Human recombinant activin-A inhibits proliferation of human fetal adrenal cells in vitro

Activins and inhibins are dimeric peptides which are structurally and functionally related to transforming growth factor-beta (TGF-beta). The mRNA for the activin and inhibin subunits is expressed in the adrenal gland. Because members of the TGF-beta superfamily have effects on mitogenesis, we examined the effect of recombinant human activin-A (rh-activin-A) on proliferation of midgestation human fetal adrenal cells in vitro. Dose-dependent growth inhibition by rh-activin-A was obtained, with an ED50 of 1 ng/ml. Rh-activin-A inhibited basal and epidermal growth factor (EGF)-stimulated fetal zone cell proliferation, but did not alter basic fibroblast growth factor (bFGF)-stimulated growth. TGF-beta combined with rh-activin-A demonstrated additive inhibition of fetal adrenal growth. These findings suggest a potential autocrine or paracrine role for activin-A in modulating the growth and/or subsequent involution of the human fetal adrenal gland.     

Adrenocorticotropin preferentially up-regulates angiopoietin 2 in the human fetal adrenal gland: implications for coordinated adrenal organ growth and angiogenesis

CONTEXT: ACTH is the key tropic hormone for the human fetal adrenal (HFA). Because vascular development must be coordinated with organ growth, ACTH may regulate local angiogenic factors, thereby influencing HFA angiogenesis. We previously demonstrated that ACTH up-regulates vascular endothelial growth factor in HFA cortical cells. A newer angiogenic factor family, the angiopoietins (Angs), also plays critical roles. Ang1 stabilizes, whereas Ang2 destabilizes vessels, increasing responsiveness to angiogenic stimuli. OBJECTIVE: The objective of this study was to investigate expression and ACTH regulation of Angs and their receptor Tie2 in the HFA. DESIGN, SETTING, AND PATIENTS: Studies were conducted involving RNA, frozen sections, and primary cell cultures from HFAs (14-24 wk) and human adult adrenal RNA. MAIN OUTCOME MEASURES: Angs and Tie2 mRNA levels were determined by real-time quantitative RT-PCR, Ang2 and Tie2 were localized by immunostaining, and ACTH regulation of Angs was investigated by real-time quantitative RT-PCR, Western blot, and ELISA. RESULTS: Mean HFA Ang2 to Ang1 mRNA ratio was 6.3-fold higher than adult adrenals (P < 0.001). Ang2 was localized predominantly in the HFA periphery, whereas Tie2 demonstrated endothelial localization. In isolated HFA cortical cells, ACTH up-regulated Ang mRNA levels in a time- and dose-dependent manner, with the balance favoring Ang2. Ang2 protein levels were elevated in ACTH-stimulated HFA cortical cells and conditioned medium. 8-Bromoadenosine cAMP and forskolin mimicked ACTH effects on the Angs. CONCLUSIONS: Higher HFA Ang2 to Ang1 ratios may reflect greater vascular remodeling than in the adult. Angs, particularly Ang2, in concert with vascular endothelial growth factor, may mediate ACTH tropic action, ensuring coordination of HFA growth, steroidogenesis, and angiogenesis.     

Adrenocorticotropin interferes with transforming growth factor-beta-induced growth inhibition of neocortical cells from the human fetal adrenal gland.

The effects of transforming growth factor-beta (TGF-beta) and ACTH on growth, as indicated by [3H]thymidine incorporation into DNA, of primary cultures of neocortical cells from the human fetal adrenal gland were studied. TGF-beta inhibited, in a dose- and time-dependent manner, the growth of fetal neocortical cells, and ACTH significantly blunted the inhibitory effects of TGF-beta on growth of these cells. ACTH did not block the inhibitory effects of TGF-beta on growth of fetal adrenal fibroblasts or liver cells; neither ACTH nor TGF-beta had any effect on growth of fetal kidney cells. Thus, it appears that growth regulation of the neocortex may differ strikingly from that of the fetal zone of the human fetal adrenal, in which ACTH and TGF-beta have been reported recently to have additive inhibitory effects on cell proliferation.     

CITED2 is expressed in human adrenocortical cells and regulated by basic fibroblast growth factor

CITED2 gene deletion in mice leads to adrenal agenesis. Therefore, we analyzed CITED2, a CBP/p300 interacting transactivator with transforming activity, in the human adrenal gland. In this study, we examined CITED2 expression in human embryonic and adult adrenal glands as well as adrenocortical carcinomas. As ACTH and basic fibroblast growth factor (bFGF) are connected to the physiology and growth of adrenocortical cells we studied the regulation of CITED2 by these factors in the NCI-H295R adrenocortical carcinoma cell line. We found CITED2 expression in the adult adrenal cortex as well in adrenocortical carcinomas. At an early stage of human adrenal organogenesis CITED2 could be located to the definitive zone of the developing adrenal gland using immunohistochemistry. In NCI-H295R cells, stimulation by bFGF led to a dose-dependent increase in CITED2 promotor activity, mRNA and protein expression while ACTH had no significant effect. The stimulatory effect of bFGF could be reduced by blocking mitogen-activated protein kinase activity using the MAPkinase kinase (MEK1)-inhibitor PD98059. CITED2 is expressed in embryonic and adult human adrenal glands as well as in adrenocortical cancer. It is connected to the signaling cascades of bFGF and its expression is modulated by mitogen-activated protein kinases. This suggests a novel role for CITED2 in human adrenal growth and possibly in adrenal tumorigenesis.     

Regulation of baboon fetal adrenal androgen formation by pituitary peptides at mid- and late gestation

A short term incubation of baboon fetal adrenal cells obtained at midgestation and near term was used to determine whether a change in the regulation of androgen formation occurs with advancing gestation. Adrenal glands were removed from baboon (Papio anubis) fetuses on day 100 (mid; n = 7) or day 170 (late; n = 5) of gestation, and cells were dispersed with 0.2% collagenase. Cells (10(5] were incubated at 37 C for 3 h in medium 199 in the presence or absence of 10 nM ACTH, 10 nM ovine PRL, 10 nM ovine GH, 50 nM hCG, or 50 nM human chorionic somatomammotropin. Dehydroepiandrosterone (DHA), DHA sulfate (DHAS), cortisol (F), and androstenedione concentrations were determined in the medium by RIA. At midgestation, ACTH, PRL, and GH elevated (P less than 0.05) DHA (168%, 169%, and 178%, respectively) and DHAS (142%, 210%, and 197%, respectively) formation. Near term, ACTH and PRL retained their ability to stimulate (P less than 0.05) DHA (307% and 220%, respectively), but not DHAS, synthesis. The fetal adrenal at late gestation, however, lost its ability to respond to treatment with GH. hCG and human chorionic somatomammotropin did not stimulate steroidogenesis at either time of gestation. F formation at midgestation was less (P less than 0.05) than that at term and not responsive to ACTH. ACTH stimulated (P less than 0.05) F secretion by 68% in fetal adrenal cells obtained near term. The secretion of androstenedione was not affected by any peptide treatment at either stage of gestation. These data indicate that the responsivity of the baboon fetal adrenal to various pituitary peptides is different at two developmentally distinct stages of gestation. We conclude, therefore, that the regulation of fetal adrenal steroidogenesis changes with advancing gestation.     

Growth hormone has no direct effect on human adrenal steroid and insulin-like growth factor-binding protein secretion

Although it is known that growth hormone (GH) exerts its growth-promoting effects mainly via Insulin-like growth factor-I (IGF-I), an increasing number of direct effects of GH has been described in many tissues. In vivo, mice transgenic for human growth hormone (hGH) show significantly elevated levels of corticosterone, enlarged adrenal glands, and altered levels of insulin-like growth factor binding proteins (IGF-BPs). Recently, we have shown that IGF's induce the secretion of cortisol and IGF-BP's in adult human adrenocortical cells. However, since human adrenal glands express the intact GH-receptor, the objective of this study was to investigate whether GH exerts a direct effect on the steroidogenesis and IGF-BP synthesis in adult human adrenocortical cells. Primary cell cultures in monolayer were incubated under serum-free conditions with human growth hormone and/or ACTH for up to 72 hours. Cortisol was measured by specific RIA and the secretion of insulin-like growth factor binding proteins was analyzed by Western ligand blotting. hGH alone was unable to stimulate basal or ACTH-induced cortisol secretion. Additionally, neither hGH alone or in combination with ACTH did significantly alter the secretion of IGF-BP's. Therefore we conclude that hGH is unable to directly stimulate cortisol secretion and IGF-BP secretion in cultured human adrenocortical cells.     

Control of bovine adrenal cortical cell proliferation by fibroblast growth factor. Lack of effect of epidermal growth factor.

Primary functional bovine adrenal cortical cell cultures have been developed to study the factors controlling adrenal cell growth. Cells were prepared by the collagenase technique and maintained in F-12 medium containing fetal calf serum and horse serum. Cells contained abundant lipid as demonstrated by staining with Oil Red O and showed strongly positive staining for delta5,3beta-hydroxysteroid dehydrogenase. ACTH inhibited DNA synthesis and stimulated steriodogenesis in these cells. Fibroblast growth factor (FGF) was shown to be a potent stimulator of the growth of normal bovine adrenal cortical cells maintained in tissue culture. The minimal effective dose of FGF was 1 ng/ml with maximal effects being observed at 100 ng/ml. The effect of FGF was dependent on the serum concentration. Inclusion of FGF in F-12 medium containing serum permitted cloning of functional bovine adrenal cortical cells from cultures seeded at low density (4 cells/cm2). ACTH inhibited the mitogenic effects of FGF. In addition to its mitogenic action, FGF is a migratory factor for bovine adrenal cortical cells. Though ACTH inhibited the mitogenic effects of FGF, it did not block the migratory activity. Epidermal growth factor did not affect the growth of either normal bovine adrenal or functional mouse adrenal tumor cells (Y-1) in tissue culture. FGF is the first direct mitogen identified for adrenal cortical cells; ACTH opposes this mitogenic action and functions directly as a differentiate function signal.     

Midkine, a heparin-binding growth factor, selectively stimulates proliferation of definitive zone cells of the human fetal adrenal gland

CONTEXT: In the human fetal adrenal gland (HFA), the inner fetal zone (FZ) secretes dehydroepiandrosterone sulfate. The function of the outer definitive zone (DZ) is less clear; however, the DZ phenotype is that of a reservoir of progenitor cells, many of which are mitotically active. Midkine (MK) is a heparin-binding growth factor with various bioactivities. OBJECTIVE: The objective of this study was to investigate expression, proliferative effects, and ACTH regulation of MK in the HFA. DESIGN AND SETTING: RNA, cryosections, and primary cell cultures from HFAs (14-24 wk) and adult adrenal RNA were used. MAIN OUTCOME MEASURES: The main outcome measures were MK mRNA levels (measured by quantitative real-time RT-PCR); MK localization (measured by immunostaining); MK proliferative effects and mechanism (measured by proliferation assays, flow cytometry, pharmacological interventions); and ACTH regulation (measured by quantitative real-time RT-PCR). RESULTS: HFA MK mRNA levels were 4-fold higher than in adult adrenals (P < 0.05) and were comparable to levels in fetal and adult brains (positive controls). MK immunoreactivity was abundant throughout the HFA. Exogenous MK caused proliferation of isolated DZ cells but not FZ cells (72 h, P < 0.05). In contrast, basic fibroblast growth factor induced proliferation of cells from both zones. Pharmacological interventions indicated that MK-induced DZ cell proliferation may be mediated by phosphatidylinositol 3-kinase, MAPK kinase, and Src family kinases. ACTH (1 nm) increased MK mRNA by 3.5-fold (48 h, P < 0.01) in isolated FZ cells. CONCLUSIONS: MK likely plays a key role in HFA development. MK's selective in vitro mitotic effects on DZ cells may provide insights into the mechanism underlying the distinct in vivo differences in mitotic activity between the DZ and FZ.     

Developmental expression of and effect of betamethasone on the messenger ribonucleic acid levels for peptide growth factors in the baboon fetal adrenal gland.

In the present study, we determined whether expression of the messenger ribonucleic acids (mRNAs) for insulin-like growth factor-II (IGF-II), and its principal IGF type-1 receptor and IGF-binding protein-2 (IGFBP-2), as well as basic fibroblast growth factor (bFGF), was developmentally regulated in the baboon fetal adrenal gland. In the second phase of this study, fetal pituitary ACTH was suppressed by the administration of betamethasone to determine the possible effect on the mRNA levels for those factors, i.e. IGF-II and IGFBP-2, shown to be expressed at high levels in the adrenal late in fetal development. Adrenals were obtained from fetuses delivered via Cesarean section on days 60 (early), 100 (mid), and 165 (late) of gestation (term=184 days) from untreated baboons and on day 165 from baboons in which betamethasone was administered to the fetus, or to fetus and mother, every other day between days 150 and 164 of gestation. Although the mRNA levels of IGF-II in the fetal adrenal were similar at early, mid and late gestation, IGF type-1 receptor mRNA levels were approximately 2- to 3-fold greater (P<0.01) at mid than at early or late gestation. In contrast, there was an increase (P<0.001) in fetal adrenal IGFBP-2 and bFGF mRNA levels in late gestation. Although fetal adrenal weights and width of the zone of definitive/transitional cells exhibiting immunocytochemical staining for Delta(5)-3beta-hydroxysteroid dehydrogenase (3beta-HSD) were markedly suppressed (P<0.01) by the administration of betamethasone, IGF-II and IGFBP-2 mRNA expression was not decreased. In summary, very different patterns of mRNA levels for IGF-II, IGF type-1 receptor, IGFBP-2 and bFGF were exhibited in the developing baboon fetal adrenal gland, which may reflect functionally important differences in their respective cellular localization within the cortex, as well as a divergence in the functional development of the fetal, transitional and definitive zones of the baboon fetal adrenal cortex.     

Development of the baboon fetal adrenal gland: regulation of the ontogenesis of the definitive and transitional zones by adrenocorticotropin.

Throughout gestation, the primate fetal adrenal gland is comprised of the fetal zone, which expresses the P-450 17alpha-hydroxylase-C17,20 lyase (P-450c17) enzyme that catalyzes the synthesis of C19 steroids used for placental estrogen production. The development of the transitional zone comprised of cortical cells that express the P-450c17 and the 3beta-hydroxysteroid dehydrogenase-isomerase (3betaHSD) enzymes for cortisol production, and the definitive zone, which expresses 3betaHSD, but not P-450c17, for mineralocorticoid synthesis, does not occur until relatively late in gestation. Although ACTH is considered essential to fetal adrenal growth and function, the role that ACTH has in the development of the transitional and definitive zones, is less clear. To answer this question, the width of these zones was determined by immunocytochemical expression of P-450c17 and/or 3betaHSD in fetal adrenal glands obtained on day 100 (mid) of gestation (term = day 184) from baboons in which ACTH was administered to the fetus on days 95-99 of gestation or on day 165 (late) of gestation from baboons in which fetal ACTH was suppressed by treatment of the mother and fetus with betamethasone on days 150-164 of gestation. At midgestation, the fetal adrenal was comprised almost exclusively of fetal zone cells and a small definitive zone (38 +/- 2 microm in width), but was essentially devoid of a transitional zone (7 +/- 2 microm). Treatment with ACTH enhanced (P < 0.05) the width of the transitional zone (67 +/- 4 microm), but not the size of the definitive zone (10 +/- 4 microm). In late gestation, the width of the definitive zone, although 2-fold greater than that on day 100, was smaller (P < 0.05) than that of the transitional zone (120 +/- 15 microm), which greatly exceeded that at midgestation. Treatment with betamethasone in late gestation eliminated the transitional zone, but had no effect on the size of the definitive zone (120 +/- 8 microm). These findings indicate that the development of the baboon fetal adrenal transitional zone late in gestation is dependent on fetal pituitary ACTH. In contrast, the ontogenesis of the definitive zone at midgestation and its growth in late gestation occur in the relative absence of ACTH.     

Corticotropin positively regulates its own receptors and cAMP response in cultured bovine adrenal cells.

Bovine fasciculata adrenal cells contain specific high-affinity (KD approximately 2.3 +/- 0.4 x 10(-10) M) and low-capacity (1910 +/- 300 sites per cell) corticotropin (ACTH) receptors. Pretreatment of cells with ACTH, caused in a time-(maximum effect at 48 hr) and dose-(ED50 approximately 10(-11) M, Vmax = 10(-10) to 10(-9) M) dependent manner an increase in ACTH binding. This was due to a 4-fold increase in the number of binding sites without modification of the binding affinity. The same pretreatment also enhanced the cAMP response to further ACTH stimulation in a dose-dependent manner (ED50 approximately 10(-11) M) and to a lesser extent the response to forskolin. However, pretreatment with higher concentrations of ACTH (10(-8) M) reduced the binding and the cAMP response when compared to the effect of 10(-9) M. These ACTH effects, which were mimicked by 8-bromoadenosine 3',5'-cyclic monophosphate, required de novo protein synthesis. Pretreatment with 10(-13) to 10(-11) M ACTH also enhanced the steroidogenic responsiveness to further hormonal stimulation. However, at higher concentrations the hormone induced an apparent steroidogenic desensitization that was probably related to a depletion of endogenous cholesterol, since cortisol production in the presence of 22-(R)-hydroxycholesterol was increased. Neither angiotensin-II nor atrial natriuretic factor alone modified ACTH receptors, but angiotensin-II partially blocked the stimulatory effect of ACTH. Thus, ACTH is one of the few polypeptide hormones having a positive trophic effect on its own receptors and target-cell responsiveness.     

Modulation of adrenocorticotropin-stimulated baboon fetal adrenal dehydroepiandrosterone formation in vitro by estrogen at mid- and late gestation

We have reported that ACTH stimulation of dehydroepiandrosterone (DHA) formation by the baboon fetal adrenal at midgestation was suppressed by estrogen. Because fetal adrenal regulation changes with advancing gestation, the action of estrogen on fetal adrenal steroidogenesis may also be dependent on the degree of fetal adrenal maturation. We examined this possibility in the present study by determining the effects of ACTH and estrogen on DHA formation by adrenal cells of fetuses obtained from baboons at mid- and late gestation and from animals administered the antiestrogen MER-25 throughout late gestation. Because low density lipoprotein (LDL) provides substrate for the fetal adrenal, we also determined whether the effect of estrogen was mediated by LDL uptake. Adrenals were removed from baboon fetuses on day 100 (midgestation; n = 7) and day 170 (late gestation; n = 6; term, day 184) of gestation from untreated animals and on day 170 from fetuses whose mothers were treated with MER-25 on days 140-170 (25 mg/kg BW.day; n = 7). Cells were dispersed with 0.2% collagenase and incubated at 37 C for 3 h in 4 ml medium 199 with 10 nM ACTH, 10(-6) M estradiol and/or 500 micrograms LDL. The secretion of DHA into medium was determined by RIA. At midgestation, mean (+/- SE) basal DHA formation (nanograms per 10(5) cells/3 h) was 5.8 +/- 2.1, and DHA was increased (P less than 0.01) by ACTH to 20.0 +/- 5.9. Although estradiol alone had no effect, estradiol prevented the increase in DHA obtained with ACTH. Basal DHA production by adrenals of late gestation (0.7 +/- 0.3 ng/10(5) cells) was lower (P less than 0.01) than at midgestation. ACTH increased (P less than 0.01) DHA in a comparable manner near term in the presence (2.0 +/- 0.4) or absence (1.7 +/- 0.4) of estradiol. Thus, in contrast to day 100, estrogen did not attenuate the action of ACTH on adrenal cells on day 170. In fetal adrenal cells obtained on day 170 from MER-25-treated baboons, DHA formation (1.4 +/- 0.6 ng/10(5) cells) was comparably increased (P less than 0.05) to 2.4 +/- 0.2 and 3.0 +/- 0.5 ng/10(5) cells by ACTH in the absence or presence of estradiol. Thus, ACTH remained effective in enhancing DHA by adrenal cells of fetuses exposed in utero to antiestrogen. DHA formation by adrenals of midgestation was increased (P less than 0.05) to 15.4 +/- 4.8 and 27.4 +/- 7.5 ng/10(5) cells, respectively, by LDL and ACTH plus LDL.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of the fetal human adrenal cortex: effects of adrenocorticotropin on growth and function of monolayer cultures of fetal and definitive zone cells

Monolayer cultures have been prepared from both definitive and fetal zones of the human fetal adrenal cortex. Cultures from each zone consist predominately of adrenocortical cells, as determined by a specific morphological retraction response to ACTH, and by ACTH-induced inhibition of DNA synthesis and cell proliferation. Cell growth was stimulated by fibroblast growth factor. ACTH stimulated steroidogenesis in cells from each zone with an ED50 of 0.4--1.0 nM and at a maximal concentration of 5 nM. Short term stimulation of less than 24 h with ACTH produced a pattern of steroid secretion that was characteristic of the zone of origin. Definitive zone cultures produced both cortisol and dehydroepiandrosterone plus its sulfate (DHA/S), with cortisol production exceeding DHA/S production. Fetal zone cultures produced more DHA/S than cortisol. 3 beta-Hydroxysteroid dehydrogenase, delta 4,5-isomerase enzyme activity was 3-fold less in fetal than in definitive zone cultures. Long term stimulation of 1--4 days with ACTH, 8-bromo-cAMP, or cholera toxin increased steroidogenic capacity in cultures from both zones. The pattern of steroid production by definitive zone cells remained constant, but cortisol production was preferentially increased in fetal zone cells. Forty-eight-hour treatment of fetal zone cells with ACTH increased 3 beta-hydroxysteroid dehydrogenase, delta 4,5-isomerase activity 5-fold. alpha-, beta-, gamma 1-, gamma 2-, and gamma 3-MSH were not effective steroidogenic agents for either zone. These studies indicate that steroidogenic agents induce in fetal zone cells steroid production characteristic of definitive and adult adrenocortical cells.     

Programming of Cells for Death under Defined Experimental Conditions: Relevance to the Tumor Problem

An attempt was made in this study to determine more precisely the nature of the factors that are involved in the programming of cells for a form of terminal cellular differentiation that results in death. These studies demonstrated that both the cytokinesins, which are potent inhibitors of plant and animal adenosine 3':5'-cyclic monophosphate phosphodiesterases, and 8-bromoadenosine 3':5'-cyclic monophosphate, which is a stable, biologically active form of adenosine 3':5'-cyclic monophosphate, are highly effective in encouraging differentiation of parenchyma cells into tracheary elements with accompanying death. Since adenosine 3':5'-cyclic monophosphate and theophylline when used together were also effective, the results reported here suggest that adenosine 3':5'-cyclic monophosphate is somehow importantly involved in the conversion of parenchyma cells into tracheary elements in this system. The possible significance to the tumor problem generally of the programming of cells for terminal differentiation, with or without resulting death, is discussed.     

Hormonal regulation of messenger ribonucleic acids for P450scc (cholesterol side-chain cleavage enzyme) and P450c17 (17 alpha-hydroxylase/17,20-lyase) in cultured human fetal adrenal cells

ACTH has acute and long term effects on adrenal steroidogenesis by week 14 of fetal life. We used human fetal adrenal cells to investigate the long term effect of physiological doses of ACTH on mRNAs for P450scc (the cholesterol side-chain cleavage enzyme) and P450c17 (17 alpha-hydroxylase/17,20-lyase). Monolayer cultures of 18- to 24-week gestation fetal zone adrenal cells were maintained in the presence and absence of 10(-9) or 10(-8) M ACTH for up to 12 days. As assessed by RNA dot blots probed with cloned homologous human cDNAs, ACTH increased P450scc and P450c17 mRNAs 4- and 9-fold, respectively, over control values on day 7 of culture. ACTH-mediated stimulation was slightly less on day 12 of culture. The ACTH-mediated accumulation of those mRNAs were time dependent. When cells were exposed to a single 10(-8)-M dose of ACTH, the amount of P450scc and P450c17 mRNA was increased by 24 h, reaching a maximum at 48 h and diminishing by 72 h. When cells were maintained in 10(-8) M ACTH continuously, mRNA for both enzymes accumulated in a similar pattern, reaching a peak at 48 h but remaining at nearly maximal values thereafter, up to 96 h. Dibutyryl cAMP (10(-3) M) mimicked these stimulatory actions of ACTH, although its effect was greater at 24 h and more stable up to 96 h. Angiotensin II (1-100 ng/mL) and hCG (1-100 ng/mL) had no effect on accumulation of P450scc and P450c17 mRNAs. The production of both dehydroepiandrosterone sulfate and cortisol also was stimulated by ACTH, suggesting that the increased mRNAs were translated into active enzymes. These results indicate that ACTH induces human fetal adrenal cells to accumulate mRNAs for both P450scc and P450c17; this effect of ACTH is probably mediated by cAMP. Chronic 96-h stimulation of human fetal adrenal cells did not diminish their responsiveness to ACTH. Together with our earlier studies of the human fetal adrenal, these data indicate that fetal adrenal tissue does not exhibit the desensitization to trophic hormone stimulation characteristic of adult tissue.     

Activation of ovine fetal adrenal function by pulsatile or continuous administration of adrenocorticotropin-(1-24). II. Effects on adrenal cell responses in vitro

We have examined 1) the effects of mode of ACTH administration to fetal sheep in vivo on the pattern of corticosteroid output by dispersed adrenal cells in vitro, and 2) the time course of fetal adrenal activation during pulsatile ACTH administration to the fetus. Fetal sheep received the same amount of ACTH either as a continuous infusion (C-ACTH; 0.5 microgram/h) or as 15-min pulses every 2 h for 72 h (P-ACTH). Other fetuses received P-ACTH until labor occurred (mean, 100 h) or saline for 72 or 100 h. Adrenal cells from fetuses that received C-ACTH for 72 h produced more corticosterone from endogenous precursors after the addition of ACTH in vitro and after the addition of 0.3 microM progesterone (P4) or pregnenolone (P5) than cells from fetuses treated with P-ACTH for 72 h. There was no difference in cortisol (F) response between the two groups, although in both groups F output was greater than in controls. Adrenal cells from control fetuses produced more P4 in vitro during incubation with ACTH plus guanosine-5'-(beta, gamma-imido)triphosphate [Gpp(NH)p] or N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate [(Bu)2cAMP] than after ACTH alone. There was no significant F response to the agonists, and F output was quantitatively less than that of P4. After 72-h P-ACTH, the mean P4 output after ACTH addition was not significantly different from that after ACTH plus Gpp(NH)p or (Bu)2cAMP. By 100 h of P-ACTH, the output of F exceeded that of P4 and was not different in response to ACTH, ACTH plus Gpp(NH)p, or (Bu)2cAMP. After 72 h of P-ACTH, incorporation of exogenous P4, but not P5, 17 alpha-hydroxyprogesterone, or 17 alpha-hydroxypregnenolone, into F was greater than that in cells from control fetuses. By 100 h of P-ACTH, incorporation of all substrates into F was greater than that after P-ACTH for 72 h. We conclude that 1) both C-ACTH and P-ACTH for 72 h increase fetal adrenal responses, but the pattern of corticosteroid output in vitro is determined by the mode of ACTH administration in vivo; 2) adrenal responsiveness is increased further between 72 and 100 h of P-ACTH, and activation probably involves changes in ACTH receptor-GTP coupling as well as enzyme activities on the pathway to F biosynthesis.     

Steroid production by definitive and fetal zones of the human fetal adrenal gland.

This study was performed to assess the relative contributions of the fetal and definitive zones of the human fetal adrenal gland to "corticoid" (cortisol and perhaps other corticosteroids) and dehydroepiandrosterone sulfate (DHAS) production, and the possible regulatory role of ACTH and the fetal pituitary in the secretion of of these steroids. Corticoid and radioimmunoassayable DHAS or total aromatizable androgen secretion by the isolated definitive and fetal zones of the human fetal adrenal gland between 10-20 weeks gestation has been studied in a superfusion system. Different functional capacities of the two zones were seen; corticoids were found to be secreted primarily by the definitive zone, while DHAS was found to be the main secretory product of the fetal zone. Addition of ACTH (250 ng/ml) or fetal pituitary homogenate produced a 2- to 5-fold stimulation of corticoid production by the definitive zone at all gestational ages studied. DHAS secretion by the fetal zone was also stimulated by ACTH. These results indicate that the definitive and fetal zones of the human fetal adrenal gland at midgestation have the capacity to respond to ACTH with increased corticoid or DHAS secretion, respectively.     

The effects of ACTH on steroid metabolomic profiles in human adrenal cells

The adrenal glands are the primary source of mineralocorticoids, glucocorticoids, and the so-called adrenal androgens. Under physiological conditions, cortisol and adrenal androgen synthesis are controlled primarily by ACTH. Although it is well established that ACTH can stimulate steroidogenesis in the human adrenal gland, the effect of ACTH on overall production of different classes of steroid hormones has not been defined. In this study, we examined the effect of ACTH on the production of 23 steroid hormones in adult adrenal primary cultures and 20 steroids in the adrenal cell line, H295R. Liquid chromatography/tandem mass spectrometry analysis revealed that, in primary adrenal cell cultures, cortisol and corticosterone were the two most abundant steroid hormones produced with or without ACTH treatment (48 h). Cortisol production responded the most to ACTH treatment, with a 64-fold increase. Interestingly, the production of two androgens, androstenedione and 11β-hydroxyandrostenedione (11OHA), that were also produced in large amounts under basal conditions significantly increased after ACTH incubation. In H295R cells, 11-deoxycortisol and androstenedione were the major products under basal conditions. Treatment with forskolin increased the percentage of 11β-hydroxylated products, including cortisol and 11OHA. This study illustrates that adrenal cells respond to ACTH through the secretion of a variety of steroid hormones, thus supporting the role of adrenal cells as a source of both corticosteroids and androgens.     

Alteration of cellular adhesion by nerve growth factor.

Nerve growth factor increases the initial rates of cell-substratum and cell-cell adhesion of sympathetic nerve cell clone PC12. It also stimulates adenosine 3':5'-cyclic monophosphate accumulation in the cells, and exogenous adenosine 3':5'-cyclic monophosphate increases cell-substratum adhesion. NGH-stimulated adhesion is energy dependent and cold-sensitive and does not require de novo protein synthesis. It is probably mediated by an effect of adenosine 3':5'-cyclic monophosphate on the plasma membrane. Stimulation of cellular adhesion may be the first step in NGF-initiated neurite outgrowth by sympathetic nerve cells.