The effects of ACTH, serotonin, K + and angiotensin analogues on 32 P incorporation into phospholipids of the rat adrenal cortex: basis for an assay method using zona glomerulosa cells

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Abstract
The effects of various concentrations of serotonin, ACTH, K+, angiotensin II (AII), angiotensin III (AIII) and [Sar1]angiotensin II (SAII) on steroidogenesis and the incorporation of 32P (after preincubation to near equilibrium with the ATP pool) into phosphatidylinositol (PI), phosphatidic acid (PA) and phosphatidylcholine (PC) in a preparation of capsular cells from rat adrenals, consisting of 95% zona glomerulosa (z.g.) and 5% zona fasciculata plus reticularis (z.f.r.) cells, were investigated. Serotonin and ACTH stimulated steroidogenesis in the usual manner but had little or no effect on 32P incorporation into any of the 3 phospholipids. AII, AIII and SAII stimulated steroidogenesis and also 32P incoporation into PA and PI (maximally to about 280% of control values) but not into PC. These results taken together with other data on effects on the cAMP output and Ca2+ fluxes of z.g. cells suggest that stimulation by ACTH and serotonin is mediated by cAMP as 2nd messenger. The angiotensins probably act through Ca2+, with associated changes in phospholipid metabolism. The 32P incorporation into PA as a function of Ig concentration of AII was linear and showed a reasonable index of precision (0.36 .+-. 0.03, 8 experiments; 0.23 .+-. 0.02 for a further 8 experiments) and correlation with steroidogenesis. The corresponding incorporation into PI showed a maximum effect and a much poorer index of precision (1.02 .+-. 0.30, 4.69 .+-. 3.7) over the same full range of AII concentration used. The effects of AIII and SAII showed similar characteristics for 32P incorporation into both PA and PI, but, as for stimulation of steroidogenesis, at higher concentrations for AIII than for AII. The effects of different doses of AII, AIII and ACTH on the corticosterone output and 32P incorporation into PA, PI and PC of a preparation of cells, consisting of more than 98% z.f.r. cells, from rat decapsulated adrenals were also studied. ACTH, at low doses, which nevertheless markedly stimulated corticosterone output, had a small (maximally to about 125% of control values) but significant effect on 32P incorporation into PA, PI and PC. The maximum effect was usually at about 10-10 M ACTH and was not significant at 10-8 M. AII had no significant effect on corticosterone output or on 32P incorporation into PC. AII had a significant effect (to about 185%) on 32P incorporation into PI, but a smaller effect on incorporation into PA. AIII gave similar results. Increased K+ concentration (from basal 3.6 to 5.9, 8.4 and 13 mM) stimulated the steroidogenesis of capsular cells but did not increase the 32P incorporation into PA at any concentration. At 8.4 mM, but not at any other K+ concentration, there was a small (about 130%) but significant increase in 32P incorporation into PI. This was not due to incorporation into PI in the contaminating cells as K+ (or serotonin) did not alter incorporation into PA or PI in z.f.r. cells. K+ does not seem to stimulate steroidogenesis by the same mechanism as do the angiotensins, that is, it uses cAMP rather than Ca2+ as messenger. The results for all the stimuli studied indicate that for a Ca2+ messenger bioassay using adrenal capsular cells, it will be preferable to measure the incorporation of 32P into PA rather than into PI and this should provide a satisfactory assay as regards specificity, sensitivity and precision.

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