Adenylate cyclase activity in membrane fractions of adrenal tissue of human anencephalic fetuses

There is greater basal and ACTH-stimulated adenylate cyclase activity in membrane fractions prepared from the neocortex of human fetal adrenal (HFA) tissue than in similar preparations from the fetal zone. In this study, the specific activity of adenylate cyclase was determined in membrane preparations of adrenal tissue obtained from anencephalic fetuses (n = 5) varying in gestational age from 17-43 weeks. The basal adenylate cyclase activity in membrane fractions of adrenals of anencephalics was 2.9 +/- 2.1 (mean +/- SEM) pmol mg protein-1 min-1, 3-5% of the average specific activity in membrane preparations of fetal zone or neocortex of normal fetuses. ACTH (10(-10)-10(-4) M) in the incubation mixture stimulated adenylate cyclase activity 2- to 5-fold in whole HFA membrane fractions. In contrast, ACTH, when added to adrenal membrane preparations of the anencephalics, did not stimulate adenylate cyclase activity. Furthermore, sodium fluoride or forskolin stimulated adenylate cyclase activity markedly in HFA membrane preparations of normal fetuses, but did not affect enzyme activity in adrenal membrane preparations of the anencephalics. In conclusion, the basal activity of adenylate cyclase in adrenal membrane preparations of anencephalics was low and unresponsive to brief exposure to ACTH, sodium fluoride, or forskolin. These findings as well as those of our previous investigations suggest that the expression of HFA adenylate cyclase may be regulated in part by ACTH.     

3 beta-hydroxysteroid dehydrogenase/isomerase in the fetal zone and neocortex of the human fetal adrenal gland

The fetal zone of the human fetal adrenal (HFA) gland is established to have decreased 3 beta-hydroxysteroid dehydrogenase/delta 4-5 isomerase (3 beta HSD) activity compared to the neocortex or definitive zone. 3 beta HSD activity, however, can be induced in primary cell culture through treatment with ACTH. Therefore, the HFA with two distinct steroidogenic zones with differences in 3 beta HSD activity as well as the capacity to increase 3 beta HSD activity in response to ACTH provides an excellent model to study the regulation of this enzyme. The presence of 3 beta HSD in the fetal and neocortex zones of the HFA was examined using a polyclonal antibody raised against purified human placental microsomal 3 beta HSD. After homogenates of the fetal and neocortical zones of the HFA were electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel and immunoblotted, the presence of the 3 beta HSD protein with a molecular size of 45 kDa could be demonstrated only in the neocortical zone. ACTH treatment (greater than 2 days) of fetal and neocortical zone explant cultures produced increases in cortisol secretion associated with the respective levels of immunodetectable 3 beta HSD protein. Cortisol and dehydroepiandrosterone sulfate were the respective principal steroid products of neocortical and fetal zone explants. After ACTH treatment, immunodetectable 3 beta HSD was induced to a greater magnitude in the neocortex. These findings provide evidence that the lack of 3 beta HSD activity in the fetal zone, previously considered to be the result of the presence of an endogenous inhibitor, is due to an absence of the protein in this portion of the gland. The lack or minimal expression of 3 beta HSD in the fetal zone of HFA may be due to the action (or lack thereof) of a tissue-specific factor regulating the synthesis of 3 beta HSD.     

Prostaglandin secretion by the neocortex and fetal zones of the human fetal adrenal gland

Previously, we reported that the human fetal adrenal (HFA) gland secretes various prostaglandins (PGs) in vitro and that PG secretion is inhibited by endogenously synthesized glucocorticosteroids. In this investigation, the neocortex (NC) and fetal zone (FZ) of the HFA gland were separated by microdissection and maintained as tissue fragments in organ culture. The rate of PG secretion into the culture medium was determined by measuring various PGs using specific RIAs in media collected at 24-h intervals. During the first 24 h in culture, the secretion rates of PGF2 alpha and PGE2 were 6- and 7-fold greater by NC [14 +/- 5 and 9.9 +/- 3 ng mg protein-1 24 h-1 (mean +/- SE)], respectively, than by FZ tissue (2.5 and 1.4 ng mg protein-1 24 h-1). The secretion rates of PGFM and PGD2 were 2-fold greater in NC tissue than in FZ tissue, but the secretion rates of thromboxane B2 were similar in both zones of HFA tissue. In another study, the patterns of secretion of PGF2 alpha and PGE2 were determined as a function of days in culture. The secretion rates of PGF2 alpha and PGE2 fell rapidly in NC from 19.0 +/- 11 and 38.3 +/- 9.7 ng mg protein-1 24 h-1, respectively, to 1.3 +/- 7.2 and 4.8 +/- 3.3 by day 4. In contrast, the secretion rates of PGF2 alpha and PGE2 rose 8- and 3-fold in FZ tissue (from 0.7 +/- 0.2 and 0.9 +/- 0.6 ng mg protein-1 24 h-1, respectively, to 5.9 +/- 0.5 and 3.1 +/- 1.2 by day 4). The addition of ACTH or dexamethasone inhibited PG secretion in both zones, but to a greater degree in FZ tissue than in NC tissue. In summary, the NC secretes larger quantities of PG than the FZ, and the patterns of secretion are different in the two zones. The secretion of PGs is inhibited more in FZ than in NC tissue by ACTH and glucocorticosteroids.     

Adenylate cyclase of human parathyroid gland.

Experiments were performed on a particulate fraction from human parathyroid glands. A high activity of adenylate cyclase was detected which was linear with time and protein concentration. The enzyme had an optimum pH in the range of 7-8 and a Km for ATP of 0.44 X 10(-3) M. Ca++ had a profound inhibitory effect; a concentration of 0.5 mM Ca++ reduced enzyme activity by 60%. Maximal enzyme activity was obtained with 5 mM Mg++; higher concentrations of this cation also inhibited enzyme activity. The effect of Mn++ was similar to that of Mg++. Enzyme activity was stimulated by NaF, catecholamines, glucagon, and calcitonin. The effect of catecholamines seems to be mediated through beta-adrenergic receptors.     

Role of hCG in regulation of the fetal zone of the human fetal adrenal gland.

It has been suggested that hCG is a trophic hormone for the fetal zone of the human fetal adrenal gland. To test this hypothesis, the isolated fetal zones of adrenals from eight fetuses (12-17-week gestation age) were superfused in the presence or absence of hCG. Dehydroepiandrosterone sulfate (DHAS) was measured in the superfusion effluent. A significant increase in DHAS production was observed in the presence of hCG. DHAS secretion decreased during the first 60 min in the control and experimental superfusions from 83 +/- 10.0 (mean +/- SE) to 71 +/- 8.0, and from 90 +/- 9.0 to 70 +/- 6.0 ng/100 mg/ml, respectively. In the presence of hCG (250 ng/ml), DHAS secretion increased significantly (P less than 0.01) over the controls to 116 +/- 12.0 at 120 min, and remained above the controls thereafter. These results support the hypothesis that hCG is one of the regulators of DHAS production by the human fetal adrenal gland early in gestation. As we found that ACTh stimulated DHAS secretion in a previous study and as there is indirect evidence for a role of ACTH in DHAS regulation late in pregnancy, these observations suggest dual regulation by hCG and ACTH early in pregnancy, and a possible transition to ACTH regulation of the fetal zone of the human fetal adrenal after midgestation.     

The role of calcium in steroidogenesis in fetal zone cells of the human fetal adrenal gland

The human fetal adrenal gland is composed primarily of fetal zone (FZ) cells, which have a high rate of steroidogenesis. The purpose of this study was to examine the role of calcium in the regulation of steroidogenesis by FZ cells. Dispersed FZ cells were incubated in Krebs-Ringers medium at 37 C for 3 h in the presence of ACTH, (Bu)2cAMP, or forskolin in addition to various drugs. The medium contents of dehydroepiandrosterone sulfate (DS), cortisol (F), and cAMP were quantified by RIA. After the addition of ACTH (10(-10)-10(-5) M), DS and cAMP secretion increased. The addition of EGTA to the medium inhibited ACTH- and forskolin-stimulated DS, F, and cAMP secretion by 50% as well as (Bu)2cAMP-stimulated steroidogenesis. The addition of calcium (10(-5)-10(-2) M) had only a slight effect on the secretion of DS or F in the absence of ACTH or (Bu)2cAMP. In the presence of ACTH and (Bu)2cAMP, however, increasing amounts of calcium resulted in a 2- to 3-fold increase in the rates of DS and F secretion. The addition of either A23187, a calcium ionophore, or verapamil, a calcium channel blocker, inhibited ACTH-stimulated DS and F secretion by 90%. The rate of cAMP formation was greater after ACTH plus verapamil treatment than after ACTH treatment alone, whereas A23187 inhibited ACTH-stimulated cAMP secretion to basal levels. Both A23187 and verapamil inhibited ACTH- and cAMP-stimulated pregnenolone secretion. The metabolism of 22R-hydroxycholesterol to pregnenolone was inhibited by A23187 and verapamil. In conclusion, our results suggest that extracellular calcium is important for activation of the human adrenal FZ cell adenylate cyclase system, while intracellular calcium plays a multifaceted role in controlling steroid production.     

Identification of definitive and fetal zone markers in the human fetal adrenal gland reveals putative developmental genes

Organogenesis is a coordinated process involving cell replication, differentiation, adhesion, and migration. We seek to understand the complex developmental signals involved in the ontogeny of the human fetal adrenal gland. The gland is comprised initially of two zones, the definitive and fetal zones. A third zone, the transitional zone, develops between them after midgestation. We have suggested that the definitive zone is comprised of a pool of progenitor cells that proliferate and differentiate into cells of the transitional and fetal zones. However, it has not been possible to demonstrate that definitive zone cells have this capacity because of the absence of protein markers unique to these cells; thus, they could not be purified or positively identified. We sought to identify definitive and fetal zone markers to facilitate cell sorting and identify molecules of biological interest in adrenal development. We performed subtractive hybridization, in situ hybridization, and immunofluorescence to identify unique markers of definitive zone cells. NovH and metallopanstimulin were identified by subtraction hybridization, primarily in the definitive zone. P-Glycoprotein, also principally on definitive zone cells, and the low density lipoprotein (LDL) receptor, predominantly on fetal zone cells, were identified by immunofluorescence. Identification of cellular markers unique to each zone of the fetal adrenal gland will enhance the ability to characterize the proliferative potential of definitive zone cells and assess their capacity to differentiate into cells of the transitional and fetal zones. Purified cells also will permit detailed molecular and mechanistic studies of regulation of human fetal adrenal development.     

Adenylate cyclase and guanylate cyclase activity in normal and leukemic human lymphocytes

Adenylate cyclase (AC) and guanylate cyclase (GC) activities were studied in normal B-enriched and T-enriched lymphocytes, in lymphocytes of children with acute lymphocytic leukemia (ALL), and in lymphocytes of adults with chronic lymphocytic leukemia (CLL). AC activity was greater in normal B than T lymphocytes (215 pmole/min/mg protein versus 80 pmole in the membrane-enriched fraction) and i both increased greatly after stimulation with isoproterenol and more so with prostaglandins E and F2 alpha. In leukemic lymphocytes, AC showed depressed activity (20 pmole in ALL cells and 55 pmole in CLL cells) and was less sensitive to hormonal stimulation: this loss of sensitivity occurred to a greater extent in ALL than in CLL lymphocytes. GC activity was greater in normal T than B cells (in membrane-enriched fraction: 10.2 pmole versus 5.3 pmole). It increased little with isoproterenol and prostaglandins stimulation, and much more with sodium azide and dehydroascorbic acid stimulation. GC activity was increased in both types of leukemic lymphocytes (23 pmole for ALL cells and 18 pmole for CLL cells) and was insensitive to stimulation. Possible derangement of cyclase and cyclic nucleotide regulation in leukemic cells is suggested.     

Low density lipoprotein binding and de novo synthesis of cholesterol in the neocortex and fetal zones of the human fetal adrenal gland

The binding of low density lipoprotein (LDL) and the de novo synthesis of cholesterol in separated zones of human fetal adrenal (HFA) tissues were investigated. The number of LDL-binding sites was 2-fold greater in membrane fractions prepared from fresh fetal zone tissue than in those from neocortex tissue. The binding capacity for LDL in fetal zone and neocortex membrane preparations of HFA tissues maintained in culture in the presence of ACTH was 2-fold greater than that in membrane fractions of control tissues. The rates of de novo synthesis of cholesterol also were determined in separated zones of HFA tissue by measuring the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase in microsomal fractions prepared from HFA tissues and by determining the rate of incorporation of tritium from [3H]water into cholesterol in HFA tissue fragments. The rate of de novo synthesis of cholesterol in fresh fetal zone tissue was twice that in neocortex tissue as estimated by these methods. When separated zones of HFA tissue were maintained in culture in the presence or absence of ACTH, the rates of de novo synthesis, as determined by the rate of incorporation of tritium from [3H]water into cholesterol, were stimulated to a similar extent by ACTH in both fetal zone and neocortex tissues. However, the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase was increased to a greater extent by ACTH pretreatment in neocortex tissues than in fetal zone tissues. In summary, fetal zone tissues of the HFA gland have a larger number of LDL-binding sites and higher rates of de novo synthesis of cholesterol than do neocortex tissues, and ACTH stimulates LDL binding and de novo synthesis of cholesterol in both zones of the HFA gland.     

Protein kinase-C in the human fetal adrenal gland

The fetal zone (FZ) of the human fetal adrenal gland undergoes rapid growth and exhibits a high rate of steroidogenesis throughout fetal life. In addition to cAMP-dependent processes regulating steroidogenesis and possibly growth of the FZ, evidence is accumulating that cAMP-independent mechanisms are also involved. The purpose of this study was to determine if the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent stimulator of protein kinase-C activity, stimulates steroidogenesis in FZ cells and to characterize protein kinase-C activity in FZ, neocortex zone, and anencephalic adrenal tissues. Adrenal glands were obtained from first and second trimester abortions and two anencephalic fetuses. The FZ was dissected from the neocortex. In some experiments, dispersed FZ cells were incubated in the presence and absence of ACTH and TPA for 3 h. TPA and ACTH stimulated steroidogenesis 2- and 5-fold, respectively. In other experiments, the separated zones and anencephalic adrenal tissues were homogenized, and the homogenates were subjected to DEAE-cellulose column chromatography. A single peak with phospholipid- and calcium-dependent activity was found. Subcellular distribution studies demonstrated greatest activity in the cytosolic fraction. The specific activity of protein kinase-C was significantly greater in FZ than neocortex zone, whether expressed per mg protein or per microgram DNA content. The activity in anencephalic tissue was low. In addition, protein kinase-C (80,000-dalton molecular size protein) was detected in adrenal tissues after electrophoresis and immunoblotting using an antibody directed against protein kinase-C. Greater amounts of protein kinase-C were detected in FZ tissue than in NC or anencephalic adrenal tissue. These results indicate that the lower activities of protein kinase-C in neocortex and anencephalic adrenal tissues were due to low amounts of enzyme rather than inactive enzyme. In summary, TPA-stimulated steroidogenesis in fetal zone cells and fetal zone cells contained greater activity and a greater amount of protein kinase-C than neocortex cells. Minimal activity and enzyme protein were found in anencephalic tissues. These results suggest that cAMP-independent mechanisms may play a role in fetal adrenal steroidogenesis.     

The role of calmodulin antagonists on steroidogenesis by fetal zone cells of the human fetal adrenal gland

The adrenal gland of the human fetus (HFA) is relatively large compared to that of the adult and exhibits an extremely high rate of steroidogenesis both in vivo and in vitro. The fetal zone cells make up 80-85% of the volume of the HFA and are the major site of steroid production during fetal development. We have recently demonstrated that calcium is involved in the regulation of steroidogenesis in fetal zone cells of the HFA. There is considerable evidence that many actions of calcium within cells are mediated by the calcium-binding protein calmodulin. The purpose of the present investigation was to determine if calmodulin also plays a role in HFA steroidogenesis. To investigate this possibility, the fetal zone was dissected from fetal adrenals of first and second trimester human abortuses. After collagenase digestion of the tissue, dispersed fetal zone cells were maintained in a Krebs-Ringers medium at 37 C for a 3-h incubation. Cells were incubated with and without ACTH (10(-8) M) in the presence of the calmodulin inhibitors trifluoperazine (TFP), chlorpromazine (CPZ), and calmidazolium (CAL) at concentrations of 5-100 microM. The media were assayed for contents of dehydroepiandrosterone sulfate (DS), cortisol (F), pregnenolone, and cAMP by RIA. The addition of ACTH stimulated F secretion 5- to 10-fold compared to that in control fetal zone cells. DS secretion increased up to 5-fold and pregnenolone about 2-fold in the presence of ACTH compared to values in control cells. ACTH also stimulated cAMP secretion by 10-fold compared to that in control cells. The addition of TFP, CPZ, and CAL significantly inhibited ACTH-stimulated DS, F, and pregnenolone secretion in a dose-related fashion to near-control levels. We observed that TFP, CPZ, and CAL inhibited cAMP accumulation as well as Bu2cAMP-stimulated steroid secretion. The metabolism of 22R-hydroxycholesterol to pregnenolone was inhibited by TFP and CPZ, but not by CAL. These studies suggest that calmodulin plays a role in regulating steroidogenesis in fetal zone cells of the HFA.     

Adenylate cyclase activity in human pancreatic adenocarcinoma cell lines

Summary Conclusion BxPC-3, Hs 766T, Capan-2, Panc-1, and Capan-1 cells possess receptors for VIP and β-adrenergic agonists that are functionally coupled to adenylate cyclase. In this respect, they resemble pancreatic duct cells. However, we speculate that the process of neoplastic transformation has either downregulated the expression of secretin receptors or led to a defect in the receptor itself, placing a question mark over the usefulness of these adenocarcinoma cell lines as models of the pancreatic ductal epithelium. Background Because of the importance of ducts in pancreatic disease, we wished to establish which duct cells receptors are functional on adenocarcinoma cell lines. Methods We investigated the expression of agonist-stimulated adenylate cyclase activity in six human pancreatic adenocarcinoma cell lines. Known stimulants of pancreatic ductal secretion, VIP, PHI, secretin, β-adrenergic, and dopamine, were tested. Results For responsive cell lines, VIP was the most effective stimulant followed by adrenaline, isoprenaline, PHI, and secretin. Dopamine was without effect. Since high concentrations of PHI and secretin were required to stimulate cyclase activity, their effect is probably mediated by VIP receptors. Based on the degree of stimulation observed with the individual agonists, Hs 766T and BxPC-3 were the most responsive cell lines, followed by Capan-2 and Capan-1, and finally Panc-1. MIAPaCa-2 cells did not respond to any of the agonists tested.     

Persistence of fetal zone function in the infant rhesus monkey adrenal gland

Plasma dehydroepiandrosterone sulfate (DHAS) concentrations increase markedly in the rhesus monkey fetus at the end of gestation. A further increase occurs in the infant. To determine whether the changes in plasma concentration between the fetus and infant represent maintenance of DHAS production by the infant adrenal gland, we measured the t1/2, distribution volume (VD), MCR, and production rate of DHAS in the late gestation rhesus monkey fetus (129-155 days gestation; term is 165 days) and infant (14-42 days of age). A single bolus dose of [3H]DHAS was injected into five fetuses and four infants, and blood samples were collected serially from 5 min to 24 h after the injection. The amount of [3H]DHAS in the circulation was measured after solvolysis, extraction, and Celite chromatography. The concentration of DHAS in each sample was measured by RIA. DHAS was cleared significantly more rapidly in the fetus than in the infant [MCR in fetus, 2.4 +/- 0.4 (+/- SE); MCR in infant, 0.6 +/- 0.2 liters day-1 kg-1]. The t1/2 of DHAS was shorter in the fetus than in the infant (1.0 +/- 0.1 vs. 3.3 +/- 0.7 h). Absolute VD values were larger in the fetus than in the infant (231 +/- 29 and 143.8 +/- 11.6 ml kg-1); however, they were similar when the fetal VD was calculated including placental weight as a component of fetal weight. The production rate of DHAS, calculated as the product of MCR and integrated plasma DHAS concentration for the duration of the experiment, was not significantly different between the fetus and the infant (1.0 +/- 0.2 and 3.3 +/- 1.2 mg kg-1 day-1) in spite of the marked differences in plasma DHAS concentrations (445.8 +/- 103.8 ng ml-1 in the fetus and 5165 +/- 1296 ng ml-1 in the infant). These results indicate that the adrenal of the infant rhesus monkey continues to secrete DHAS at a rate at least as high as that in the late gestation fetus. Since the infant maintains DHAS production similar to that of the fetus in the absence of the placenta, a corollary of these studies is that the elevated DHAS secretion in the rhesus infant is independent of the placenta or the hormonal milieu of pregnancy. The maintenance of a functional fetal zone in the adrenal gland makes the rhesus infant a suitable model to use in studying the regulation of DHAS secretion and fetal zone morphology.     

Expression and regulation of adenylyl cyclase isoforms in the human adrenal gland

The aim of the present study was to identify which adenylyl cyclase isoforms were expressed in the human adrenal gland and to determine which isoform(s) may be coupled to ACTH action. Our results indicate that, in both glomerulosa and fasciculata zones, adenylyl cyclase 1 was detected in cells at the membrane level, adenylyl cyclases 3 and 2 in both the cytoplasm and the plasma membrane, whereas adenylyl cyclase 5/6 and adenylyl cyclase 4 were found mainly in cytoplasm. The levels of expression of each isoform were similar between the two adrenocortical zones, except for adenylyl cyclase 5/6, which had a lower level of expression in the zona fasciculata. We next evaluated the role of the various adenylyl cyclase isoforms during ACTH-stimulated cAMP production in both glomerulosa and fasciculata cell preparations. Corroborating with previous observations, we found that calcium had a biphasic effect on cAMP production. Interestingly, pertussis toxin treatment increased cAMP production, indicating that, in addition to Gs, ACTH is coupled to a Gi protein. Incubation with the betagamma-subunit sequestrant peptide QEHA decreased cAMP production, as did incubation with inhibitory antibodies against either adenylyl cyclase 2 or adenylyl cyclase 5/6. Inhibitory adenylyl cyclase 3 antibodies interfered with ACTH action only in the zona fasciculata. Altogether these data indicate that adrenocortical cells express one or two isoforms of each class of adenylyl cyclases and, thus, have the ability to produce cAMP in response to various regulatory, intracellular mediators. Importantly, our results indicate that in the human adrenal gland, ACTH acts mainly through adenylyl cyclase 5/6 and adenylyl cyclase 2/4, whereas the effect of ACTH on adenylyl cyclase 3 activity may be a consequence of calcium influx.     

Localization of G protein alpha-subunits in the human fetal adrenal gland

The aim of the present study was to investigate the presence and localization of the main G protein alpha-subunits in the human fetal adrenal gland during the second trimester of gestation. Immunofluorescence studies conducted on sections from frozen glands obtained immediately after therapeutic abortion indicated that the alpha s subunit of the heterotrimeric Gs protein was detected in all adrenal cell types, except for endothelial cells. The other alpha-subunits had a more specific pattern of distribution. Indeed, the alpha il-2 protein was restricted to the definitive zone, whereas alpha i3 labeling was mainly expressed in the fetal zone. The alpha q protein subunit was localized in vascular endothelial cells at the periphery of the adrenal gland and in fetal cells at the center. Finally, chromaffin cells expressed alpha s, alpha q, and alpha o1, but not alpha o2 nor alpha i. Altogether, these results indicate that the human fetal adrenal gland is not only unique in its particular morphology and expression of steroidogenic enzymes, but also by the differential expression of G protein alpha-subunits. Such cell specific distribution in glands from midgestational fetuses may account for the absence or the different responses to stimuli, when compared with the adult adrenal gland.     

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.     

Lipoprotein utilization and cholesterol synthesis by the human fetal adrenal gland

A model proposed for regulation of steroidogenesis, lipoprotein utilization and cholesterol metabolism in HFA tissue is presented in Fig 17. We envision that the role of ACTH and cAMP in steroidogenesis and cholesterol metabolism is as follows. ACTH binds to specific receptors on the surface of the cells of the HFA gland and as a consequence, adenylate cyclase is activated, leading to increased formation of cAMP. cAMP causes activation of protein kinase that leads, presumably, to phosphorylation of specific proteins. This leads to the initiation of reactions that give rise to increased activity of key enzymes and levels of proteins involved in adrenal cholesterol metabolism. Presumably, the action of ACTH causes an increase in the activity of cholesterol side chain cleavage, the rate-limiting step in the conversion of cholesterol to steroid hormones. We suggest that once the mitochondrial cholesterol side-chain cleavage system is fully activated by ACTH, the supply of cholesterol to the mitochondria becomes rate-limiting for steroidogenesis. To meet this demand for cholesterol, a further action of ACTH results in an increase in the number of LDL receptors. LDL binds to specific receptors on the cell surface that are localized in coated pits. LDL is internalized by a process of adsorptive endocytosis and the internalized vesicles fuse with lysosomes and the protein component of LDL is hydrolyzed by lysosomal proteolytic enzymes to amino acids. The cholesteryl esters of LDL also are hydrolyzed to give rise to fatty acids and cholesterol. The liberated cholesterol is available for utilization in the biosynthesis of steroid hormones and other cellular processes. In addition, ACTH stimulates the activity of HMG CoA reductase and, thus, the rate of de novo cholesterol biosynthesis. In this way sufficient cholesterol is obtained to provide for precursor cholesterol to maintain the high rate of steroid synthesis by the HFA. HDL is not utilized as a source of cholesterol by the HFA. Because of the rapid rate of utilization of LDL by the HFA, fetal plasma levels of LDL are low and the activity of the HFA is a primary determinant of these levels. Thus, in the case of anencephaly, in which the activity of the adrenal is very low, plasma levels of LDL are 2--3 times higher than in normal fetuses, whereas plasma HDL levels are similar. In addition, in the normal neonate plasma LDL levels rise rapidly after birth, and this event is coincident with the involution of the fetal zone of the adrenal. The fetal liver is likely to be the major source ultimately of the LDL-cholesterol utilized by the HFA. Consequently, factors that regulate cholesterol and lipoprotein synthesis in the fetal liver may, in turn, affect the steroidogenic activity of the HFA through regulation of the supply of cholesterol precursor. Thus, if trophic factors for the HFA other than ACTH exist, an important site of their action might be the fetal liver, rather than a direct action to influence the rate of synthesis of steroids by the fetal adrenal.