36s Biochemical Society Transactions ( 1 99 1 ) 20 18-hydroxycortisol stimulation in isolated guinea pig adrenocortical cells parallels that of both aldosterone and cortisol Alison M. Creedy, Daniel Burt, Christopher R.W. Edwards, and Brent C. Williams Department of Medicine, Western General Hospital, Crewe Road, Edinburgh, Scotland EH4 2XU. 180HF is a ZGEF structural hybrid because of the hydroxyl groups at both C17 and C18 positions. In virro it can be synthesised from F by the ZG enzyme p450CMO [ 11. This blurring of zonal function has led to the suggestion that conditions, such as dexsuppressible hyperaldosteronism, where plasma 180HF is high are a result of transition zone hyperplasia[2,3]. An alternative hypothesis is that excess 180HF is produced as a result of a defective ZF whereby an enzyme eg ~ 4 5 0 1 1 0acquires aldo synthase activity as a result of either a mutation or a change in microenvironment[4]. 180HF production has been shown to be stimulated by ACTH, and suppressed by dex in man[5]. It was also stimulated by a reduced dietary sodium intake which suggests a link with the p450CMO enzyme system, although there was no increased output in response to A11 infusion. The purpose of this study was to use isolated guinea pig adrenocortical cells as a model to investigate control of 18OHF secretion in virro. 6 adult male Dunkin-Hartley guinea pigs (500-900g BW) were killed by cervical dislocation and the adrenal glands swiftly removed into 0.9% (w/v) saline on ice. Glands were carefully trimmed of adhering fat, and kidney and liver tissue, weighed, and sliced into approximately 2mm2 size pieces with a scalpel in lOml of MEM equilibrated under 95%02/5%C02 in a petri dish on ice. Tissue fragments were transferred to a Teflon beaker and the supernatant removed and replaced with lOml digestion medium (MEM containing 1.5mg)ml collagenase). A Teflon paddle was attached to the lid of the beaker and the whole apparatus was incubated at 37oC with the addle stining the tissue fragments while they were gassed with 95k02/5%c02. After intervals of 15, 20, 30, 30 and 30 minutes, the supernatant was removed and the digestion medium replaced. All but the first of the supernatants were stored individually on ice. The 4 volumes of cell suspension were then centrifuged at 12000rpm for 5 minutes to obtain a pellet of cells. The resultin supernatants were discarded and each re laced with loml IF-KRb (0.3%BSA) equilibrated under 95%02/5kCO2. The pellets were gently resuspended using a wide-mouthed glass Pasteur pipette and incubated in a shaking water bath at 37OC for 15 mii.utes. The cell suspensions were then centrifuged again for 5 minutes, the supernatants discarded and replaced with another loml IF-KRB (0.3%BSA), resuspended again and filtered carefully through 100lm nylon gauze, thus pooling all the suspensions. This final cell suspension was centrifuged once more, supernatant discarded, and the pellet of cells resuspended gently in M199 (0.5%BSA;0.2% glucose) and preincubated at 37W for 30 minutes before incubates were set up in triplicate. 4 0 0 ~ 1aliquots of cell suspension (10~cells) were dispensed into 1.5ml conical microcentrifuge tubes on ice containing either 10-8M A11 or lO-9M ACTH (final concentrations) or raised K+ concentration (3.68.4mM) and vortex mixed. Incubation was for 1 hour at 37W in a shaking water bath, after which tubes were placed in ice to prevent further steroidogenesis. Tubes were then centrifuged at 20,OOOg for 2 minutes and the supernatant collected and stored at -2OW prior to steroid assays. F, 180HF, and aldo were all measured by radioimmunoassay procedures. Data was analysed by a paired Students r-test. lO-9M ACTH caused a 700% increase in secretion of F and 18OHF, but only a 50% increase in aldo secretion. 10-8M A11 had no effect on F or a 50% Abbreviations used: 180HF, 18-hydroxycortisol; F, cortisol; aldo, aldosterone; ZG, zona glomerulosa; ZF, zona fasciculata; p450CM0, cytochrome methyl oxidase enzyme; ACTH, adrenoconicotrophic hormone; AII, angiotensin 11; BW, body weight; MEM, minimum Essential Medium Eagle (Modified) with Earle's Salts; IF-KRB, Krebs-Ringer Bicarbonate buffer minus C a b and M g b ions; BSA, bovine serum albumin; M199, modified Medium 199 with Earle's salts; dex, dexamethasone.

Figure 1. % stimulation of aldosterone, 18-hydroxycortisol. and cortisol in response to ACTH and A11 (LHS)and increased K+ concenmtions (RHS). % stimulation +/- sem. n=3; * pe0.05

Increased K+ concentrations caused a dose-dependent increase in aldo secretion, and a similar though smaller increase in 180HF secretion, while having no effect on F. These results suggest that 18OHF secretion in virro shares characteristics with both that of aldo and F depending upon the stimulus. 1. Ulick, S., Chu, M.D. & Land, M. (1983) J. Biol. Chem. 258, 5498-5502 2. Connell, J.M.C., Kenyon, C.J., Corrie, J.E.T., Fraser, R., Watt, R. & Lever, A.F. (1986) Hypertension 8,669-676 3. Gomez-Sanchez, C.E., Gill, J.R., Ganguly, A. & Gordon, R.D. (1988) J. Clin. Endocr. & Met. 67,444-448

4. Ulick, S., Chan, C.K., Gill, J.R., Gutkin, M., Letcher, L., Mantero, F. & New, M.I. (1990) J. Clin. Endocr. & Met. 71, 1151-1157

5. Come, J.E.T., Edwards, C.R.W., Jones, D.B., Padfield, P.L. & Budd, P.S. (1985) Clin. Endocr. 23, 579-586