysis. Stimulation of MIT Mg2+-ATPase plays an essential but not exclusive role in cardioprotective effect provided by both DZX and DIA. Grants: VEGA 2/0101/12, 2/0094/12, APVV 0102-11, KEGA 003 UK-4/2012.