Preparation and evaluation of sustained-release matrix tablets based on metoprolol and an acrylic carrier using injection moulding.

Sustained-release matrix tablets based on Eudragit RL and RS were manufactured by injection moulding. The influence of process temperature; matrix composition; drug load, plasticizer level; and salt form of metoprolol: tartrate (MPT), fumarate (MPF) and succinate (MPS) on ease of processing and drug release were evaluated. Formulations composed of 70/30% Eudragit RL/MPT showed the fastest drug release, substituting part of Eudragit RL by RS resulted in slower drug release, all following first-order release kinetics. Drug load only affected drug release of matrices composed of Eudragit RS: a higher MPT concentration yielded faster release rates. Adding triethyl citrate enhanced the processability, but was detrimental to long-term stability. The process temperature and plasticizer level had no effect on drug release, whereas metoprolol salt form significantly influenced release properties. The moulded tablets had a low porosity and a smooth surface morphology. A plasticizing effect of MPT, MPS and MPF on Eudragit RS and Eudragit RL was observed via DSC and DMA. Solubility parameter assessment, thermal analysis and X-ray diffraction demonstrated the formation of a solid solution immediately after production, in which H-bonds were formed between metoprolol and Eudragit as evidenced by near-infrared spectroscopy. However, high drug loadings of MPS and MPF showed a tendency to recrystallise during storage. The in vivo performance of injection-moulded tablets was strongly dependent upon drug loading.

Adrenocortical tissue formed by transplantation of normal clones of bovine adrenocortical cells in scid mice replaces the essential functions of the animals' adrenal glands.

Xenotransplanted adrenocortical tissue of clonal origin was formed in immunodeficient (scid) mice by using techniques of cell transplantation. The experiments reported here used a single clone of bovine adrenocortical cells, but 5 of 20 other randomly selected clones also formed tissue. Most adrenalectomized animals bearing transplanted cells survived indefinitely, demonstrating that the cells restored the animals' capacity to survive in the absence of sodium supplementation. Formation of well-vascularized tissue at the site of transplantation was associated with stable levels of cortisol in the blood, replacing the mouse glucocorticoid (corticosterone). Ultrastructurally, the cultured cells before transplantation had characteristics of rapidly growing cells, but tissue formed in vivo showed features associated with active steroidogenesis. These experiments show that an endocrine tissue can be derived from a single, normal somatic cell.

Presence of double-strand breaks with single-base 3' overhangs in cells undergoing apoptosis but not necrosis.

Apoptotic cells in rat thymus were labeled in situ in paraffin-embedded and frozen tissue sections by ligation of double-stranded DNA fragments containing digoxigenin or Texas red. Two forms of double-stranded DNA fragments were prepared using the polymerase chain reaction: one was synthesized using Taq polymerase, which yields products with single-base 3' overhangs, and one using Pfu polymerase, which produces blunt-ended products. Both types of fragment could be ligated to apoptotic nuclei in thymus, indicating the presence in such nuclei of DNA double-strand breaks with single-base 3' overhangs as well as blunt ends. However, in nuclei with DNA damage resulting from a variety of nonapoptotic processes (necrosis, in vitro autolysis, peroxide damage, and heating) single-base 3' overhangs were either nondetectable or present at much lower concentrations than in apoptotic cells. Blunt DNA ends were present in such tissues, but at lower concentrations than in apoptotic cells. In contrast, in all of these forms of DNA damage, nuclei contained abundant 3'-hydroxyls accessible to labeling with terminal deoxynucleotidyl transferase. Thus, although single-base 3' overhangs and blunt ends are present in apoptotic nuclei, the specificity of the in situ ligation of 3'-overhang fragments to apoptotic nuclei indicates that apoptotic cells labeled in this way can readily be distinguished from cells with nonapoptotic DNA damage. These data are consistent with the involvement of an endonuclease similar to DNase I in apoptosis, which is predicted to leave short 3' overhangs as well as blunt ends in digestion of chromatin.

Hormonal control of adrenocortical cell proliferation. Desensitization to ACTH and interaction between ACTH and fibroblast growth factor in bovine adrenocortical cell cultures.

A primary bovine adrenocortical cell culture system responsive to physiological concentrations of ACTH has been established. When added to cultures, ACTH inhibited DNA synthesis and cell division over the same concentration range required for stimulation of fluorogenic steroid production (0.01-10 nM). With chronic exposure to ACTH, cells became desensitized to the growth inhibitory effects of ACTH. Though cell growth was initially completely inhibited by ACTH, cells ultimately began to grow in its continued presence. The lag time to initiation of cell growth, the rate of growth, and the final density achieved depended on the ACTH concentration. Desensitization to ACTH(1-39) was not induced by monobutyryl cyclic AMP nor by ACTH(11-24). Specificity of desensitization was apparent because cells which had become desensitized to ACTH(1-39) remained fully responsive to monobutyryl cyclic AMP, prostaglandin E(1), and cholera toxin. Though the effects of ACTH on cell growth were readily reversible upon hormone removal, the desensitized response to readdition of ACTH persisted for at least 8 h. Fibroblast growth factor (FGF) stimulated both the growth rate and saturation density achieved. FGF did not alter the growth inhibitory effects of ACTH nor the reduced growth rate observed in desensitized cells maintained in ACTH. However, FGF greatly increased the saturation density achieved by cultures maintained with ACTH. Through the process of desensitization, adrenocortical cells are able to grow in the presence of high concentrations of ACTH and to respond to the effects of a growth factor by increasing the cell density achieved. This pattern of response may be a general one for growth control under the combined effects of antimitotic and mitotic factors.

Expression of p21(WAF1/CIP1/SDI1) and p53 in apoptotic cells in the adrenal cortex and induction by ischemia/reperfusion injury.

p21(WAF1/CIP1/SDI1), an inhibitor of cyclin-dependent kinases, is expressed at varying levels in human adrenal glands removed during surgery or organ recovery. In glands with p21 mRNA, nuclear p21 immunoreactivity, which was occasionally extensive, colocalized with p53 immunoreactivity and DNA damage, as evidenced by in situ end-labeling. Many cells showed morphological features of apoptosis when observed by fluorescent DNA dye staining and electron microscopy. This pattern was also associated with high levels of cytoplasmic heat shock protein 70. To address the question of the origin of p21 expression in some human adrenal glands, rat adrenal glands were subjected to 30 min of ischemia followed by 8 h of reperfusion. Cells with nuclear p21 and p53 appeared in the adrenal cortex together with DNA damage detected by in situ end-labeling. Nuclear p21 immunoreactivity was also produced in adrenal tissue fragments incubated at 37 degrees C in vitro. However, in this case, p21 expression was confined to the cut edge of the tissue. In contrast, p21 in human adrenal glands, as in ischemic rat glands, was within the inner regions of the cortex, supporting an origin of the protein in vivo rather than postmortem. The p53/p21 pathway of reaction to cellular injury, potentially leading to apoptosis, may play a role in tissue damage such as that resulting from ischemia/reperfusion. In the human adrenal cortex this process may be a precursor of adrenal failure.

Formation of functional tissue from transplanted adrenocortical cells expressing telomerase reverse transcriptase.

We report the first use of human telomerase reverse transcriptase (hTERT) expression in experimental xenotransplantation. Previously, we showed that bovine adrenocortical cells can be transplanted into severe combined immunodeficient (SCID) mice, and that these cells form functional tissue that replaces the animals' own adrenal glands. We cotransfected primary bovine adrenocortical cells with plasmids encoding hTERT, SV40 T antigen, neo, and green fluorescent protein. These clones do not undergo loss of telomeric DNA and appear to be immortalized. Two clones were transplanted beneath the kidney capsule of SCID mice. Animals that received cell transplants survived indefinitely despite adrenalectomy. The mouse glucocorticoid, corticosterone, was replaced by the bovine glucocorticoid, cortisol, in the plasma of these animals. The tissue formed from the transplanted cells resembled that formed by transplantation of cells that were not genetically modified and was similar to normal bovine adrenal cortex. The proliferation rate in tissues formed from these clones was low and there were no indications of malignant transformation.

Adenovirus-delivered DKK3/WNT4 and steroidogenesis in primary cultures of adrenocortical cells.

The Wnt family molecules Dickkopf-3 (DKK3) and WNT4 are present at higher concentrations in the zona glomerulosa than in the rest of the adrenal cortex. In order to study direct effects of these proteins on adrenocortical cell function, we created adenoviruses encoding human DKK3 and WNT4. When added to cultured human adrenocortical cells, DKK3 inhibited aldosterone and cortisol biosynthesis, either alone or together with cyclic AMP. WNT4 increased steroidogenesis when added alone but decreased it in the presence of cyclic AMP. A control adenovirus encoding GFP had no effect. RNA was prepared from cultured cells and was assayed by real-time PCR. CYP11A1 (cholesterol side-chain cleavage enzyme), HSD3B2 (3beta-hydroxysteroid dehydrogenase type II), CYP17 (17 alpha-hydroxylase), CYP21 (21-hydroxylase) and CYP11B1 (11 beta-hydroxylase) mRNAs were all increased by cyclic AMP, whereas CYP11B2 (aldosterone synthase) was unaffected. DKK3 decreased cyclic AMP-stimulated CYP17. WNT4 increased both CYP17 and CYP21 in the absence of cyclic AMP. Both DKK3 and WNT4 increased the level of CYP11B2. These data show that these Wnt signaling molecules have multiple actions on steroidogenesis in adrenocortical cells, including effects on overall steroidogenesis (aldosterone and cortisol biosynthesis) and distinct effects on steroidogenic enzyme mRNA levels. The co-localization of DKK3 and WNT4 in the glomerulosa and their stimulation of CYP11B2 imply an action on glomerulosa-specific function.

Early events in the formation of a tissue structure from dispersed bovine adrenocortical cells following transplantation into scid mice.

The early events that follow the transplantation of dispersed bovine adrenocortical cells into scid mice were investigated. We introduced adrenocortical cells into a small cylinder inserted beneath the kidney capsule, where they form a tissue structure that becomes vascularized and secretes steroids that replace those from the animal's own adrenal glands, which are removed during the transplantation surgery. We studied cell proliferation, cell survival, apoptosis, and the role of p21WAF1/CIP1/SDI1 over the first 6 days following transplantation. Additionally we examined the invasion of the tissue by host macrophages and endothelial cells. The data show that there is healthy survival of most of the transplanted cells, and that this is related to their position in the cell transplantation cylinder. In the layer of cells that was adjacent to the kidney parenchyma there was a higher rate of cell proliferation and a lower rate of apoptosis than in cells that were located in the upper part of the cylinder. In the lower layer cells were more likely to have nuclear p21, and macrophages and endothelial cells were observed only in this region. Cells that incorporated bromodeoxyuridine administered to animals 2 or 4 days following transplantation were not more likely than other cells to be undergoing a second division when the animals were killed at 6 days, suggesting that proliferation in the lower layer is not confined to a small subpopulation of cells. Among different animals, the extent to which the spaces between the transplanted cells became lined by host endothelial cells was correlated with higher levels of proliferation and nuclear p21, suggesting that vascularization is the critical step for the continued survival and proliferation of the transplant. The present experiments show that bovine adrenocortical cells transplanted into scid mice form a useful model for the study of tissue formation from dispersed cells and the interaction of the transplanted cells with the host.

Expression of 11 beta-hydroxylase and 21-hydroxylase in long-term cultures of bovine adrenocortical cells requires extracellular matrix factors.

In order to elucidate mechanisms for the loss of expression of 11 beta-hydroxylase and 21-hydroxylase, induction of these genes in long-term cultures of bovine adrenocortical cells was reassessed and compared with induction of 17 alpha-hydroxylase. We previously showed that both 11 beta- and 21-hydroxylases require insulin-like growth factor-I (IGF-I) as well as cAMP for induction; these are the only factors needed by primary cultures. Cells at population doubling level 10 grown on fibronectin-coated polystyrene dishes and incubated with cholera toxin and IGF-I did not express 11 beta-hydroxylase and 21-hydroxylase. They showed a truncated steroidogenic pathway, converting 25-hydroxycholesterol to some 11-deoxycortisol but little cortisol. However, when population doubling level 10 cells were grown for 5 days in extracellular matrix Matrigel, cholera toxin and IGF-I induced a complete steroidogenic pathway to cortisol. Northern blotting also showed that expression of 11 beta-hydroxylase messenger RNA (mRNA) after cholera toxin/IGF-I induction was observed only in cultures grown in Matrigel and was undetectable in cultures grown on plastic. 21-Hydroxylase mRNA was observed in cultures grown on plastic but was greatly enhanced by Matrigel; however, 17 alpha-hydroxylase mRNA was induced to a similar extent with and without Matrigel. In other middle passage cultures, whether grown as mass cultures, SV40 T antigen-transfected clones, or normal (nontransfected) clones, cells did not express 11 beta-hydroxylase except when grown in Matrigel; 21-hydroxylase was low and expression was enhanced by Matrigel, whereas 17 alpha-hydroxylase expression was unaffected. As previously determined, in late-passage cells and clones only side-chain cleavage enzyme and 3 beta-hydroxysteroid dehydrogenase activities were detected and 17 alpha-hydroxylase was not expressed. In such cells 11 beta-hydroxylase and 21-hydroxylase were also not expressed, even in the presence of Matrigel. Thus, prior to the previously described loss of expression of 17 alpha-hydroxylase, Matrigel permits the cholera toxin/IGF-I-induced expression of a complete steroidogenic pathway in bovine adrenocortical cells in long-term culture.

Dual regulation of 3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, and dehydroepiandrosterone sulfotransferase by adenosine 3',5'-monophosphate and activators of protein kinase C in cultured human adrenocortical cells.

The relationship between cAMP and protein kinase C in the regulation of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), 17 alpha-hydroxylase, and sulfotransferase was examined in human fetal adrenocortical cells under defined serum-free conditions in culture. Forskolin induced 3 beta HSD and 17 alpha-hydroxylase in a dose-dependent manner, with maximal effects at 10 microM. 12-O-Tetradecanoyl phorbol 13-acetate (TPA) at 1 nM depressed the induction of 17 alpha-hydroxylase activity by forskolin by more than 95% and increased the stimulation of 3 beta HSD activity by forskolin by 4- to 5-fold. Increases were maximal at 48-72 h of incubation. Dehydroepiandrosterone sulfotransferase activity increased over 48 h when cells were transferred to serum-free defined medium. Addition of 10 microM forskolin stimulated sulfotransferase activity only when cells remained in 10% serum. TPA at 1 nM inhibited the increase in sulfotransferase activity. The concentration of TPA required for inhibition of forskolin-stimulated 17 alpha-hydroxylase and sulfotransferase activity was similar to that required for enhancement of forskolin-induced 3 beta HSD activity, suggesting that comparable levels of C kinase activation are involved in these events. Angiotensin II, carbachol, epidermal growth factor, and fibroblast growth factor had actions similar to those of TPA on one or more of these enzyme activities. TPA also had similar actions on enzyme activities when they were stimulated by cAMP analogs rather than by forskolin. These studies suggest that adrenal steroid biosynthesis is under dual regulation by cAMP and protein kinase C. cAMP induces enzymes required for synthesis of 17 alpha-hydroxylated steroids, including the adrenal androgens. Activation of protein kinase C may play a complementary role by enhancing the induction of enzymes required for non-17 alpha-hydroxylated steroid biosynthesis and inhibiting those involved in the synthesis of androgens.

TPA inhibits the synthesis of androgens and cortisol and enhances the synthesis non-17 alpha-hydroxylated steroids in cultured human adrenocortical cells.

Previously, we showed that 12-O-tetradecanoyl phorbol 13-acetate (TPA), an activator of protein kinase C, is a mitogen for fetal human definitive zone adrenocortical cells in culture. In the present experiments, TPA inhibited forskolin-stimulated cortisol, dehydroepiandrosterone (DHEA), and dehydroepiandrosterone sulfate (DHEAS) synthesis, and enhanced forskolin-stimulated progesterone and corticosterone synthesis. These changes in the pattern of steroidogenesis were shown to result from changes in enzyme activities after forskolin treatment. TPA increased forskolin-stimulated 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) 2-fold, while depressing forskolin-stimulated 17 c-hydroxylase to basal values DHEA sulfotransferase increased 3-fold on transfer of human adrenocortical cells from serum-containing to defined, serum-free medium; TPA inhibited this increase. Experiments in which TPA was added at various times during the time course of forskolin stimulation of 17 alpha-hydroxylase showed that TPA prevents the increase in the level of 17 alpha-hydroxylase, and does not have a direct inhibitory effect on 17 alpha-hydroxylase activity. TPA also inhibited stimulation of 17 alpha-hydroxylase by cAMP analogs, indicating that the inhibition of 17 alpha-hydroxylase by TPA is not due to an effect on adenylate cyclase. Previous experiments have shown that stimulation of intracellular cAMP is sufficient for androgen synthesis by the human adrenocortical cell, under defined, serum-free conditions, and that its high rate of androgen synthesis likely results from the relative levels of 3 beta-HSD, 17 alpha-hydroxylase, and DHEA sulfotransferase in the cell. Enzyme induction by cAMP results in increased production of both androgens and glucocorticoids, whereas activation of protein kinase C changes the balance of enzymes, resulting in increased non-17 alpha-hydroxylated steroid synthesis and decreased androgen and cortisol biosynthesis.

Regulation of responsiveness of cultured adrenal cells to adrenocorticotropin and prostaglandin E1: cell density, cell division, and inhibitors of protein synthesis.

In cultured bovine adrenocortical cells, responsiveness to ACTH, as assessed by the maximal rate of ACTH-stimulated cAMP production, has been found to depend on cell density and cell proliferation, while the maximal rate of prostaglandin E1 (PGE1)-stimulated cAMP production was constant. The combination of low cell density and normal cell proliferation caused a specific decline in responsiveness to ACTH. Responsiveness did not decline at any density when proliferation was inhibited by mitomycin C treatment. Specific declines in responsiveness to ACTH were also seen when cultures were treated with cycloheximide or sodium butyrate. When protein synthesis was completely inhibited by cycloheximide treatment, responsiveness to ACTH declined with a half-life of 20.5 h and responsiveness to PGE1 declined with a half-life of 75h. Because PGE1-stimulated cAMP production indicates intact adenylate cyclase catalytic activity, changes in ACTH-stimulated cAMP production appear to be due to specific changes in functional ACTH receptors.

Characterization of adult bovine adrenocortical cells throughout their life span in tissue culture.

The characteristics of adult bovine adrenocortical cells were studied throughout their life span of 55-65 generations in monolayer culture. Over this period, the cells maintained the capacity to synthesize steroids when tested with repeated maximal doses of ACTH, prostaglandin E1, monobutyryl cAMP, or cholera toxin. Prostaglandin E1 stimulated cAMP production and steroidogenesis, and inhibited DNA synthesis, as measured by incorporation of [3H]thymidine, with dose-response characteristics that did not vary over the first 50 generations in culture. In contrast, the maximal rate of cAMP production stimulated by ACTH declined exponentially at a rate of 7% per generation. In primary and secondary cultures, ACTH stimulated steroidogenesis maximally and inhibited [3H]thymidine incorporation into DNA completely at a half-maximal effective concentration (ED50) of 0.08 nM which was two orders of magnitude less than the ED50 of 8 nM for stimulation of cAMP production. As the ACTH-stimulated maximal rate of cAMP production fell with increasing generation number, the ED50 for ACTH stimulation of steroidogenesis and inhibition of DNA synthesis increased. From about the 20th generation onward, the ability of ACTH to inhibit DNA synthesis maximally declined so that by the 40th generation, cells were completely resistant to the growth-inhibitory effects of ACTH. High-dose ACTH continued, however, to stimulate steroid production maximally over the 50 generations studied. In late passage cells, the ED50 for ACTH stimulation of steroidogenesis was 8 nM, identical to that for cAMP production. Although ACTH-stimulated cAMP production was related to both stimulation of steroidogenesis and inhibition of DNA synthesis, higher cAMP levels appeared required for inhibition of DNA synthesis than for stimulation of steroidogenesis. Mitogenic responses to fibroblast growth factor and to angiotensin II were retained throughout long term growth in culture. The progressive loss of ACTH-responsiveness was specific and a function of aging of bovine adrenocortical cells in culture.

Regulation of 11 beta- and 17 alpha-hydroxylases in cultured bovine adrenocortical cells: 3', 5'-cyclic adenosine monophosphate, insulin-like growth factor-I, and activators of protein kinase C.

The induction of steroid 11 beta-hydroxylase and 17 alpha-hydroxylase was studied in bovine adrenocortical cell cultures in serum-free medium. In the absence of insulin-like growth factor (IGF)-I or insulin, cholera toxin failed to increase 11 beta-hydroxylase enzyme activity or messenger RNA (mRNA) levels; cholera toxin increased 11 beta-hydroxylase activity and mRNA only in the presence of 10 nM IGF-I or of higher concentrations of insulin. 17 alpha-Hydroxylase enzyme activity and mRNA, in contrast, were increased maximally by cholera toxin in the absence of insulin or IGF. We also compared the induction of 11 beta-hydroxylase and 17 alpha-hydroxylase by intracellular second messengers. When cultures were incubated with cholera toxin, cAMP analogs, forskolin, ACTH, or prostaglandin E1 in defined medium with insulin, all agents increased the mRNA levels for 11 beta-hydroxylase and 17 alpha-hydroxylase. 11 beta-Hydroxylase enzyme activity was detectable in control (insulin only) cultures and was increased to varying extents by the different agents. 17 alpha-Hydroxylase enzyme activity was undetectable in control cultures and was increased more than 50-fold by all agents. We compared the sensitivity of induction of 11 beta-hydroxylase and 17 alpha-hydroxylase enzyme activities by cAMP using serial dilutions of an equimolar mixture of N6-monobutyryl cAMP and 8-bromo cAMP. For both enzymes, the response curve was biphasic, with a maximal response in the range of 20 to 100 microM each analog, but the decline in response at higher cAMP concentrations was much more marked for 11 beta-hydroxylase than for 17 alpha-hydroxylase. The effects of activation of protein kinase C were studied in cultures incubated with 12-O-tetradecanoylphorbol-13-acetate (TPA) together with a cAMP analog mixture. TPA decreased cAMP-induced 11 beta-hydroxylase mRNA; TPA also decreased the induction of 17 alpha-hydroxylase mRNA, as previously reported. TPA caused a dose-dependent decrease in cAMP-induced 11 beta-hydroxylase enzyme activity. Angiotensin II at 0.1 to 10 microM also decreased induction of 11 beta-hydroxylase. Induction of 11 beta-hydroxylase and 17 alpha-hydroxylase is coordinately regulated by cAMP, protein kinase C, and IGF-I/insulin, but responses to these regulators differ in various respects between these two cytochrome P450 enzymes.

Mechanism of angiotensin II-induced proliferation in bovine adrenocortical cells.

The peptide hormone angiotensin-II (AII) is a potent vasoconstrictor and major regulator of aldosterone synthesis. In addition, AII also has growth-promoting effects. We have recently shown that the lipoxygenase (LO) pathway of arachidonic acid plays a major role in AII-induced aldosterone synthesis in adrenal glomerulosa cells. The LO pathway is also involved in the vasopressor and renin-inhibitory effects of AII. However, the role of LO products in AII-induced mitogenic effects have not yet been investigated. In the present studies we have evaluated the role of the LO pathway in AII-induced proliferative responses in a bovine adrenocortical cell clone termed AC1 cells. In addition, the potential receptor type and mechanism of AII-induced proliferation was studied by evaluating the effect of specific nonpeptide type 1 and type 2 AII receptor antagonists and the role of protein kinase-C (PKC). AII-induced DNA synthesis was significantly attenuated by two structurally dissimilar LO inhibitors, baicalein and phenidone. In addition, the LO product 12-hydroxyeicosatetraenoic acid (12-HETE) itself caused a significant increase in DNA synthesis, suggesting that the 12-LO pathway in part plays a role in AII-mediated mitogenesis. AII-induced proliferative responses were blocked by the type 1 AII receptor antagonist. Both AII- and 12-HETE-induced increases in DNA synthesis were markedly inhibited by two PKC blockers, staurosporine and sangivamycin. Further, both AII and 12-HETE could activate PKC by translocating it from the cytosol to the membrane fraction, as determined by Western immunoblotting. These results suggest that both 12-LO activation and protein kinase-C have an important role in AII-induced adrenal cell proliferation.

Morphological correlates of adrenocorticotropin-stimulated steroidogenesis in cultured adrenocortical cells: differences between bovine and human adrenal cells.

ACTH causes dramatic morphological changes in cultured adrenocortical cells of several species. In the present study, the morphological changes induced by ACTH in primary bovine and human fetal adrenocortical cells have been examined. Both cell types responded similarly in their ACTH-stimulated increase in steroidogenesis and ACTH inhibition of cell proliferation. However, they differed in their morphological responses. Epithelial-like human fetal adrenal cells became round and lost substrate attachment as a result of ACTH treatment, whereas similarly treated cultured bovine adrenal cells did not. Observation of actin distribution using 7-nitrobenz-2-oxa-1,3-diazolephallacidin and fluorescence microscopy revealed a loss of stress fibers in the human cells treated with ACTH, but little or no effect in identically treated bovine cells. Because of the similar biochemical responses but different morphological responses in these cells, we suggest that the steroidogenic and retraction responses to ACTH are separable events.

The response of 21-hydroxylase messenger ribonucleic acid levels to adenosine 3',5'-monophosphate and 12-O-tetradecanoylphorbol-13-acetate in bovine adrenocortical cells is dependent on culture conditions.

The regulation of 21-hydroxylase mRNA and enzyme activity by cAMP and 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied as a function of different culture conditions in bovine adrenocortical cells. Induction of 21-hydroxylase mRNA and enzyme activity was assessed after cultures were incubated with cAMP analogs. Basal 21-hydroxylase mRNA and enzyme activities were high; cAMP addition resulted in some increase in the level of mRNA, but little increase in enzyme activity. When cultures were made mitotically quiescent by using a higher starting cell density or by allowing cells to grow for a longer period before incubation with cAMP, basal mRNA and enzyme activity were low and increased greatly in response to cAMP. Other agents that raise intracellular cAMP (ACTH, prostaglandin E1, cholera toxin, and forskolin) increased 21-hydroxylase mRNA in quiescent cultures. As previously demonstrated for 11 beta-hydroxylase,21-hydroxylase mRNA induction by cholera toxin or cAMP analogs was dependent on the presence of insulin-like growth factor-I or insulin in the culture medium. When cultures were not quiescent, 21-hydroxylase mRNA and enzyme activity were highly variable, but characterized by 1) high basal levels, 2) small increases or actual decreases in response to cAMP, and 3) variable increases in response to TPA, not observed in quiescent cultures. This last effect was observed within a very narrow range of TPA concentrations (0.3-3 nM); at higher concentrations 21-hydroxylase activity decreased to values below those in the control culture. The protein kinase inhibitor staurosporine at 1-5 nM inhibited basal 21-hydroxylase enzyme activity in nonquiescent cultures by up to 80%. The probable existence of controlling factors for 21-hydroxylase expression other than cAMP suggests that the regulation of this gene differs substantially from that of the other steroid hydroxylases.

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.

The role of ascorbic acid in the function of the adrenal cortex: studies in adrenocortical cells in culture.

To investigate the role of ascorbic acid in the function of the adrenal cortex, we studied the effects of ascorbate on the regulation of 11 beta-hydroxylase in culture. When primary bovine adrenocortical cells were cultured in a serum-free defined medium in the absence of ACTH, 11 beta-hydroxylase activity declined with a half-time of about 40 h. When 50 microM cortisol, which acts as a pseudosubstrate for 11 beta-hydroxylase, was added to such cultures, 11 beta-hydroxylase activity declined with a half-time of about 6 h. Ascorbate (5 mM) markedly reduced the rate of loss of 11 beta-hydroxylase activity in the presence of cortisol. Previous studies showed that phenolic and sulfoxide antioxidants, which also prevent loss of 11 beta-hydroxylase activity, inhibited the enzyme at concentrations somewhat higher than those required for protective activity. However, ascorbate at concentrations from 10 microM to 5 mM did not inhibit 11 beta-hydroxylase. The same range of ascorbate concentrations added to cells during a 24-h preincubation with cortisol showed increasing prevention of loss of 11 beta-hydroxylase activity. Ascorbate and a lowered concentration of oxygen were synergistic in their protective action. At 2% oxygen, 5 mM ascorbate almost completely prevented loss of 11 beta-hydroxylase activity in the presence of 50 microM cortisol. 11 beta-Hydroxylase activity was reinduced over a period of 5 days in third passage cultures by addition of 1 microM ACTH in defined lipoprotein-free medium. Addition of ascorbate enhanced the reinduction about 2-fold. The action of ascorbate in prevention of pseudosubstrate-mediated loss of activity and in enhancing reinduction of 11 beta-hydroxylase is specific; neither alpha-tocopherol nor selenium prevented loss of 11 beta-hydroxylase in the presence of cortisol or enhanced reinduction of 11 beta-hydroxylase in the presence of ACTH. As an additional test of specificity, it was shown that reinduction of 17-hydroxylase activity was completely unaffected by ascorbate, selenium, or alpha-tocopherol, and addition of cortisol to cultures with high 17-hydroxylase did not result in any loss of enzyme activity. Thus, a major function of ascorbate in the adrenal cortex is as a protective compound for cytochrome.

Steroidogenic enzyme activities in cultured human definitive zone adrenocortical cells: comparison with bovine adrenocortical cells and resultant differences in adrenal androgen synthesis.

The activities of 3 beta-hydroxysteroid dehydrogenase, 17-hydroxylase, 21-hydroxylase, 11 beta-hydroxylase, C17,20-lyase, and dehydroepiandrosterone sulfotransferase were measured in cultured human fetal definitive zone adrenocortical cells with and without prior exposure to 1 microM ACTH for 48 h. Enzyme induction and measurements of activity were performed using serum- and lipoprotein-free conditions. ACTH induced increases of 5- to 100-fold in the activity of all of these enzymes. Although 3 beta-hydroxysteroid dehydrogenase activity was increased 15-fold, its activity was still an order of magnitude less than that of the hydroxylases. In contrast, when similar experiments were performed using bovine adrenocortical cells, 3 beta-hydroxysteroid dehydrogenase activity was similar to that of the hydroxylases after induction with ACTH. The lower activity of 3 beta-hydroxysteroid dehydrogenase in human cells compared to that in bovine cells resulted in different sequences of transformation of [3H]pregnenolone. The initial product in human cells, before or after induction with ACTH, was 17-hydroxypregnenolone, which was then converted about equally to cortisol (via 17-hydroxyprogesterone and 11-deoxycortisol) and dehydroepiandrosterone sulfate (via dehydroepiandrosterone). In contrast, bovine cells converted pregnenolone to progesterone, with or without prior exposure to ACTH, which was then converted to 17-hydroxyprogesterone, with minimal formation of dehydroepiandrosterone. Adrenal androgen synthesis by human adrenocortical cells thus results from low 3 beta-hydroxysteroid dehydrogenase, which is an intrinsic cell property. Since these experiments were performed using serum-free conditions, cells were not exposed to hormones other than ACTH. The results support the hypothesis that human adrenal androgen synthesis does not require a special hormone.