Bovine Adrenocortical Cells Research Articles

Bradykinin stimulates aldosterone release from cultured bovine adrenocortical cells through bradykinin B2 receptors.

The adrenal cortex contains a kallikrein-like enzyme that may lead to bradykinin (BK) formation. This study was designed to determine whether BK acts on adrenocortical cells to stimulate steroid secretion. BK, Lys-BK, a specific BK 2 (B2) receptor agonist, and desArg9-BK, a specific BK 1 (B1) receptor agonist, all stimulated aldosterone secretion from cultured bovine adrenal zona glomerulosa cells. BK and Lys-BK were equipotent (EC50 = 2 x 10(-9) M), whereas desArg9-BK was 1000-fold less potent. The maximal effects of BK and BK analogs were comparable to the maximal effects of adrenocorticotropin or angiotensin II. A B2, but not a B1, receptor antagonist inhibited BK-stimulated aldosterone release. Verapamil and N,N-diethylamino-octyl-3,4,5-trimethoxybenzoate, which reduce intracellular calcium concentrations, reduced BK-stimulated aldosterone secretion. Although BK stimulated both prostacyclin and aldosterone production, indomethacin abolished prostacyclin production without affecting aldosterone secretion. In cultured adrenal fasciculata cells, high concentrations of BK stimulated cortisol release, but B1 or B2 receptor agonists were not effective. BK-stimulated cortisol secretion was reduced by N,N-diethylamino-octyl-3,4,5-trimethoxybenzoate but not by indomethacin. In summary, BK stimulates aldosterone release from cultured adrenal glomerulosa cells via high affinity B2 receptors. The effect is calcium-dependent and independent of prostaglandins. BK also increases cortisol release; however, this stimulation requires high concentrations of BK and may be mediated by an unknown receptor or by a receptor-independent mechanism.

Bradykinin stimulates aldosterone release from cultured bovine adrenocortical cells through bradykinin B2 receptors

Bradykinin stimulates aldosterone release from cultured bovine adrenocortical cells through bradykinin B2 receptors

Regulation of c-fos, c-jun and jun-B messenger ribonucleic acids by angiotensin-II and corticotropin in ovine and bovine adrenocortical cells

Regulation of c-fos, c-jun and jun-B messenger ribonucleic acids by angiotensin-II and corticotropin in ovine and bovine adrenocortical cells

Regulation of c-fos, c-jun and jun-B messenger ribonucleic acids by angiotensin-II and corticotropin in ovine and bovine adrenocortical cells.

Previous work has shown that corticotropin (ACTH) and angiotensin-II (A-II), in addition to their acute steroidogenic effects, exert long-term influences on adrenal cell differentiated function, stimulatory or inhibitory, respectively. Certain nuclear proto-oncogenes have been implicated in the regulation of gene expression in many cell systems. We have investigated the effects of ACTH and A-II on the levels of c-fos, c-jun, and jun-B messenger RNAs (mRNAs), in bovine and ovine (OAC) adrenal fasciculata cells. In both cell types ACTH produced time- (maximum at 1 h) and dose-dependent (ED50 congruent to 10(-12) M) increase in c-fos (2- to 4-fold) and jun-B (10- to 20-fold) mRNA levels but did not affect c-jun. The concentrations required to induce half-maximal mRNA accumulation and cortisol production were similar. A-II also produced a dose-dependent increase in c-fos and jun-B mRNAs but also in c-jun in both cell types, despite the fact that OAC are resistant to the steroidogenic action of the hormone. The stimulatory effects of A-II on c-fos mRNA were higher than those produced by ACTH, whereas the effects on jun-B were similar but ACTH abolished (OAC) or decreased (bovine adrenal fasciculata cells) the stimulatory effects of A-II on c-jun mRNA. The effects of ACTH and A-II on cortisol production and proto-oncogene mRNAs were in part mimicked by 8 Bromo-cAMP and the phorbol ester phorbol-12-myristate-13 acetate plus calcium ionophore A23187, respectively. In the presence of cycloheximide, which blocks the steroidogenic effects of both hormones, proto-oncogene mRNAs were superinduced by both hormones. This result, together with the fact that dexamethasone failed to affect the mRNA levels suggests that the stimulatory effects of ACTH and A-II on proto-oncogene expression were not related to an autocrine/intracrine action of cortisol. Taken together, these findings show that the proto-oncogene mRNAs in normal adrenal cells are regulated by ACTH and A-II, acting through different intracellular pathways. They also demonstrate differential responsiveness of the Jun family to both hormones. Thus, the opposite long-term action of ACTH and A-II on adrenal cell differentiated function could be mediated by its different initial effects on proto-oncogene expression, in particular in the members of the Jun family.

Cytochrome P-450 17 α,lyase -mediating pathway of androgen synthesis in bovine adrenocortical cultured cells

Cytochrome P-450 17 α,lyase -mediating pathway of dehydroepiandrosterone (DHA) formation from pregnenolone was investigated in primary cultures of bovine adrenocortical fasciculata-reticularis cells. To determine whether DHA formation proceeds predominantly by successive monooxygenase reactions without 17α-hydroxypregnenolone leaving P-450 17 α,lyase the cells were incubated with [ 14C]pregnenolone and 17α-[ 3H]hydroxypregnenolone in the presence of Trilostane. Results of the double-substrate double-label experiments indicate that in the presence of high concentration of pregnenolone most of DHA was formed directly from pregnenolone by the successive reactions. Since the concentration of pregnenolone usually exceeds that of 17α-hydroxypregnenolone in the adrenal glands, DHA is concluded to be formed predominantly by successive reactions from pregnenolone without 17α-hydroxypregnenolone leaving P-450 17 α,lyase in vivo. By chronic ACTH treatment, the activities of 17α-hydroxylation and DHA formation in adrenocortical cultured cells became higher concomitantly with the increase of P-450 17 α,lyase content. Most of DHA was found to be formed by successive reactions from pregnenolone even under such conditions.

Human recombinant activin-A modulates the steroidogenesis of cultured bovine adrenocortical cells.

The effect of human recombinant activin-A on adrenal steroidogenesis was studied in cultured bovine adrenocortical cells. Activin-A significantly reduced cortisol output from ACTH (10nmol/l)-stimulated adrenocortical cells incubated for 24 hours in a dose-dependent manner (10, 100 and 500ng activin-A/ml suppressed cortisol secretion by 19, 33 and 40%), although no significant effect was observed in the case of 3 h incubation. Dehydroepiandrosterone (DHEA) secretion from ACTH-stimulated adrenocortical cells incubated for 24 h was also decreased by the addition of activin-A in a dose-dependent manner. (10, 100 and 500ng activin-A/ml suppressed DHEA secretion by 22, 56 and 58%). These inhibitory effects of activin-A (100ng/ml) on cortisol and DHEA secretion were partially blocked by the addition of follistatin/FSH-Suppressing Protein (200ng/ml). In contrast, activin-A treatment resulted in no significant decrease in aldosterone secretion. There were no significant effects of activin-A on basal secretions of cortisol, DHEA or aldosterone from adrenocortical cells. These results suggest that activin-A has a direct inhibitory effect on ACTH-stimulated bovine adrenocortical steroidogenesis.

Cortisol secretion induced by substance p from bovine adrenocortical cells and its inhibition by calmodulin inhibitors

When primary cultured bovine adrenocortical cells were treated with substance P (SP) at concentrations higher than 10 pM, cortisol output increased in a dose-dependent fashion. Although other neurokinins, such as neurokinin A (NKA) and neurokinin B (NKB), were also effective in secreting cortisol, SP was the most potent among the tested neurokinins, the potency order being SP > NKA > NKB. This suggests that the NK-1 type receptor on adrenocortical cells may be the site of action of SP on cortisol secretion. The maximal response in SP-induced cortisol secretion was comparable to that elicited by adrenocorticotropic hormone (ACTH). SP-induced cortisol secretion was dependent upon extracellular Ca 2+ concentrations, and 45Ca 2+ uptake into adrenocortical cells treated with SP was long-lasting. While, in the case of ACTH, 45Ca 2+ uptake proceeded transiently, the increase in intracellular cAMP content was much greater compared with that of SP. Although KT-5720, an inhibitor of protein kinase A, inhibited potently ACTH-induced cortisol secretion, SP-induced secretin was not affected by this inhibitor at all. On the other hand, calmodulin inhibitors, such as calmidazolium, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, were not more effective in inhibiting SP-induced cortisol secretion than secretion induced by ACTH. The present study indicates that SP may be one of the physiological stimulants of cortisol secretion and that an increase in intracellular Ca 2+ concentration and the subsequent activation of calmodulin may precede SP-induced cortisol secretion.

Reversible uncoupling of phosphoinositidase C activation and steroidogenesis in cultured bovine adrenocortical zfr cells.

ACTH and adr stimulate F secretion from primary cultures of bovine zfr cells through increased AC activity, while All and ACh stimulate steroidogenesis through activation of PIC [ l ] . Although F secretion to all agonists is maximal after 48 h of culture, cells are consistenly less responsive to All and ACh after 24 h than when freshly isolated. We have investigated whether this reflects changes in second messenger formation, or alterations in the coupling of this signal to steroidogenesis. Bovine zfr cells were isolated and maintained in culture as previously described [2] in 12 well culture plates at a density of 333000 cells/well. Immediately following isolation, and after 24, 48, 72 and 96 h of primary culture, F secretion, cAMP production and PIC activity were determined in response to ACTH (O.lnM), Adr (1uM). All (O.luM), ACh (O.lmM), 8-Br cAMP (1 mM) and a combination of A231 87/PMA (both 1 OuM). All incubations were performed in triplicate, and experiments repeated at least twice. Details of the agonist incubations and assay procedures can be found in [3]. Mean basal values for F secretion were 6.80, 7.48, 13.10, 1 1.76 and 15.1 2 pmol/well/h-/I OOug protein for cells at 0, 24, 48, 72 and 96 h respectively. ACTH and Adr-stimulated F secretion increased over the first 72 h (pcO.Ol), and declined thereafter (n-fold stimulation ratios : 20.4, 32.1, 51.4,69.9 and 35.5 for ACTH, and 1.0, 2.1, 10.9, 17.4 and 9.1 for Adr, for cells at 0, 24, 48, 72 and 96 h respectively). These changes were accompanied by a corresponding increase in cAMP formation over the sal ile time period, followed by a decrease at 96 h (see fig 1 for ACTH data). The F secretory response to 8-Br cAMP also increased from initial isolation to 72 h, and decreased thereafter (n-fold stimulation ratios : 8.6,28.3,44.2, 69.0, 32.1 at 0, 24, 48, 72 and 96 h respectively). In contrast, the F secretory response to All and ACh was reduced after 24 h (All: pc0.01; ACh pc0.05 in 2 experiments) or unchanged (1 experiment), and rose to a maximum after 48 h before again declining (n-fold stimulation ratios : 43.1, 22.3, 127.1, 68.7 and 21.6 for All, and 4.5, 2.5, 25.1, 11.6 and 2.3 for ACh, for cells at 0, 24, 48, 72 and 96 h respectively). However, the PIC response to All and ACh over this time period did not parallel the changes in F secretion, but was significantly increased (pc0.05) for both agonists at 24 h, rising further at 48 h, before declining by 72 h. (Mean basal [3H]inositol phosphates labelling was 345, 657, 723, 346 and 321 dpm1lOOug protein at 0. 24, 48, 72 and 96 h respectively. Corresponding n-fold stimulation

Calcium is essential for ATP-induced steroidogenesis in bovine adrenocortical fasciculata cells.

The effect of extracellular ATP on steroidogenesis in primary cultured bovine adrenocortical fasciculata cells was investigated. I observed that in the presence of extracellular Ca2+, ATP caused a dose-dependent elevation of intracellular Ca2+ ([Ca2+]i) and induced steroidogenesis concentration- and time-dependently. However, in the absence of extracellular Ca2+, ATP had no effect on steroidogenesis. In the presence of extracellular Ca2+, calmodulin inhibitors inhibited the ATP-induced steroidogenesis, but dihydropyridine calcium channel blockers did not. Furthermore, ATP did not cause an elevation of cyclic AMP in bovine adrenocortical fasciculata cells even if extracellular Ca2+ existed. These results suggest that extracellular ATP might have an influence on bovine adrenocortical cells via the purinoceptor (P2Y) in connection with calcium mobilization, open the non-selective calcium channel and induce steroidogenesis by means of an elevation of [Ca2+]i via the calcium-calmodulin system.

Calcium Is Essential for ATP-Induced Steroidogenesis in Bovine Adrenocortical Fasciculata Cells

The effect of extracellular ATP on steroidogenesis in primary cultured bovine adrenocortical fasciculata cells was investigated. I observed that in the presence of extracellular Ca2+, ATP caused a dose-dependent elevation of intracellular Ca2+ ([Ca2+]i) and induced steroidogenesis concentration- and time-dependently. However, in the absence of extracellular Ca2+, ATP had no effect on steroidogenesis. In the presence of extracellular Ca2+, calmodulin inhibitors inhibited the ATP-induced steroidogenesis, but dihydropyridine calcium channel blockers did not. Furthermore, ATP did not cause an elevation of cyclic AMP in bovine adrenocortical fasciculata cells even if extracellular Ca2+ existed. These results suggest that extracellular ATP might have an influence on bovine adrenocortical cells via the purinoceptor (P2Y) in connection with calcium mobilization, open the non-selective calcium channel and induce steroidogenesis by means of an elevation of [Ca2+]i via the calcium-calmodulin system.

Demethylation of satellite I DNA during senescence of bovine adrenocortical cells in culture.

Over the finite proliferative life span of cultured bovine adrenocortical cells, satellite I DNA shows a progressive and extensive loss of methylation at CCGG sites. This was shown by Southern blotting after digestion with the methylation-sensitive enzyme HpaII alone, which provides a sensitive indicator of methylation loss, or digestion with the combination of EcoRI and HpaII, which provides a quantitative indication of loss of methylation. Bovine tissues, including adrenal cortex, all showed a much higher level of satellite methylation than cultured adrenocortical cells. After adrenocortical cells are placed in culture, some demethylation of satellite I is seen as early as 10 population doublings. By 80 population doublings, loss of satellite DNA methylation is extensive. The loss does not appear to prevent continued cell division, since an extended life span clone of bovine adrenocortical cells transfected with SV40 T antigen showed a similar pattern of extensive demethylation. Satellite demethylation has been reported in aging in vivo and the present cell culture system may provide an in vitro model for this form of genetic instability.

Mechanisms of insulin inhibition of ACTH-stimulated steroid secretion by cultured bovine adrenocortical cells

Results of previous studies indicated that insulin at levels comparable to those in humans during hyperinsulinemia decreased ACTH-stimulated cortisol and androstenedione secretion by bovine adrenal fasciculata-reticularis cells in primary culture. In the present studies this inhibitory action was examined further by comparing the effects of insulin on ACTH-stimulated corticosteroid secretion with its effects on 8-(4-chlorophenylthio)-cAMP (cpt-cAMP), forskolin- and [ 5val]angiotensin II (Ang II)-stimulated corticosteroid secretion. Effects on corticosteroid secretion were correlated with effects on cAMP accumulation and rates of cAMP production. Monolayers were incubated for 24 h in the absence or presence of each agonist alone or in combination with insulin. Insulin (1.7 × 10 −9 or 17.5 × 10 −9 M) caused about a 50% decrease in cortisol and androstenedione secretion in response to ACTH (10 −11 or 10 −8 M). Insulin also decreased ACTH-stimulated aldosterone secretion by cultured glomerulosa cells. Cpt-cAMP (10 −4 or 10 −3 M)-stimulated increases in cortisol and androstenedione secretion were inhibited by insulin, but to a lesser extent than those in response to ACTH. The inhibition of cpt-cAMP-stimulated steroid secretion was not related to increased degradation of the cyclic nucleotide. Increases in cortisol and androstenedione secretion caused by a submaximal concentration (10 −6 M) of forskolin were decreased 50–70% by insulin. In contrast, insulin failed to significantly affect cortisol or androstenedione secretion caused by a maximal concentration (10 −5 M) of forskolin. The secretory responses to Ang II (10 −8 M) were also unaffected by insulin. The effect of insulin to inhibit ACTH-stimulated steroid secretion was accompanied by a reduction in cAMP accumulation as well as an apparent inhibition of adenylate cyclase activation. These data indicate that the effect of insulin to attenuate ACTH-stimulated corticosteroid secretion results from both an inhibition of ACTH-stimulated adenylate cyclase activity and an antagonism of the intracellular actions of cAMP.

O-453 - Ca2+-induced cortisol secretion in permeabilized bovine adrenocortical cells

O-453 - Ca2+-induced cortisol secretion in permeabilized bovine adrenocortical cells

Effect of Ca(2+)-channel antagonists on the corticoidogenesis in primary cultured bovine adrenocortical cells

We investigated the effect of Ca(2+)-channel antagonists, Nicardipine(N), Verapamil(V) and Flunarizine(F), on the corticoidogenesis(CG) in primary cultured bovine adrenocortical cells. To examine the effect on receptor operated Ca(2+)-channel(ROC) and voltage operated Ca(2+)-channel(VOC), involved in corticoid synthesis, adrenocorticotropic hormone(ACTH) and high-K+ were used respectively. With or without the antagonists, cells were incubated at 37 degrees C for 1h in the presence of ACTH (100pM) or K+ (30mM). Corticoid was measured fluorometrically using cortisol as the standard. N and V inhibited not only ACTH-induced CG, but high K(+)-induced CG in a concentration-dependent manner. However, F inhibited only high K(+)-induced CG, and did not affect ACTH-induced CG. These inhibitions were observed at the low micromolar concentrations (below 40 microM) of the antagonists. In the regulation of corticoid synthesis, we indicate that F has an inhibitory effect on VOC without ROC; on the other hand, N and V inhibit both ROC and VOC.

O-086 - Steroidogenesis induced by the dibutyryi cyclic AMP in dispersed bovine adrenocortical cells

O-086 - Steroidogenesis induced by the dibutyryi cyclic AMP in dispersed bovine adrenocortical cells

Effects of ageing on the growth and differentiated function of transfected human thyrocytes

Senescence in primary cultures of mammalian cells is characterised by cessation of growth after a number of cell divisions; this may be associated with loss of some differentiated functions. Recent studies on bovine adrenocortical cells have suggested that expression of simian virus-40 (SV40) early region in these cells may prevent phenotypic losses due to senescence. We report here data on growth and differentiated function of two human thyrocyte cell lines (SGHTL-34 and -45) generated by the transfection of primary thyrocytes with the plasmid pSV3 neo which contains the SV40 early region. Growth was assessed by fluorometric DNA estimations and calculation of cell population doubling time; function was assessed by binding studies using 125I-bovine thyrotrophin (TSH) and measurement of cyclic adenosine 3',5'-monophosphate (cAMP) response to stimulation with TSH, forskolin and cholera toxin. After 3–12 months in stable culture there was a gradual increase in the doubling time of both cell lines over a 3-month period (SGHTL-34 cells, early 34.5 ± 4.5 h, late 301 ± 111.6 h; SGHTL-45 cells, early 53.4 ± 4.4 h, late 148.3 ± 26.3 h; mean ± SEM). Scatchard analysis demonstrated a loss of the high affinity TSH receptor over the same time period. The increase in cAMP in response to 1000 μU/ml TSH declined until the cells became unresponsive (SGHTL-34 early, cAMP 10.3 ± 0.7 pmol/well; late, cAMP −0.4 ± 0.3 pmol/well; SGHTL-45 early, cAMP 11.3 ± 1.1 pmol/well, late, cAMP 0.3 ± 0.1 pmol/well). The cAMP responses to forskolin and cholera toxin were unaffected. This loss of response to TSH in SGHTL-45 cells was unaffected by the removal of TSH from the culture medium. We conclude that pSV3 neo transfected human thyrocytes, whilst having a longevity of great use for biochemical, endocrinological and immunological studies, are not immortal and that they demonstrate parallel loss of growth and differentiated function during ageing.

ADRENALINE STIMULATES CHOLESTEROL SIDE-CHAIN CLEAVAGE CYTOCHROME P450 mRNA ACCUMULATION IN BOVINE ADRENOCORTICAL CELLS

The effect of adrenaline on the accumulation of mRNA encoding cholesterol side-chain cleavage cytochrome P450 (P450scc) and cortisol secretion was studied in bovine adrenocortical cells in primary culture. Treatment of cultured cells with adrenaline resulted in a 2-fold increase in mRNA encoding P-450scc, as revealed by Northern blot analysis. Under these conditions the maximal stimulation with ACTH resulted in a 6-fold accumulation of mRNA encoding P450scc. The effect of adrenaline on the expression of P450scc was abolished by the beta-blocker propranolol, while propranolol had no effect on ACTH-induced P450scc mRNA accumulation. Adrenaline stimulated the secretion of cortisol in a dose-dependent manner with a median effective dose of 0.5 mumol/l. The adrenaline-stimulated cortisol secretion amounted to 42% of the effect of ACTH (0.1 nmol/l). Upon adrenaline treatment, cAMP concentration in the culture medium increased about 50-fold over the basal value. It is concluded that the stimulatory action of adrenaline upon cortisol formation requires beta-adrenergic receptors and is due, at least in part, to a cAMP-mediated increases in the accumulation of mRNA encoding P450scc.

The origin and significance of 18-hydroxycortisol: Studies in hyperaldosteronism and in bovine adrenocortical cells in vitro

18-Hydroxycortisol has been suggested as a marker compound for a transitional zone between the adrenocortical zonae glomerulosa and fasciculata. The control of secretion of 18-hydroxycortisol has been compared with those of cortisol and aldosterone in normal subjects and patients with primary hyperaldosteronism. Comparisons were also made in isolated bovine zona glomerulosa and zona fasciculata cell preparations. Although there was considerable cross-contamination between fractions, 18-hydroxycortisol secretion occurred with equal facility in both fractions but depended on the availability of cortisol as substrate. Changes in secretion during stimulation following those of cortisol. It is concluded that, in vitro, 18-hydroxycortisol derives mainly from the zona fasciculata. The relevance of these findings to primary hyperaldosteronism and to the nature of the transition is discussed.

Angiotensin-II activation of cAMP and corticosterone production in bovine adrenocortical cells: Effects of nonpeptide angiotensin-II antagonists

The ability of angiotensin-II (A-II) to increase cAMP production in adrenocortical cells is not widely accepted due to numerous conflicting reports. The recent observation that rat adrenal cells exhibit multiple subtypes of A-II receptors raises the possibility that a specific subtype could be responsible for controlling cAMP stimulation. In the present study we characterize in detail the effects of A-II on cAMP production in bovine adrenocortical zona fasciculata cells (BAC) cells and determined which A-II receptor subtype is responsible for stimulating both cAMP production and steroidogenesis. A-II (100 nM) increased the medium content of cAMP by 5- to 10-fold. The magnitude of A-II stimulation, while significant, was considerably less than that observed following treatment with ACTH (100 nM) (10-fold vs. 500-fold). The A-II stimulation of cAMP was both concentration and time dependent with a significant increase in cAMP observed in the presence of 1 nM A-II and a maximal response observed using 100 nM A-II. Stimulation was also seen using the decapeptide, A-I, and the heptapeptide, A-III. Of the angiotensin analogues tested, the order of potency was A-II > A-III > A-I. The A-II antagonist, [Sar 1, Ala 8]-A-II (saralasin), reversed the stimulatory effect of A-II. The superior potency of A-II and the ability of saralasin to inhibit cAMP production suggest a specific receptor mediated mechanism. In order to determine which A-II receptor subtype was responsible for stimulating cAMP, we performed experiments using the nonpeptide A-II antagonists DuP-753 and PD-123319 which are specific antagonists for A-II receptor subtypes AT 1 and AT 2, respectively. DuP-753 (10–10,000 nM) caused a concentration dependent inhibition of A-II (100 nM) stimulation of corticosterone and cAMP production. PD-123319 did not affect the level of either corticosterone or cAMP production in response to A-II. These results give additional support toward the ability of A-II to stimulate BAC cell cAMP production. Furthermore, the A-II stimulation of cAMP and corticosterone synthesis is regulated by the AT 1 receptor subtype.

Prostaglandin E2Is a Positive Regulator of Adrenocorticotropin Receptors, 3β-Hydroxysteroid Dehydrogenase, and 17α-Hydroxylase Expression in Bovine Adrenocortical Cells*

The maintenance of optimal steroidogenesis in adrenocortical cells primarily depends on the chronic action of cAMP. Herein we examine the effects of prostaglandin E2 (PGE2) on the differentiated functions of bovine adrenocortical (BAC) cells in primary culture. PGE2 (10 microM) treatment for 3 h stimulated steroidogenesis and cAMP production by over 100-fold. In addition, the cAMP antagonist Rp-cAMP (1 mM) inhibited PGE2 stimulation of steroidogenesis by 60%. This observation suggests that the cAMP second messenger system is responsible for much of the PGE2-activated steroid hormone synthesis. Chronic treatment of BAC cells with PGE2 caused induction of 3 beta-hydroxysteroid dehydrogenase and steroid 17 alpha-hydroxylase cytochrome P-450 expression as determined by the examination of enzyme activity, enzyme levels by immunoblotting, and specific messenger RNA (mRNA) levels by Northern analysis. The positive effects of PGE2 on expression of 3 beta-hydroxysteroid dehydrogenase and 17 alpha-hydroxylase cytochrome P-450 were similar to the effects seen after ACTH treatment of BAC cells. In addition, treatment of BAC cells with PGE2 for 3 days caused a 3-fold induction of ACTH receptors as determined by increased cell binding of [125I]ACTH. Finally, we determined that BAC cells produced PGE2 and that the level of synthesis increased 10-fold after treatment with the hormone angiotensin II. Taken together these data indicate that PGE2 is a positive regulator of BAC cell differentiation acting on ACTH receptors, steroid metabolizing enzymes, and steroidogenesis. The ability of BAC cells to produce PGE2 leaves open the possibility for paracrine and autocrine regulation within the adrenal.