The effects of ischaemic preconditioning, diazoxide and 5-hydroxydecanoate on rat heart mitochondrial volume and respiration

Abstract

Studies with different ATP-sensitive potassium (K(ATP)) channel openers and blockers have implicated opening of mitochondrial K(ATP) (mitoK(ATP)) channels in ischaemic preconditioning (IPC). It would be predicted that this should increase mitochondrial matrix volume and hence respiratory chain activity. Here we confirm this directly using mitochondria rapidly isolated from Langendorff-perfused hearts. Pre-ischaemic matrix volumes for control and IPC hearts (expressed in microl per mg protein +/- S.E.M., n = 6), determined with (3)H(2)O and [(14)C]sucrose, were 0.67 +/- 0.02 and 0.83 +/- 0.04 (P < 0.01), respectively, increasing to 1.01 +/- 0.05 and 1.18 +/- 0.02 following 30 min ischaemia (P < 0.01) and to 1.21 +/- 0.13 and 1.26 +/- 0.25 after 30 min reperfusion. Rates of ADP-stimulated (State 3) and uncoupled 2-oxoglutarate and succinate oxidation increased in parallel with matrix volume until maximum rates were reached at volumes of 1.1 microl ml(-1) or greater. The mitoK(ATP) channel opener, diazoxide (50 microM), caused a similar increase in matrix volume, but with inhibition rather than activation of succinate and 2-oxoglutarate oxidation. Direct addition of diazoxide (50 microM) to isolated mitochondria also inhibited State 3 succinate and 2-oxoglutarate oxidation by 30 %, but not that of palmitoyl carnitine. Unexpectedly, treatment of hearts with the mitoK(ATP) channel blocker 5-hydroxydecanoate (5HD) at 100 or 300 microM, also increased mitochondrial volume and inhibited respiration. In isolated mitochondria, 5HD was rapidly converted to 5HD-CoA by mitochondrial fatty acyl CoA synthetase and acted as a weak substrate or inhibitor of respiration depending on the conditions employed. These data highlight the dangers of using 5HD and diazoxide as specific modulators of mitoK(ATP) channels in the heart.

Figure 1

Perfusion protocols used for measuring the effects of ischaemic preconditioning (IPC), diazoxide and 5HD, alone or in combination, on the mitochondrial matrix volume. Values above bars denote time elapsed in minutes.

Figure 3. The relationship between matrix volume and rates of ADP-stimulated respiration in mitochondria from control, end-ischaemia and reperfused hearts with or without IPC

For individual preparations of mitochondria from control (squares), end ischaemia (circles) and reperfused (triangles) hearts with IPC (filled symbols) or without IPC (open symbols) the matrix volume is plotted against the State 3 rate of succinate (A) or 2-oxoglutarate plus malate (B) oxidation expressed as ratios relative to the rate of ascorbate TMPD oxidation. The rationale behind this is explained in the text whilst full data are presented in Table 1. In C data for the pre-ischaemia and end-ischaemia mitochondria are plotted as their mean values ±s.e.m. (error bars) in conjunction with previously published data for heart mitochondria whose matrix volumes were varied by changing the osmolarity (Halestrap, 1987).

Figure 2. The effects of ischaemic preconditioning, diazoxide and 5HD, alone or in combination, on the rate pressure product of hearts subject to 30 min global ischaemia followed by reperfusion

Data are presented as means ± s.e.m. (error bars) of 6-8 separate hearts. Full functional data for pre-ischaemic, end-ischaemic and reperfused hearts are given in Table 2. Horizontal bars indicate the composition of the perfusion medium: KHS, Krebs-Henseleit buffer alone; 5HD, KHS containing 5HD at 100 or 300 μm (5HD100 or 5HD300); Diaz, KHS containing 50 μm diazoxide.

Figure 4. The relationship between matrix volume and rates of ADP-stimulated respiration by mitochondria isolated from control, IPC, diazoxide- and 5HD-treated hearts

Data are presented as in Fig. 3 and as means ±s.e.m. (error bars) of 6-8 hearts for each treatment as indicated. The arrows indicate the distinctive effects of IPC and diazoxide. Full data are presented in Table 3 with parallel haemodynamic data in Table 2.

Figure 5. The effects of diazoxide and 5HD on respiration by isolated heart mitochondria

In A rates of ADP-stimulated respiration in the presence of 5 mm succinate + 1 μm rotenone, 5 mm 2-oxoglutarate + 1 mm l-malate, 5 mm l-glutamate + 1 mm l-malate or 50 μm palmitoyl carnitine + 1 mm l-malate were measured in the presence and absence of diazoxide or 5HD at the concentrations shown. Data are presented as the mean percentage inhibition of respiration cause by the reagent ±s.e.m. (error bars) of 6 separate mitochondrial preparations. In B rates of oxygen uptake by isolated heart and liver mitochondria were measured in the presence of 0.2 mm l-malate supplemented with 200 μm 5HD or decanoate as indicated. In the three left bars, respiration was stimulated by addition of 0.4 mm ADP whilst in the three right bars 1 mm ATP, 1 μm oligomycin, 0.1 mm coenzyme A and 1 mm l-carnitine were added to enable extramitochondrial activation of the fatty acid (see Fig. 6) and oxidation was initiated by addition of uncoupler (0.2 μm FCCP). Data are presented as means ±s.e.m. (error bars) of 3-6 separate mitochondrial preparations. Significant increases or decreases in respiration are indicated (*P < 0.05; **P < 0.01).

Figure 6. Activation of 5HD to 5HD-CoA by isolated heart mitochondria

The fatty acyl CoA synthetase activity of intact heart mitochondria was measured by a coupled enzyme assay linked to NADH oxidation as illustrated schematically and described under Methods. Usually 0.1 mm coenzyme A was present at the start and the assay initiated by addition of the fatty acid indicated at 50 μm. However, in the bottom trace of B, 50 μm 5HD was present at the start and the assay initiated by addition of 0.1 mm coenzyme A. Abbreviations used: PPi, pyrophosphate; PEP, phosphoenolpyruvate; Pyr, pyruvate; Lac, lactate.