Biochimica et Biopkysica Acta, 1134(1q92) 143-148 1992 Elsevier Science PublishersB.V. All rishls re~erved 0167-4889/92/$05.00


BBAMCR 13115

Cytochrome P-450~7~,~ya~e-mediatingpathway Üf androgen synthesis in bovine adrenocortical cultured cells Takcshi Yamazaki, Keiko Nawa, Shire Kominami and Shigeki Takemori Faculty of InteRbred ~rt$ and Sciences, Hiroshimu Univer~ily, Hiroshi~e (~apan)

(Received3 October1991)

Key words: Androgen synthesis; Primaw cell cullexc; BOvineadrenal; Cytochxomep-450y~.,r,~: Sieroid~gezle~s

Cytochromc P-450 eT~.lya~=mediating pathway of dehydro~piandrostcrone (DFIA) formation from pregnenolonc was investigated in primary cultures of bovine adreno¢orticai fascieulata-rcticular[s cells. To detelmine whether DHA formation proceeds p~dominantiy by suecessive monooxygenas~ reactions without tT~,-bydroxypregnc,olun© kaving P-450t~..b.~ the ceils were incubated with [~4CIpregneonlone and 17u-[SH]hydroxypregnenolone in the presence of Trilmtaae. Results of the double-substrata double-label ~pcriments indicate that in the presence of high concentratien of pregnenokme rncsz of DHA was formed directly from pgegnenolone by the successive reactions. Since the concentration of pregnenolone usually exceeds that of 17a-hydroxypregnenotone in the adrenal slands, DHA is concluded to be formed predominantly by successive reactions from presneno]one without 17a-hydrmypregneonlonc leaving P-450~7,.~~ in rive. By chronic ACTH treatment, the activities of 17u-hydroxTlation and DHA formation in adrenocortical cultured cells became higher concomilamly with i11¢ increase of P-'~5(~i~a.l~e.~ content. Most of DHA was found to bc formed by successive reactions from presnenoloa~ ~ under such conditions.

Iatredut'lion T h e adrenal cortex synthesizes adt.'nal androgens other than corticoids from cholesterol through actions of cytochromes P-450 and 3/]-HSD [1]. Cholesterol in mitochondrial inner m e m b r a n e s is converted to pregnenolone by P-450s= and pregnenolono is transferred to the endoplasmic reticulum where it is convetted to some extent to androgens such as D H A and andmstenedione by reactions of 3p-HSD and P4-~!7~,1~a~e. P-45017~,~a.~, catalyzing 17a-bydroxylation and the C17-C20 bond cleavage (C17,C20-1yase) reac-

Abhreviatiorm: DHA, dehydroepiandrmterene; 3~-HSD, 3~-hydlexy-ds-stereid:NAD~ d:~-~isomerase; ELISA~enzTme-tinkedimmuaosot'benta.ssa.V;P-45017~.~p~e, c~lochmmeP-450havins steroid lTee-hydt'o~fflaseand C17-C20]yas~ activities(P-450XVlIAI); P450~, cylcehromeP450 Ilavhlgcbokst=rol ~id=chain ckpage acth,ity (P-450XIAI): P-45On:~,cytcehmme P-450 havinssteroid I IJ~-hydrex~aseactivily(P-450XIBl~,P-4..qOc~i, ~tochrome P-450 having 21-hl~dm~las=activity(P-45-XX1AI); P4~,~m, cyt~chgemcP-450havingaromata,~ aclivity(P-450XIXAD; P'45014DM,~ochroule P'45U h~rirl~ lanosterel 14.demetb,ylaseactivity (P-450LD; P-450a (P-.4.5OIICL3).

Cml'espondence:S, Takemofl,Faculty.of Integrated Aa'tsand Sciences, Hiroshima University, Higachiseada-Machi,Naka-Ka, Himshima 730, Japan.

tion, situates at a coneeivaMe regulatory point between formations of ~drogens and slucocmttcoids [2-4]. Production of adrenal a n d m s e n and glucocortieoid as reported to be controlled scpatatehr in the adrenals bet the details of the intraccllular mcebanisrn have not been clarified yet [5]. D H A formation from presnenolone by P-450,7,,.tme may p r o ~ e d in two alternative reaction pathways as shown in Scheme L In pathway 1, pregnenolone is 17a-bydroxylated and the produced 17a-bydrozypregnone[one dissociates fxom P-4~lT~jxa~ into the reaction medium. After accumulation of 17a-bydro~ypregnenolone, it is subsequently metabelized to D H A by the C17,C20-1yase reaction of P-450,7=~,,~. In pathway 2, pregnenolone is convened to 1 7 a - l ~ s x t e n olone, some of w h k h is converted suco:asiveb" into D H A wit;~OIdtL L d . ~ l i i ~ g ~ u . l t ~ active site of F450tT,,~e. In the previous studies on andrc~en formation in reaction systems with P450wo.~m-pmteolipesomes and with •zinea-pig adrenal ~ the rates o f andreetenedioue formation f, v m progesterone were not changed by the addition of P450c2rPmteo]iposomes, althouBh a considerable amount of produced 1 7 a - b y d ~ t e r o n e in the medium was decreased by the r e a c t i ~ of added P-45Uc~t, suggesting that the pathway 2 is predominant for androgen formation from progesterone [6,7]. Some detailed stud-




in KRBG buffer supplemented with 1.5 me/ml eollagenase (Waho Pure Chemical, Osaka) and 0.l mg/ml DNase Type I (Sigma, St. Louis) and incubated for digestion in a shaking water bath at 37~C for 30 rain. The turbid supernatant was discarded by decantation and the tissue fragments were resuspended in the same buffer for digestion and incubated for an additional 30


rain. T h e enzyme-treated tissues w e r e resuspended in

2 ~JP~gV


Schematicdiagram of reaclion sequence from pragnehelenato DHA catalyzedby P-45017~,1~~. 111pathway1, an intermediate, 17ce-h?droxypregneno]oneis releasedfromcymchrome/'-450. ~heme 1,

Cytochrome P-450 binds again to the intermediate to produce DHA. In pathway 2, the intermcdLate is ant r©leascd from the active site of P-450 daring conversion to DHA. P-450, P-¢501?~,~r~,~;Preg, pregne-

nolonc; i%OH-P, 17tx-hydmxyprcgncnoloac.

ins have been carried out on the mechanism of the successive monooxygenase reactions in testicular mi. crosornal ¢ytochrome P-450 [8,9]. Successive monooxygcnase reactions without release of intermediate from cytochrome/'-450 have important roles in steroid metabolic pathways: cholesterol side-chain eleava?e by P-450~ [10], aldosterone synthesis from D e c by P450jr p [111, estrogen production by P-450 . . . . [12], Ianosterol 14-demethylation by P-450t4DM [13] and progcsterone oxidation by P-450, [14]. These successive monooxygenase reactions have been well investigated in the reconstituted system and in the organella levets, bet little have been done in cellular systems. in order to clarify whether such successive monooxygenase reactions of P-450tT~,ty~s. are also functioning predominantly in cellular systems, DHA formation from pregnenolone was studied in primary cultures of bovine adrenocortical fasciculata-reticularis cells. The chronic effect of ACTH on DHA formation was also investigated. Materials and Methods

Preparation of cells Two or three fresh adrenal glands of Holstein-Friesian cows were obtained from a local slaughterhouse and transported within 30 rain to the laboratory in ice-cold phusphate-buffered saline. Following removal of fat, the glands were bisected. The medulla was scraped off and zonae fasciculata-reticularis were separated from the zona glomerulosa and capsule with a blade. The zonae fasciculata-reticularia were finely minced with scissors and washed several times in Kxebs-Ringer-bicad~onate buffer containing 5 m.M glucose and 2 mg/ml bovine serum albumin (KRBG buffer). The fragments thus obtained were suspended

KRBG buffer and pipetted up and down about 50 times using a wide bore pipette. The dispersed cells were then separated from undigested tissue by filtration through three layers of cheesecloth. The cells were collected by c e n t r i f u g a t i o n at 150 x 8 f o r 4 rain and

washed four times with the KRBG buffer. The cells suspended in 5% dimcthylsulphoxide were frozen at -8(PC prior to use. Proliferation and steroidogenic activity of the celts were not affected significantly by the freeze-thawing. After thawing, the ceils were washed three times in KRBG buffer and suspended in Ham's F-12 medium containing 10% fetal calf serum (Riggs, Dubuque) and antibiotics (2.5 mg/l amphotericin B, 25 mg/i gentamiein, Sigma, St. Louis; 100 mg/I streptomycin, 10000 U / I penicillin, Meiji Seika, Tokyo). (5-7). l0 s cells suspended in 2 ml of medium were cultured in 35-ram diameter wells of 6-well tissue culture plates (Iwaki Glass, Tokyo) in a 032 incubator (atmosphere: 95% air, 5% CO 2) at 37~[3. The medium was replaced each day. The cells had become confluent after 2 or 3 days cultivation, and then the monolayer cells, (1-1.5)" 106 per a well, were used for the experiments. In some experiments, the grown ceils were incubated with the medium containing 0.2 )tM ACTH 1-24 (Cortrosyn, Daiichi Pharmaceutical, Tokyo) for several hours as described in the text.

Steroid metabolism The labeled steroids of 17a-hydroxy[7.SH]pregnenolone (4.9 Ci/mmol; Amcrsham) and [7-3I-I]pregnenolone (22.6 Ci/mmol; New England Hudear Research Products, Boston) were purified by HPLC prior to use and diluted with non-labeled 17¢-b.ydroxypregnenolone (Sigma, St. Louis) and pregnenolone (Naealai Tesque, Kyoto), respectively, at a specific radioactivity of 0.33 Ci/mmol. The purchased [4-]4C]pregnenolone and [4-14C]progesterone (57.2 and 60.0 mCi/mmol, respectively; New England Nuclear Research Products, Boston) were used without further purification. The medium in the cultured plate was changed to 1.5 ml KRBO buffer and the plate was placed in a water bath incubator at 3"PC In experiments of P450=?,.~y== reaction, 3/3-HSD activity was inhibited by the addition of 2 ~M Trilostane (kindly provided by Dr. C.R, Jefcoate, University of Wisconsin, and Mochida Pharmaceutical Industries, Tokyo) to each welt before starting the reactions. The reaction was initiated by addition of substrates, i.e. [3H]pregne-

145 nolone, 17et-[3H]hydroxypregneflolone or mixture of [t4C]pregnenolone and 17a~[3H]hydroxyprcgnenolone, previously dissolved in 5 ~l of dimetlwlsulphoxide. After incubation at 37°C, the reaction buffer of 0.5 ml in the well was pipetted. The steroids secreted into the buffer were extracted with 1 ml chloroform containing 150 nmol pregnenolone, 45 nmol 17a-hydro~pregnenolone, 1130 nmol DHA and 2.5 nmol progesterone in order to get appreciable peaks on UV-detector of HPLC. The chloroform layer was evaporated to dryness under a air stream at room tem=,'Jerature and dissolved in a 0.1 ml mobile phase of the HPLC system. The extraction efficiency was estimated from total radioaetivities in medium and extract, which was more than 90%. The cells in each well were counted with hemocytometer after resuspension in phosphate buffered-saline containing 2 m g / m l trypsin. In the assay for DOC formation by the cells, 5 t~M bifonazole (Sigma, St. Louis) and 50 p,M melyrapone (Sigma, St. Louis), inhibitors of P-450tT,~,lyas: and P450H#, respectively, were added to the reaction buffer instead of Trilostane. Reaction was started by addition of [J4C]pregnenolone and [3H]progesterone. The incubation and extraction procedures were essentially the same as above, except that the steroid carriers were 75 nmol pregnenoione, 2.5 umol progesterone and 5 nmoi DOC in l ml chloroform. The determinations of enzymatic aeiti,,ity were repeated for two or three times using the typical preparation of the cells. In one determination of the activity, the datum at each point was the average of those from three dishes. HPLC analysis of steroids To determine the amounts of metabolized steroids, the extracted steroids were separated and collected by HPLC (Tosuh CCPE and UV-8011) equipped with a Tosoh AS-48 atttosampler and Gilson fraction collector Model 21)2 Pregnenolone, 17a-bydro~pregnenoione and DHA were chromatographed on a LiChrospher RP-18 column (5 p.m, 250 × 4 ram; Merck, Rahway). using a mobile phase of aqueous acetonitrile at a flow rate of 0.5 ml/min at the following concentrations; 45% isocratie for 30 rain, a linear gradient 45-65% for 5 rain and then 65% isocratic for 30 rain. Authentic steroids of 17a-hffJroxypregnenoione, DHA, progesterone and pregneno!0ne were eluted at retention times of 27, 3~, 48 and 53 rain, respectively. Pregnenolone, progesterone and D e c were separated on the same column with an isocratlc mobile phase of 60% aqueous acetonitxile at a flow rate of 0.5 ml/min. The retention times were 14 rain for D e C , 27 rain for progesterone and 31 rain for preBnenolonc. Steroids were detected by ultraviolet absorbance at 220 nm and were collected. Radioactivities of the fractions were measured in 2.5 ml selmillator (Clear-sol; Nacalal Tesque, Kyoto)

using an Aloka LSC-701 liquid scinti|larion spectrometer with 50% efficiency for 3H and 95% for ~*C EL1SA assay

P'450tT~.L~.~ content in the cultured cells was estimated by ELISA assay using 96-well carboplate (Sumitomo bakelite, Tokyo) and anti-P-450~7 ~ ~ rabbit ]gG~ prepared as descn'bed previously ['15]. The assay was conducted essentially as desen'bed by Friguet et al. [16]. The carboplate was activated by 10% let hyl-3-(3-dimethylaminopropyl)carbod iimide hydrochloride for 5 h prior to use, which was the cross-linker between proteins and the plate. The cells were scraped from the cu|mred plate, solubilized with phosphatebuffered saline conlaining 1% Tween 20, diluted to a protein concentration of 0.15 mg/ml and cross-linked 1o the carboplate overnight at 4°C. After blocking the remaining binding sites on the plate with skim walk (Difco, Detroit), the plate was treated with anti-P45017o.tyL~ IgG for 4 h, and then with anti-rabbit lgC_.,(H + L) goat.-lgG conjugated with horse-radish pe~xidase (Bio-Rad, Richmond) for 4 h. Horse-radish peroxidase activity was measured using 2,2'-azino-di(3-cthytbenzthiazoline sulphonate) and H202 as substratus. Results

DIdA formaJion in bo~,ine adrenoconical celia For precise measurement of DHA formation, the reaction of 3/~-HSD must be blocked. 2 ~tM Triiostane was sufficient to inhibit this activity completely but had no effect on that of P-4$0~,,jr,~. In subsequent experiments with bovine cultured cells, 2 p.M Tdlostane was added to the ruction medium ~x~pt for the experiment on the conversion of pregnenolone to D e c . After bovine adreno~ortical fasciculata-reticulatis cells had been incubated v,ith [SH]pregnenoione for 20 rain, the steroid metabolites were s~paxated by HPLC. As shc~vn in Fig. 1, only two radiaactive metabolites corresponding to l T a - b y d ~ r e g n c n o l o n e and DHA could be detected on the chromatogram. The fraotions of DHA and 17a~bydroxyp~gnenoione were further chromatographed with a few different separation conditions including different type of HPLC columns but no sign of further separations waa ob~rved,indicating the peaks to be b e e from contamination by other radioactive mctabolites. Water soluble DHA-sulphate formation was aegligible under the reaction conditions, since radioactivity in the water layer was less than 0.4% that of DHA after three extractions with dichloromethane. Fig. 2a shows the time dependence for the formation of 17,,-hydroxypregnenolone and DHA from 1 ~M pregnenolone in adrenal cultured cells. Concentrations of the products increased linearly with time up to



ill i 1;'

,;ill, ' ill- o=;'


Ot - - t ~ m ~ t ~ m - - J 17-0H-P DttA




Fig. I. HPLC patterns of steroid melabolites produced from0.5 FM [31"I]prcgneaulnnein bovineadrcnu¢orli~a* cultured ccl~s fur 21}rain at 37~Cin the presence or 2 /~M Trilostane. Incubationpr~:edare and method for HPLC analysiswere as described ia Materials and Methods. Radioactivily of elaled steroids from HPI.C was detected using a Beckman model 171 redioisot~pe detector. At'ro~sindicate positionsof authenticsteroids. I, 17c~-hydroa~pregnenolone;2, DHA; 3, progesterone;4, pregnenolone.

20 rain. The rate of DHA formation from pregnenolone was about one tenth that of 17~-hydroxypregnenolone. Fig. 2b shows the time-course of DHA production by C17,C20-lyase reaction from 1/zM 17a-hydroxypregnenolouc in the adrenal cultured cells, The rate was basically the same as that from pregnenolone, as shown in Fig. 2a, although the concentration of 17a-hydroxypregnenolone in the medium of Fig, 2a was much tess than that in Fig. 2b.

Double-substrate double-;abel erpenment DHA formation from pregnenolone may proceed in two alternative reaction pathways shown in Scheme l. To determine which reaction pathway is dominant for DHA production in cultured cells, the cells were incubated with [t4C]prvgnenolone and 17a-[aH]hydroxy-

,oaol/ 0


10 T i m e (rain}


o Q





Time (min)

Fig. 2. Time-courseof metabolismof 1 ,~M laHlpregneaolone (a) and I t~M |7¢-[~H]hydro~pregncnolonc(b) by bovineadronucortical cultttred e~llsin the presenceof 2 ~M Txilostane,Incubationand analytical procedures are described in Materials and Methods. Values are the means c~ triplicate determinalions using ~t Wpical preparation of cells. SD is smafier than saqnbol size. IT-OH-P, 17a-hydmxypxegnenolon¢, • •, 17a-hydroxypregnenolone; ~, DHA.rightaxison (a).






P¢oge- ru-d-



Fig. 3. Double-substratedoable-label experimentsin bovine adrenocortical cidlu~ed cells. The ceils were incubated for 20 rain with I #.M [14CIpregnenolone, t p-M 17a-[aHlhydro:oypregnenoloneand 2 tJ.M Triloslanc in (a) and idsowith 1 /rM [31-I]prcgnenulune,1 p.M [~4C]'prognsterone,5/tM bifona2n]nand 50 o.M m~*,.,ral~mein (b). Incubation and analytical pmoeduresate described in Materials and Methods. Black bo~s indicate 14C-labeled steroids. Pars with dots indicate all-labeled steroids. Values are the means of duplicate determinationsusinga typicalpreparationof cells, iV-OH-P,l?e~-hydroxTpregnenn~nne.

pregneno|one at t h e same concentration. As shown in Fig. 3a, the concentration of 17a-[ 14C]hydrox~regnenalone produced from [14C]pregnenolone was much less than that of 170~-[aH]hydroxypregnenolone. If most of the DHA was produced by C17,C20-1yasc reactions from the released 17~.hydroxypregnenolone, the ~H/t4C ratio for D H A ought to be similar t o that for 17a-hydroxypregnenolone. However, the 3H/14C ratio for DHA was much lower than that for 17a-hydroxypregnenolone. Thus, in the cultured cells, most [t4C]DHA was produced from pregnenolone directly by the pathway 2, without the intermediate leaving P.450tT,,lya~. Fig. 3b shows results of the double-substrate double-label experiment on D e C formation from pregnenolon¢ in cultured cells. Pregnenolone is convetted to D e c via progesterone as an intermediate by two different enzymes, 315-HSD and P-450c21, in the presence of bffonazole and metyrapone, inhibitors of P-450=7,,~ty=~: and P-450=t0, respectively. After incubating the cells with [aH]pregnenolone and [14CJprogesterone at the same concentration for 10 rain, the 14C/all ratio for D e c appeared similar to that for progesterone as shown in Hg. 3b. This shows that the significant difference in the ratios of ' * C / ~ H for the intermediate from that for the final product can not occur in reaction pathway 1 of Scheme I. After incubation of the cells with various concentration of 17¢-[3H]hydroxypregnenolone and a constant concentration of [ i, C]pregnenolone, DHA and 17a-hydroxypregnenolone produced in the medium were determined as shown in Fig. 4. In the absence of 17a[31-1~hydroxypregnenolone, the amount of produced 17~-[l*C]hydroxa,pregnenolone was about 200 nM unde: this condition. It can be estimated from the dependence of [aH]DHA production on 17a-[al-I]lJydroxy-

147 droxylatiotl by 17=-hydroxypregnenolone, since competition has been observed for P-450t~¢,~,~ reaction between progesterone and 17a-hydroxyprosesterone [4,7]. However, the amount of [3[-]]DHA did not exceed that of [t4C]DHA even when the cells were incubated with t7a-[~H]hydro~pregncnolone and ['4C]pregnenulouc at the same concentration.



20 I


-,..., 0


0.5 1.0 13HI17-OH-P (1JM) Fig. 4. Steroid metabolismin bovine adrellooortical cultured cells as a functionof [Ta-[~H~hydroxy'prcg~enoloonconcenlyation.Th~ cells were incubatedfor 10 rain with I ~M [14C~'pregnano~oneand various concenlratlans Of ]Tn-[SH]bydro,'~preg~nenulone in the presence of 2 /.~M Trilcelan¢. Incubatiun and analytical pr~:edu~es ~re des~ibcd

in Materialsand Methods. Values ~_~ !he mca,s of duplicate determ|nations.FI-cluation of data are smaller Ihan symbolsiz¢.The data were reproducible in four individual experiments with cells from different adrenals. ]7-OH-P, 17~-hydroxypregaenolone~~ 3 ~ o , [t~C]DHA; n [3, I~I-I]DHA; • e, []~C]lTa-h~d~o~pregnenolone(risht exis~ pregnenolone concentration that the maximum prodoclion of [t4C]DHA from 200 nM 17a.[t4C]hydrosypreg. nenolone was about ],/5 of the observed amount of [uC]DHA. Thus, most of [t4C]DHA can be considered to be produced directly from [l*C]hydroxypregnenoione by pathway 2. With an increase in the concentration of lTa-[SH]hydroxypregnenolone, the formation of [3H]DHA increased but ~[:at ,~f [14C]DHA and 17~[t4C]hydroxypregnenolone de.creased. T h e decreases can be attributed to competitive inhibition of 17a-hy-

toe e











i, 24


AClrH Tme, t m e m t { h o u r )

Fit 5. Effecls o[ ACTH on the formationel slemkl metabolitesand P-4501.~l~e.,¢ contetlt in bovine adrenocortifal ¢ttltered cells. Cells were ipcubated with 0.2 p.M ACI'H fo~ several hums as shown ia the fleers. Asses were conductedwith I FM [14C]pll.~gllenoloneand 0.5 tim 17a-lsl-I~roxypregne unions in the presence of :2 /.~M Trilostane. lneubatioa and analyticalprocedures for the mctabolize¢l steroids and P-450 am described in Materialsand Melhods.VaJues are the meansof duplka~ determiantionsu~inga lypicalple0at tins of cellS. Flsftuati0~ of the data are smaller than symbolsize. The data were reproducible i0 two individual experiments ruing cells from different adrenals. 17*OHoP,17a-hydto~qfpresnonokme;P4.~0, p-450t?e,tmte.• 0, [t4e~l? a-hydroxsrp~egneaolone; ~ o, [14C]DI-IA; [3

,12, [SH]DHA; • axis),

A, (right

Effects o f ACTH on DHA [ornmtion The chronic ACTH treatmem of bovine adrenocortical cells has been shown to result in increased P-45017,~jy~= content and activity [17]. As shown in Fig~ 5, P-450~7,," ~ content and pregnenolone 17a-hydroxylas¢ activity in the cells increased concomitantly by treatment with 2 /aM ACTH for several hours. T h e degrees of increase in successive reactions (pathway 2) and ClT,C2~lyas¢ activity by A C T H treatment were determined by incubating the cells with [laC]pregnenolone and 17a-[31-1]hydroxyprcg ncnolone. 17a-itydroxylation, C17,C20-[yase reaction and successive D H A production (pathway 2) were also enhanced by this treatment. D H A was formed predominantly by the pathway 2 in the cells even with ACTH treatment. Discussion There are two possible pathways for the synthesis of DHA from pregnenulone, as shown in Scheme I. When the concentration of pregnenolone was higher than that of 17~-hydroxypregnenoloue, pathway 2 (without the intermediate leavin8 P-450) proceeded predominantly for D H A formation in bovine adrenocortical cultured cells, as shown in Fig. 4. The concentration of pregnenolonc has been reported as ll-16-fuld higher than that of 17a-hydroxsq)regnenoione in guinea-pig adrenal glands [18], and a few times higher in the cultured medium of b o v i ~ ~drenocorticat cells [19]. The present results thus pm~qde strong indication that most D H A is produced from pregnenolone by pathway 2 in the adrenal cells. McCarthy and Waterman, however, found for D H A to be formed from pregnenolone by reaction pathway 1 Lq bovine adrenal m i ~ e s [20]. They incubated the microsmnes with a low concentration of pregnenolone (0.1 p.M) in the presence of cyanoketone for a Iomg period of time ((30 rain). Under this condition, nearly all the i;re~gnenolone was consumed and the accumulated 17a-hydroxs~presnennines was further converted to DHA by the CIT, C2Olyase activity of P-'4501Ter.b~ , TO apply double-substrata double-label experiments on a cellular system, it is required for exogenous ste.,oids to be diffused rapidly into intracellular membrane and mixed well with endogepous steroids. The transfer rate of steroids across biological membrane is adequately rapid, since the diffusion of steroids between aqueous medium and lipids bilaycr was reported

148 to be very fast compared with the reaction rates of

P-450,7,,,~ and P-450cH in the proteolipos~ma! system [6,21]. Furthermore, the lateral diffusion of lipidlike molecules in biological membranes is faster than 10 -9 em2/s [22]. Since the rate of electron transfer from NADPH-cytochrome P-450 reducta~ to cytochrome P-450 is less than l0 s - i in microsomes [23], the steroid left from cyto0hrome P-450 should be diffused over the membrane of the endoplasmie reticulum prior to the next reaction. Homogeneous distribution of endogenous and exogenous steroids is evident from the resulta of experiments on reactions from pregnenolone to D e c via progesterone which are catalyzed by 3~-HSD and P-450c2 ~ in the endoplasmic reticulure. In this reactions, the t+C/ZH ratio for D e c secreted from the ceils was almost the same as that for progesterone in the medium as shown in Fig. 31>. This shows that exogenous [ mC]progesterone in the medium Jiffuses rapidly into the endopla.~nic reticulum in the cells and mixes well with endogenous [SH~rogesterone prior to their conversion, This study indicates that pathway 2 of Scheme 1 (successive reactions without the intermediate leaving P-450~o.~a ~) is dominant for the formation of adrenal androgen. However, regulation of the successive androgen formation has not been clarified yet. Thus, the effects of ACTH on the successive DHA formation was investigated in the bovine adrenal cultured cells. As shown iu Fig, 5, 17~-hydroxylatinn and DHA formation were stimulated similarly by chronic ACFH treatment of the cells. This activation is due to the increaee of P-450~o.~a ~ content, by a factor of more than 10 foflowing ACTH treatment for 24 h. Preliminary egperimcr~ts indicated that NADPH-cytechrome P-450 reductase content in the cells increases only 1.6-fold even after 24 h trcatmem (data not shown). Dee et aL [24] found the reductase to increase slightly under almost the same experimental conditions. Although the ratio of the reductase to P-45017,~,~e decreased by ACI'H treatment, that of 17a-hydroxTlation to D H A formation remained essentially constant. Thus, in bovine adrenocorticai cultured cells, the ratio of the reductase to P-450~7~.iya~e may not have a significant effect on the regulation of DHA formation. Admowledgments

The authors are grateful Dr. A. Kawahara (Hiroshima University) for his suggestion regarding the

cell cultures. This work was supported in part by a Grant-in-Aid from the Ministry of Science, Education and Culture of Japan.

References L Takemori, S+ and Komtnaml, S.

Cytochrome P-450(17 alpha,lyase)-mediating pathway of androgen synthesis in bovine adrenocortical cultured cells.

Cytochrome P-450(17 alpha,lyase) mediating pathway of dehydroepiandrosterone (DHA) formation from pregnenolone was investigated in primary cultures of...
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