The Effect of Ether Anaesthesia Stress on Cholesterol‐Side‐Chain Cleavage and Cytochrome P450 in Rat‐Adrenal Mitochondria

Abstract

Cholesterol side‐chain cleavage has been studied in intact adrenal mitochondria from rats subjected to stress by ether anaesthesia as a means of raising the plasma adrenocorticotrophin levels. Control rats were either injected with cycloheximide or kept in a quiescent state. After initiation of cholesterol side‐chain cleavage by addition of isocitrate, pregnenolone formation from endogenous cholesterol in intact rat‐adrenal mitochondria follows a biphasic time‐course of formation with an initial rapid phase lasting 3–5 min followed by a much slower rate of formation.Pregnenolone formation from [4‐14C]cholesterol is linear if the tracer substrate is added to the mitochondria together with isocitrate. Preincubation of the mitochondria with [4‐14C]cholesterol prior to addition of isocitrate results in a biphasic [4‐14C]pregnenolone formation; the rate of the initial rapid phase depending on the duration of preincubation.The effect of stress is to increase 2–3 times the rate of pregnenolone formation in the initial phase compared to the rates observed in mitochondria from quiescent or cycloheximide‐treated rats.Depletion of cholesterol from adrenal mitochondria follows a similar pattern to pregnenolone formation, but the initial cholesterol content of the mitochondria is not affected by the pretreatment.On the basis of these results it is suggested that only a fraction of the total mitochondrial cholesterol is readily available for cholesterol side‐chain cleavage and that this pool is increased by stress.The cytochrome P450 type II spectral changes induced by addition of pregnenolone and isocitrate to the intact rat adrenal mitochondria have also been studied. The effect of stress was to increase pregnenolone‐induced difference spectrum in adrenal mitochondrial two‐ to three‐fold compared with cycloheximide‐treated animals. Similar results were found for the isocitrate‐induced type II difference spectrum.The spectral changes are interpreted to mean that stress causes an increase in the cholesterol complex of side‐chain cleavage cytochrome P450. Metabolism of the remainder of the cholesterol in the mitochondria is limited by the rate of transport, or binding, to this reactive centre. It is proposed that the acute effect of stress, mediated by adrenocorticotrophin, is to increase the proportion of mitochondrial cholesterol in the readily available form perhaps by an increase in the rate of transport or binding of cholesterol to sites from which it can be readily metabolised.