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 I1 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 isocitrateinduced type I1 difference spectrum.The spectral changes are interpreted to mean that stress causes an increase in the cholesterol complex of side-chain cleavage cytochrome P460. 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.Side-chain cleavage of cholesterol is the first step in the production of corticosteroids in the adrenal cortex. The initial products of the reaction are pregnenolone and a six-carbon fragment usually isolated as isocaproic acid side-chain is thought to involve hydroxylation a t (3-20 and C-22 [2,3] but the exact nature and sequence of formation of intermediates still has to be established [4,5].I n common with other hydroxylases, cholesterol side-chain cleavage requires NADPH and molecular oxygen [6]. The enzyme system occurs in the mitochondria of...

The effect of calcium ions on the metabolism of exogenous cholesterol by rat adrenal mitochondria

“…It can be seen that in agreement with previous reports from this laboratory [6,10] the ether stress, which raises blood ACTH levels, caused the stimulation of pregnenolone production in these adrenal mitochondria in comparison of those from cycloheximide treated animals. On the addition of exogenous cholesterol to the mitochondria, from ether-stressed and cycloheximide-treated animals, it can be seen that pregnenolone production is stimulated.…”

“…Cholesterol side chain cleavage activity was determined as follows: incubations were carried out at 37°C in a buffer, pH 7.4, consisting of 250 mM sucrose, 20 mM KC1, 15 mM triethanolamine hydrochloride, 10 mM potassium phosphate, 5 mM MgC12, 0.2 mM EDTA, 0.1% bovine serum albumin (Type F, Sigma) and 0.2 mM NADP ÷, at a mitochondrial protein concentration of about 1 mg/ml and a total volume oi" 1 ml. Cyanoketone (2t~-cyano-4,4,17a-trimethyl-17/~-hydroxy-5-androsten-3-one), 6/aM, was included in the incubation to prevent the metabolism of pregnenolone to progesterone and further products [ 10]. Cholesterol was added in 10/al acetone to a final concentration of 100 #M 3 min prior to initiation of the reaction with 10 mM DLisocitrate.…”

“…Mitochondria were prepared from the adrenal glands as previously described [10]. The mitochondria were washed once with 0.25 M sucrose and finally resuspended in the same medium at a protein concentration of about 4 mg/ml.…”

“…Protein concentrations were estimated using the method of Lowry [12]. Mitochondrial cholesterol content was determined as described previously [10].…”

Specific Regulation of StAR Expression in the Rat Adrenal Zona Glomerulosa: an In Situ Hybridization Study

“…StAR protein is an important factor in steroidogenesis, because there is evidence that the delivery of cholesterol to the inner mitochondrial membrane is the ratelimiting step of steroidogenesis (Simpson et al 1972;Jefcoate et al 1987). Although regulation of this protein is well documented in vivo for the zona fasciculata, its regulation by factors stimulating aldosterone production has thus far been investigated only in vitro.…”