Ubiquinone and cytochrome b in isolated rat heart

Abstract

The relationship between changes in steady state redox states of the mitochondrial respiratory carriers, cytochrome <i>b</i> (cyt. <i>b</i>) and ubiquinone (Q), was investigated in the isolated, perfused rat heart. O₂ consumption, rate of coronary flow and peak developed pressure in the aorta were recorded for the spontaneously beating heart (100.min⁻¹) retrogradely perfused with glucose-supplemented Krebs solution at 24°C. Cyt. <i>b</i> redox changes were continuously monitored from the heart surface at 430 nm-410 nm using a dual wavelength spectrophotometer modifed for reflectance measurements. Following freeze clamping of the heart in liquid N₂, Q redox status was chemically determined. Inhibitors of cyt. <i>b</i> and Q oxidation or reduction were utilized to demonstrate that spectral interference from other heme proteins does not preclude monitoring of changes in cyt. <i>b</i> redox status in the intact rat heart. Increases in contractile rate to 150.min⁻¹ and 200.min⁻¹ induced by electrical stimulation resulted in reduction of both cyt. <i>b</i> and Q; the higher rate being associate with a greater degree of reduction. This reduction of the carriers was associated with increased oxygen consumption and decreased peak developed pressure. As Ca²⁺ concentration in the perfusion medium was increased in the range from 0 to 2.5 mM, both cyt. <i>b</i> and Q showed smaller changes, but the overall trend was towards oxidation. The oxidation of the carriers was associated with increased oxygen consumption and increased peak developed pressure. A linear relationship between steady state log (Q/QH₂) and log (cyt.<i>b</i>³⁺/<i>b</i>²⁺) was observed in both groups of hearts despite the fact that no metabolic inhibitor was employed to allow for equilibration of the redox states of the two respiratory carriers. These results suggest the redox states of Q and of cyt. <i>b</i> in the intact heart shift during such transitions so as to remain in close equilibrium. Furthermore, the reduction of cyt. <i>b</i> and Q associated with increased contractile frequency in the intact heart is compatible with the near equilibrium hypothesis of respiratory control. On the other hand, the responses of the carriers to changes in external Ca²⁺ concentration appear to be more complex.