THE INFLUENCE OF THE SIDE CHAIN ON STEROL SIDE-CHAIN CLEAVAGE IN RAT ADRENAL GLANDS
IAIN F. CRAIG ª, J. IAN MASON b .* , KEITH E. SUCKLING ª and GEORGE S. BOYD ª
” Department of Biochemistry, University of Edinburgh Medical School, Edinburgh, EH8 9XD (U.K.) and ” The Cecil H. and Ida Green Center for Reproductive Biology Sciences and Departments of Biochemistry and Obstetrics-Gynecology, University of Texas Health Science Center, Dallas, TX 75235 (U.S.A.)
(Received October 2nd, 1981)
The cholesterol side-chain cleavage enzyme system of rat adrenal cortex, the enzyme catalyzing a rate-limiting step of adrenal steroidogenesis, was shown to metabolize a series of cholesterol analogues to pregnenolone. In the presence of Ca2+, rat adrenocortical mitochondria converted the analogue with two less methylene groups (C25) than cholesterol into pregnenolone at a faster rate than cholesterol. The analogues with one or three less methylene groups (C26 or C24) were metabolized at a similar rate to cholesterol. Lengthening the non-polar side chain produced analogues that did not appear to be metabolized. Studies of the metabolism of these analogues in isolated rat adrenocortical carcinoma cells showed that the C24 and C25 analogues were converted into pregnenolone much more efficiently than was cholesterol or the C26 sterol. The experimental findings are explained in terms of the differing ability of each exogenously added sterol to gain access to the active site of the sterol side-chain cleavage enzyme by passage through the membranes of the adrenal cell.
Introduction
A rate-limiting step for adrenal steroidogenesis is at the level of the cholesterol side-chain cleavage enzyme situated in the mitochondria of the adrenal cortex [1]. This reaction is catalyzed by a mixed function oxidase with a specific cytochrome P-450 (cytochrome P-450gcc) as the terminal electron acceptor and substrate binding component [2]. The reaction involves sequential hydroxylations at C22 and C20, followed by cleavage of the triol [3,4].
Several reports have indicated that Ca2+ is involved in the response of adrenal cortical tissue to ACTH [5,6]. Particularly Ca2+ might stimulate pregnenolone production from exogenous sterols by facilitating the access of substrate to the reac- tive center of the mixed function oxidase on the inner cristae [7,8]. Previously it has been shown that the nature of the sterol side chain is important for the ability of ACTH to regulate the activity of
the mitochondrial sterol side-chain cleavage en- zyme [9,10]. Cholesterol was shown also to be the preferred substrate for the liver microsomal cy- tochrome P-450-linked cholesterol 7a-hydroxylase [11]. We reported previously that a reduction in the length of the non-polar cholesterol side chain did not influence markedly the rate of pregnen- olone production in rat adrenal mitochondria ob- tained from non-stressed animals [12]. A similar conclusion has also been reached recently using a purified, reconstituted cytochrome P-450scc system [21]. A study of the metabolism of exogenous sterols by adrenocortical cells of normal rat adrenal cortex is difficult, due to the high endogenous cholesterol content of such cells. However, rat adrenocortical carcinoma Snell 494 cells have a relatively low cholesterol content and appear to be suitable models for the study of exogenous sterol metabolism in adrenal cells. In the present study we have investigated the ability of the sterol side- chain cleavage system in rat adrenal mitochondria and rat adrenal carcinoma cells to interact with and metabolize various nonpolar side-chain ana-
* To whom correspondence should be addressed. Abbreviation: ACTH, adrenocorticotropin.
R
R
R
R
C22
C24
C25
C26
R
R
R
C27
C28
C29
R =
HO
logues of cholesterol in the presence of Ca2+ . The structures of the analogues are illustrated in Fig. 1. The following abbreviations are used in the text for these analogues: C22, 23,24-bisnor-5-cholen- 3B-ol; C24, 5-cholen-38-ol; C25, 24-methyl-5- cholen-3B-ol; C26, 26-nor-5-cholesten-38-ol; C27, cholesterol; C28, 26 - nor- 27,27 - dimethyl - 5 - cholesten-3B-ol; C29, 26-nor-27-(n-propyl)-5- cholesten-38-ol.
Materials and Methods
Female rats (150-200 g) of the Wistar strain obtained from the Small Animal Breeding Station, University of Edinburgh, were subjected to diethyl ether anaesthesia for 10 min, a process which increases blood ACTH levels [13]. A mitochondrial fraction was prepared from the adrenal glands as previously described [10] and washed once with 0.25 M sucrose. The resultant pellet was resus- pended in the same medium at a protein con- centration of about 5 mg protein/ml.
Protein concentrations were estimated by the method of Lowry et al. [14], using bovine serum albumin as standard. Sterol side-chain cleavage activity was determined in a buffer of pH 7.4, containing the following constituents: 250 mM sucrose, 20 mM KCI, 15 mM triethanolamine hy- drochloride, 10 mM potassium phosphate, 5 mM MgCl2, 0.2 mM EDTA, 0.2 mM NADP+, 1 mM CaCl2 and 0.1% bovine serum albumin (essentially fatty acid-free) at a mitochondrial protein con- centration of about 1 mg protein/ml in a total
volume of 1 ml. The steroid dehydrogenase inhibi- tor, cyanoketone (2a-cyano-4,4,17a-trimethyl-178- hydroxy-5-androstene-3-one), was included in in- cubations (final concentration, 6 M) to prevent further metabolism of pregnenolone. The side- chain cleavage reaction was initiated with 10 mM DL-isocitrate, after the mitochondria had been in- cubated at 37℃ for 15 min with the sterols (final concentration, 100 uM) normally added in 10 ul ethanol. Aliquots (0.2 ml) of the reaction medium were transferred into 4 ml methanol to terminate the reaction at 2, 5 and 10 min intervals after the addition of isocitrate. 5 ml chloroform and 2 ml water were added to the extract. The organic layer was assayed for pregnenolone using the radioim- munoassay method described previously [10], based closely on that described by Abraham et al. [15].
A dispersed cell preparation of rat adrenocorti- cal carcinoma cells was obtained from a rat adren- ocortical Snell 494 carcinoma grown in 4-week-old male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA). These procedures have been described in detail previously [16]. Adrenocortical tumor cells (106 cells per 0.5 ml) were incubated in Krebs-Ringer phosphate buffer, pH 7.4, containing 0.5% bovine serum albumin (fraction V) and 0.2% glucose in the presence of 20 nM ACTH and 6 uM cyanoketone. Sterols were added in ethanol of final concentration less than 1%. The media including cells were assayed di- rectly for pregnenolone using the radioimmunoas- say procedure.
The various sterol analogues were synthesized as described in detail previously [17]. [7a-3H]- Pregnenolone (17-20 Ci/mmol) was supplied by New England Nuclear Chemicals (GmbH, Dreieichenhain, F.R.G.). All other chemicals were obtained from Sigma Chemical Company, London U.K .; BDH Chemicals, Poole, Dorset, U.K .; or Koch-Light Laboratories, Colnbrook, Bucks, U.K.
Results and Discussion
The adrenal mitochondrial production of preg- nenolone from the sterol side chain analogues in the presence of 1 mM Ca2+ is illustrated in Fig. 2. The endogenous rate of pregnenolone production was low, and after 2 min was negligible. This was due presumably to the reduction in endogenous
Pregnenolone Formation (nmol· mg-1 protein)
30
C25
C27
20
C26
C24
C22
10
C28
Endo
C29
0
O
5
10
Time (min)
adrenal mitochondrial cholesterol previously noted for diethyl ether-stressed rats [6]. It may be seen that the C25 sterol was metabolized more effi- ciently than C24 and C26 sterols, which were metabolized at a similar rate to cholesterol. The C22 sterol was metabolized at a reduced rate com- pared to cholesterol. The C28 and C29 sterols were not metabolized to any significant extent. In previ- ous experiments a marked biphasic response of pregnenolone production from exogenous cholesterol was observed [12]. This was attributed to depletion of the available substrate cholesterol pool in mitochondria. A monophasic response was observed in the presence of Ca2+ .
There are at least two possible explanations for the pattern of pregnenolone production from the exogenous side chain analogues. (a) The relative affinity of each analogue for the Ca2+ -treated, ACTH-stimulated enzyme may be different; (b) the relative translational mobilities of each ana- logue in the membrane may be different. The translational rates may themselves be affected by changes in the physical state of the membrane brought about by Ca2+. As our previous results [12] have shown that C22, C24, C26 and C27 sterols were equivalent substrates for the enzyme from
unstimulated mitochondria, it is unlikely that the difference in rates of metabolism shown in Fig. 2 are due to differences in the affinity of the enzyme for each substrate.
The current evidence favors the concept that the availability of cholesterol to the enzyme regu- lates pregnenolone synthesis. Simpson et al. [8] have postulated the existence of a cholesterol pool that is associated specifically with cytochrome P- 450scc and suggest that a possible rate-determining step for cholesterol side-chain cleavage is the ease with which the remaining cholesterol within the mitochondria forms an active substrate cleavage adduct with cytochrome P-450scc. The effect of Ca2+ on the lateral mobility of membrane compo- nents is well known [18]. The presence of Ca2+ can induce phase separation of lipids by formation of ‘crystalline’ patches of calcium-bound phospholi- pid with a consequent extrusion of phosphati- dylcholine from these rigid areas of lipid [19]. The lipid phase separation could result in the displace- ment of cholesterol from one region of the mem- brane to a second region which is more accessible to the cytochrome P-450-linked enzyme. The dif- ferential metabolism of the various cholesterol side -chain analogues could then be explained in terms of their relative abilities to migrate in the plane of the Ca2+ -treated mitochrondrial membrane.
Pregnenolone Formation (nmol /106 cells)
6
C25
5
4
C24
3
2
C26
-C27
1
Endo
0
0
2
4
Time (hr)
We have reported previously that addition of polar sterols such as 25-hydroxycholesterol to rat adrenocortical carcinoma cells results in marked stimulation of pregnenolone production [20]. In contrast, a similar stimulation was observed with cholesterol only when it was added as a lipopro- tein complex [20]. We have examined the ability of these rat adrenocortical carcinoma cells to metabolize the various sterol analogues to preg- nenolone on addition of the sterols in ethanolic solution. The results of such a study are shown in Fig. 3. It can be seen that addition of only C24 and C25 sterols results in a stimulation of pregnenolone formation. It would appear that these two sterols can traverse the plasma membrane directly and reach the mitochondrial site of metabolism whereas the C26 sterol and cholesterol require specific transport systems such as those involved in lipo- protein uptake in order to transverse the plasma membrane [16].
The results presented demonstrate that the rat cholesterol side-chain cleavage enzyme system of adrenal cortex can metabolize a variety of sub- strate analogues to pregnenolone, indicating an imprecise structural requirement for the sterol side chain, as has also been reported for a purified reconstituted preparation of cytochrome P-450g SCC [21]. It is interesting to note that in the recon- stituted system [21], as in intact adrenal mitochondria [10], hydroxy sterols such as 25- hydroxycholesterol were metabolized more rapidly than cholesterol. Because of its greater polarity 25-hydroxycholesterol cannot take up the same environment in the phospholipid bilayer of the mitochondrial membrane as cholesterol. The in- fluence of the mobility of the sterol in the mem- brane will, therefore, not be of significance here. In the present work in the intact cells the C25 and C24 sterols were metabolized more rapidly than cholesterol. In contrast, these sterols were oxidized at the same rate as cholesterol, or slightly slower, by the reconstituted system [21]. These compounds are taken up into membranes in a way essentially similar to cholesterol, but they do not interact with phospholipids to the same extent [17,22], resulting in a more fluid membrane. It is likely that the presence of the whole cellular apparatus, including membrane and transport systems, can have a sub- stantial effect on the rate of metabolism of endog-
enous sterols. Comparison of the present studies, in which all these effects may operate, with those in the purified system suggests that the rate of sterol movement within the cell may indeed be important and needs to be considered when cells and membrane-bound systems are examined. It is likely that in other membrane enzyme systems the rate of transport of a membrane-soluble substrate, which may be a membrane component like cholesterol, may be rate-limiting, rather than the enzyme-catalyzed reaction itself. For example, re- cent experiments show that both in a reconstituted preparation and in rat liver microsomes the rate of cholesteryl ester formation catalyzed by acyl-CoA: cholesterol acyltransferase can depend entirely on the rate of delivery of cholesterol to the enzyme through the membrane [23].
Acknowledgments
This work was supported in part by Grant CA-30253, awarded by the National Cancer In- stitute, DHHS (J.I.M.) and by a Medical Research Council Programme Grant. We thank Ms. Lydia Morris for expert editorial assistance.
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