K(ATP) channel-independent targets of diazoxide and 5-hydroxydecanoate in the heart

Abstract

Diazoxide and 5-hydroxydecanoate (5-HD; C10:0) are reputed to target specifically mitochondrial ATP-sensitive K(+) (K(ATP)) channels. Here we describe K(ATP) channel-independent targets of diazoxide and 5-HD in the heart. Using submitochondrial particles isolated from pig heart, we found that diazoxide (10-100 microM) dose-dependently decreased succinate oxidation without affecting NADH oxidation. Pinacidil, a non-selective K(ATP) channel opener, did not inhibit succinate oxidation. However, it selectively inhibited NADH oxidation. These direct inhibitory effects of diazoxide and pinacidil cannot be explained by activation of mitochondrial K(ATP) channels. Furthermore, application of either diazoxide (100 microM) or pinacidil (100 microM) did not decrease mitochondrial membrane potential, assessed using TMRE (tetramethylrhodamine ethyl ester), in isolated guinea-pig ventricular myocytes. We also tested whether 5-HD, a medium-chain fatty acid derivative which blocks diazoxide-induced cardioprotection, was 'activated' via acyl-CoA synthetase (EC 6.2.1.3), an enzyme present both on the outer mitochondrial membrane and in the matrix. Using analytical HPLC and electrospray ionisation mass spectrometry, we showed that 5-HD-CoA (5-hydroxydecanoyl-CoA) is indeed synthesized from 5-HD and CoA via acyl-CoA synthetase. Thus, 5-HD-CoA may be the active form of 5-HD, serving as substrate for (or inhibiting) acyl-CoA dehydrogenase (beta-oxidation) and/or exerting some other cellular action. In conclusion, we have identified K(ATP) channel-independent targets of 5-HD, diazoxide and pinacidil. Our findings question the assumption that sensitivity to diazoxide and 5-HD implies involvement of mitochondrial K(ATP) channels. We propose that pharmacological preconditioning may be related to partial inhibition of respiratory chain complexes.

Figure 1. Effects of diazoxide and pinacidil on mitochondrial membrane potential and electron transport chain activity

Application of 100 μm diazoxide (A) or 100 μm pinacidil (B) to TMRE-loaded ventricular myocytes had no effect on TMRE fluorescence. The relative magnitude of the fluorescence intensity is plotted against time; 0 indicates baseline (resting) fluorescence, 1 indicates the fluorescence measured after dissipation of the mitochondrial membrane potential with 100 μm DNP. C, diazoxide (10-100 μm) inhibited succinate oxidation in submitochondrial particles (n = 5 for each data point). D, pinacidil inhibited NADH oxidation. Each symbol represents 6 experiments.

Figure 2. Effects of diazoxide and malonate on flavoprotein fluorescence

A, lack of effect of diazoxide (100 μm) on flavoprotein fluorescence in a ventricular myocyte. DNP (100 μm) was used to obtain maximal flavoprotein fluorescence. The myocyte was continuously excited (485 nm) and fluorescence (530 ± 15 nm) was measured at 1 point s⁻¹ (symbols are spaced by 5 s). B, increase in flavoprotein fluorescence induced by the specific succinate dehydrogenase inhibitor malonate (2 mm).

Figure 3. Synthesis of 5-HD-CoA from 5-HD via acyl-CoA synthetase

A, representative chromatograms of samples taken before (top) and after (bottom) addition of acyl-CoA synthetase (EC 6.2.1.3). Injection volume was 10 μl and UV absorption was performed at 260 nm (mUA indicates milliabsorbance unit). The inset in the bottom chromatogram compares the UV spectra of CoA (continuous line) and 5-HD-CoA (dashed line). B, using electrospray ionization mass spectrometry, the reaction product 5-HD-CoA (mass/charge (m/z) ratio ≈938) was detected when 5-HD was exposed to acyl-CoA synthetase in the presence of CoA and ATP.

Figure 4. Schematic diagram showing mitochondrial sites of action of diazoxide, pinacidil and 5-HD

Diazoxide and pinacidil, as well as volatile anaesthetics, inhibit the electron transport chain at the sites depicted. Nicorandil may also inhibit the electron transport chain via the production of NO (nitric oxide). 5-Hydroxydecanoate serves as substrate for the enzyme acyl-CoA synthetase. The principal product of this reaction, 5-HD-CoA (an acyl-CoA ester), may serve as substrate for acyl-CoA dehydrogenase or, possibly, inhibit this enzyme.