Opposite effects of angiotensin-II and corticotropin on bovine adrenocortical cell steroidogenic responsiveness.

The long-term effects of angiotensin-II (A-II) and corticotropin (ACTH) on bovine adrenal fasciculata cells (BAC) were studied. Cells were pretreated for 3 days with either A-II or ACTH followed by an examination of the acute steroidogenic response to both hormones as well as the ability to convert several steroid precursors to cortisol and corticosterone. ACTH pretreatment caused a marked increase in cortisol output associated with a decrease in corticosterone secretion in response to both hormones leading to a 50-fold decrease in the corticosterone/cortisol ratio compared to control cells. After incubation with saturating concentrations (5 X 10(-5) M) of 22 R-hydroxycholesterol, pregnenolone or progesterone, ACTH-pretreated cells produced more cortisol than corticosterone whereas the contrary was observed in control cells. However, the conversion of 17 alpha-hydroxyprogesterone and 11-deoxycortisol to cortisol by ACTH-pretreated cells was lower than by control cells. Thus, the main effects of ACTH were a marked increase of 17 alpha-hydroxylase and a small but significant decrease of 21-hydroxylase and 11 beta-hydroxylase activities. A-II pretreatment produced, in a concentration-dependent manner, a down-regulation of its own receptors and homologous and heterologous steroidogenic desensitization. At maximal concentrations (10(-6) M) A-II reduced by 70% its own receptors while the steroidogenic response to A-II and ACTH was reduced by 95% and 75%, respectively. However, the coupling of A-II receptors to phosphoinositide pathway and to Ca2+ influx, as well as its potentiation effect on ACTH-induced cAMP production were similar in control and A-II pretreated cells. Moreover, the conversion of several steroid precursors to corticosterone was similar in control cells and A-II-pretreated cells, whereas the conversion to cortisol was reduced by approximately 30% due mainly to a decrease of 17 alpha-hydroxylase activity. Thus, the marked steroidogenic desensitization induced by A-II is most likely related to some alteration located beyond the activation of the two branches of the phosphoinositide pathway and before the first steps of steroidogenesis.     

Serotonin receptor-mediated stimulation of bovine smooth muscle cell prostacyclin synthesis and its modulation by platelet-derived growth factor.

Serotonin (5-hydroxytryptamine; 0.5 microM and above) stimulated the synthesis of prostacyclin (as measured by radioimmunoassay of 6-ketoprostaglandin F1 alpha) by bovine aortic smooth muscle cells in culture. This effect was structurally specific; a similar response was not elicited by the other indoles (tryptophan, n-acetylserotonin, 5-hydroxytryptophan, melatonin, or 5-hydroxyindoleacetic acid) or by the amines phenylephrine, isoproterenol, dopamine, or histamine). The response was reversible and was saturable at serotonin concentrations of 10 microM or higher. An increase in prostacyclin synthesis was elicited by the addition of a serotonin agonist, quipazine (1 microM and above), and antagonized by the serotonin receptor blockers cyproheptadine, methysergide, or methiothepin but not by other aminergic receptor-blocking drugs (e.g., phentolamine or propranolol). This effect was selective for cell type because serotonin or quipazine (100 microM) did not increase prostacyclin synthesis by bovine aortic endothelial cells. The addition of platelet-derived growth factor (PDGF) to cultures of smooth muscle cells dramatically enhanced prostacyclin synthesis in response to the coadministration of serotonin. PDGF greatly increased the maximum response to serotonin without altering the half-maximal effective concentration for serotonin. This synergistic interaction was blocked by the addition of a serotonin-receptor blocking agent. Taken together, these data suggest that serotonin stimulates smooth muscle prostacyclin synthesis through a specific receptor-mediated mechanism that can be modulated by PDGF.     

The effects of suramin on human adrenocortical cells in vitro: suramin inhibits cortisol secretion and adrenocortical cell growth.

Suramin, a polycyclic and polyanionic drug, has been successfully used in the therapy of inoperable adrenocortical cancer. The present study was undertaken to investigate the effects of suramin on normal human adrenocortical cells in primary monolayer cultures. The proliferation and the basal, as well as the adrenocorticotropin (ACTH)-stimulated, cortisol secretion of these cells were studied. The data show that suramin decreases basal, as well as ACTH-stimulated, cortisol secretion in a dose-dependent manner (P less than .05 from 300 mumol/L upward). At a suramin concentration of 3 mmol/L, cortisol secretion was inhibited by 70% +/- 4% in ACTH-stimulated cells and by 42% +/- 6% in unstimulated cells. The proliferation of adrenocortical cells in response to fetal calf serum was also inhibited by suramin at concentrations from 300 mumol/L upward, maximal suppression (71% +/- 6%, P less than .01) being observed at a concentration of 10 mmol/L. Both inhibition of cortisol secretion and inhibition of adrenocortical cell proliferation were not due to toxicity of the compound, as could be shown by restimulation of cortisol secretion in suramin-treated cells with ACTH. Our results indicate that suramin exerts an inhibitory influence on the cortisol secretion and on the proliferation of normal human adrenocortical cells. Suramin may not only be useful in the treatment of adrenocortical cancer, but may also have an ameliorative effect on other malignant conditions with augmented steroid hormone production, resistant to conventional forms of therapy.     

Real-time detection of insulin-like growth factor-1 stimulation of the MAC-T bovine mammary epithelial cell line

Binding of growth factors by cell-surface receptors is an essential means by which cell regulate normaltissue growth and differentiation. Exposure to growth factors is often transient, and our goal was to determine whether short-term exposure to insulin-like growth factor-1 (IGF-1) would lead to activation, assayed as cell proliferation, of mammary epithelial cells. The MAC-T cell line is an immortalized bovine mammary epithelial cell line, chosen as our model mammary cell line because of its known sensitivity to IGF-1. Using the Cytosensor Microphysiometer System, a biosensor capable of measuring extracellular acidification, we were able to measure activation of the cellsowing to IGF-1 addition in real time and found that peak acidification occurred in only 14 min. We show that this rapid response to IGF-1 is dose dependent and specific for IGF-1. A significant increase in [³H]thymidine incorporation by cells after a similar short-term exposure to IGF-1 suggests that the measured increase in extracellular acidification following IGF-1 addition is physiologically relevant. This technology offers a new, novel, and rapid means for the study of IGF-1 activity, as well as the screening of IGF-1 inhibitors, in mammary epithelial cells.     

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.     

Steroidogenic potential of lyophilized mitochondria from bovine adrenocortical tissue.

When incubated with [3H]cholesterol, a bovine adrenocortical mitochondrial pellet obtained by centrifugation at 12,000 x g yielded, as expected, only the C21O2 metabolites progesterone and pregnenolone. However, the steroidogenic potential of the 12,000 x g pellet fraction was augmented significantly by lyophilization. After lyophilization, the 12,000 x g pellet converted the sterol into C19 androgens and corticosteroids, in addition to C21O2 pregnane derivatives. Leaching the lyophilized mitochondrial fraction with either hexane or acetone increased substantially the yields of the metabolites. It did not change qualitatively the array of metabolites formed during in vitro incubation, but 5 alpha-reductase activity was unmasked by the washings, particularly with acetone. Thus, the fraction sedimented at 12,000 x g contains the complete complement of steroidogenic enzymes required for the biosynthesis of the aforementioned adrenal hormones. These results cast doubt upon the widely held belief that the various enzymes required for adrenocortical steroidogenesis are distributed between two different subcellular compartments, the mitochondrion and the endoplasmic reticulum.     

Vasopressin stimulation of mouse 3T3 cell growth.

Vasopressin is shown to be a potent mitogen for Swiss 3T3 cells. The hormone (1--10 ng/ml) causes a striking shift of the dose--response curve for the effect of serum on thymidine incorporation by cultures of 3T3 cells arrested in the G1/G0 phase of the cell cycle. In the absence of added serum, the effect of vasopressin on DNA synthesis is greatly potentiated by insulin, epidermal growth factor, and a factor isolated from medium conditioned by simian virus 40-infected baby hamster kidney cells. The mitogenic effect of vasopressin is dependent on time and hormone concentration. In the presence of insulin, the half-maximal effect elicited by the peptide is obtained at 0.6 ng/ml. [Arg]Vasopressin and [Lys]vasopressin are equally potent. The vasopressins are 10(3)-fold more potent than oxytocin. In the presence of a low (2.5%) concentration of serum, vasopressins stimulate cell proliferation.     

Prostaglandin-dependent in vitro stimulation of adrenocortical steroidogenesis by interleukins.

The effects of interleukins on adrenal steroidogenesis and their mode of action were studied using cultured rat adrenal cells. The addition of rat interleukin-1 alpha (IL-1 alpha) or rat IL-2 increased corticosterone levels in the medium in a concentration-dependent manner during 24 h of incubation. The minimum, half-maximum, and maximum effective concentrations of both rat IL-1 alpha and rat IL-2 were almost same (approximately 3, 10, and 100 U/ml, respectively). After a latent period, the effect became apparent after 12 h of incubation. Human IL-1 beta and human IL-6 also showed a stimulatory effect on corticosterone production, whereas human IL-2 was inactive in this system. To clarify the cellular mechanism of these stimulatory effects, we measured the levels of prostaglandin E2 (PGE2) and cAMP in the cells and media as well as the corticosterone levels. Corticosterone production stimulated by IL-1 alpha or IL-2 was accompanied by intracellular and extracellular cAMP and PGE2 accumulation. Although the stimulation of both cAMP and corticosterone was observed only after 12 h of incubation, PGE2 levels increased during the first 4 h of incubation. Corticosterone, cAMP, and PGE2 production stimulated by ILs was almost completely blocked by the addition of 0.1 mM aspirin, a cyclooxygenase inhibitor. Lipoxygenase inhibitors, i.e. AA-861, nordihydroguaiaretic acid, and 5,8,11,14-eicosatetrynoic acid, did not abolish corticosterone production stimulated by ILs. Submaximal doses of IL-1 alpha and IL-2 synergistically stimulated PGE2 production, but did not have even additional effects on cAMP and corticosterone levels. On the other hand, submaximal doses of ACTH, which did not significantly affect PGE2 levels, acted synergistically with IL to increase cAMP and corticosterone levels in these cells. These results indicate that 1) IL-1 alpha and IL-2 directly stimulate glucocorticoid synthesis in a dose- and time-dependent manner; 2) a half-maximum effective concentration of ACTH acts synergistically with IL in stimulating glucocorticoidogenesis; 3) the stimulatory process initially requires PGs, followed by the activation of the adenylate cyclase system; 4) although the profiles of steroidogenic action of IL-1 alpha and IL-2 are quite similar, they may exert their effects through different mechanisms in their early steps of PGE2 production; and 5) the low effective concentrations of both cytokines suggest possible physiological or pathophysiological roles of circulating cytokines in the glucocorticoidogenesis under certain conditions.     

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

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

Transplantation of bovine adrenocortical cells encapsulated in alginate

Current treatment options for adrenal insufficiency are limited to corticosteroid replacement therapies. However, hormone therapy does not replicate circadian rhythms and has unpleasant side effects especially due to the failure to restore normal function of the hypothalamic–pituitary–adrenal (HPA) axis. Adrenal cell transplantation and the restoration of HPA axis function would be a feasible and useful therapeutic strategy for patients with adrenal insufficiency. We created a bioartificial adrenal with 3D cell culture conditions by encapsulation of bovine adrenocortical cells (BACs) in alginate (enBACs). We found that, compared with BACs in monolayer culture, encapsulation in alginate significantly increased the life span of BACs. Encapsulation also improved significantly both the capacity of adrenal cells for stable, long-term basal hormone release as well as the response to pituitary adrenocorticotropic hormone (ACTH) and hypothalamic luteinizing hormone-releasing hormone (LHRH) agonist, [D-Trp6]LHRH. The enBACs were transplanted into adrenalectomized, immunodeficient, and immunocompetent rats. Animals received enBACs intraperitoneally, under the kidney capsule (free cells or cells encapsulated in alginate slabs) or s.c. enclosed in oxygenating and immunoisolating βAir devices. Graft function was confirmed by the presence of cortisol in the plasma of rats. Both types of grafted encapsulated cells, explanted after 21–25 d, preserved their morphology and functional response to ACTH stimulation. In conclusion, transplantation of a bioartificial adrenal with xenogeneic cells may be a treatment option for patients with adrenocortical insufficiency and other stress-related disorders. Furthermore, this model provides a microenvironment that ensures 3D cell–cell interactions as a unique tool to investigate new insights into cell biology, differentiation, tissue organization, and homeostasis.Adrenal insufficiency is the failure of adrenocortical cells to produce adequate amounts of glucocorticoids and/or mineralocorticoids. These steroid hormones play a central role in the body’s homeostasis of energy, salt, and fluid; thus, adrenal insufficiency is a potentially life-threatening condition. The most relevant causes of adrenal insufficiency are autoimmune disorders (up to 80%); infectious diseases; hereditary factors; traumatic, metabolic, or neoplastic conditions; or surgical bilateral adrenalectomy, sometimes due to a compulsory therapeutic strategy in the treatment of adrenal tumors or congenital adrenal hyperplasia (CAH).CAH due to 21-hydroxylase deficiency is the most common form of inherited adrenal insufficiency, presenting with clinical symptoms of neuroendocrine perturbations, virilization, and metabolic diseases in later life. Patients may suffer from hypotensive crises, hypoglycemia, acne, and infertility (1, 2). Current options of treatment consisting of replacement therapy with glucocorticoids, mineralocorticoids, and/or androgens can reverse the symptoms only partially, exhibit the unpleasant side effects of inappropriate glucocorticoid substitution, and leave the patients without the diurnal rhythm of glucocorticoid release. Furthermore, adrenomedullary functions, including catecholamine and neuropeptide secretion, also are disrupted (2), which correlates with cardiovascular risks, hypoglycemia, and physical disability in these patients (1, 3).Adrenal gland transplantation could and would be a desirable therapeutic alternative for these patients if it was available and practical (4). Transplanted organs restore and maintain normal hormonal levels, adequately respond to functional demands, and regulate steroid production in response to endogenous and exogenous stimulation, including the circadian rhythm of hormone secretion. However, the application of this strategy is currently extremely limited due to the lack of human donor organs, the surgical difficulties of adrenal transplantation, and the required chronic immunosuppression.For the correction of adrenocortical insufficiency, transplantation of whole adrenal glands might not be mandatory and the transplantation of isolated adrenal cells may be sufficient. An additional advantage of transplantation using isolated cells is the availability of various immunoisolating materials and methods for immune protection of such transplants. Application of these materials not only allows avoidance of chronic immunosuppression but also allows the transplantation of xenogeneic cells (5, 6).Sodium alginate is one of the clinically approved immunoisolating biopolymers; it has already been widely used in a variety of biomedical applications (5–10). Alginates have several unique structural and chemical parameters, appropriate not only for immune isolation but also for the creation of 3D cellular scaffolds that allow artificial organs to function long term in vitro and in vivo (7, 8, 11). The potential of xenogeneic adrenocortical cells to replace adrenal gland function has already been tested in animal models but requires acquisition of immunodeficiency (12, 13). Alginate encapsulation may protect xenogeneic adrenocortical cells from destruction by immunological processes (6). Alternatively, similar to pancreatic islets (9, 14), adrenocortical cells can be transplanted within special oxygenating and immunoisolating devices, thus reducing the risk of an immunological host versus graft response.Creating a long-lasting, immune-isolated, and functional bioartificial adrenal was the main aim of this research. The objectives of our work included testing of primary bovine adrenocortical cells (BACs) as a potential source of cells, defining optimal conditions, and long-term monitoring. Because adrenocortical cells also express receptors for luteinizing hormone-releasing hormone (LHRH), the effect of the LHRH agonist [D-Trp6]LHRH on adrenocortical steroidogenesis in encapsulated BACs (enBACs) was tested. We characterized bioartificial adrenals in vivo and investigated their functionality and efficacy after implantation into bilaterally adrenalectomized rats.     

Angiotensin II receptor-mediated calcium influx in bovine adrenal glomerulosa cells.

The cytoplasmic calcium ([Ca2+]i) response to angiotensin II (AII) in bovine adrenal glomerulosa cells is characterized by an initial transient peak due to intracellular Ca2+ mobilization, followed by a sustained plateau phase that is dependent on Ca2+ entry from the extracellular fluid. In Fura-2 loaded cells, the calcium channel antagonists, nifedipine (1 microM) and verapamil (20 microM), only partially reduced the cytosolic calcium profile induced by AII. The dihydropyridine agonist, Bay K 8644, caused a moderate increase in [Ca2+]i when added in concentrations of 50-100 nM, but did not enhance the AII-induced rise in [Ca2+]i. These results indicate that most of the AII-stimulated Ca2+ influx is through channels that are insensitive to dihydropyridines and verapamil. In contrast, the inorganic Ca2+ channel blocker, LaCl3 (10 microM), inhibited the AII-induced plateau phase by more than 50%. The AII-induced Ca2+ signal was not affected by treatment with pertussis toxin (100-300 ng/ml for 12 h). The prior addition of specific AII-antagonists (DuP 753, a nonpeptide antagonist, and three peptide analogs, [Sar1,Thr8]AII, [Sar1,Ala8]AII, and [Sar1,Ile8]AII) completely inhibited the AII-induced Ca2+ signal. However, addition of up to 25,000 molar excess of these antagonists at intervals from 10 sec to 5 min after AII caused progressively less attenuation of the sustained Ca2+ signal. After 5 min, addition of antagonists did not inhibit the agonist-induced Ca2+ response for up to 20 min. The progressive loss of ability of the antagonists to inhibit the sustained elevation of [Ca2+]i could reflect prolonged activation of the receptor or of a subsequent process that maintains Ca2+ influx despite receptor blockade. It is possible that sequestration and/or endocytosis of the AII-receptor complex is accompanied by continued generation of intracellular signals that contribute to the maintenance of the [Ca2+]i response.     

Angiotensin II stimulation of Na+/K+ATPase activity and cell growth by calcium-independent pathway in MCF-7 breast cancer cells

Here we demonstrated, by RT-PCR analysis, the expression of both angiotensin II (Ang II) receptor subtypes, AT1 and AT2, in a breast cancer epithelial cell line, MCF-7. Ang II was not able to affect the intracellular Ca2+ concentration in Fura-2 loaded cells suggesting that AT1-mediated phospholipid hydrolysis is not involved in its intracellular transduction pathway. Ang II modulated the activity of the Na+/K+ATPase in a dose- and time-dependent manner and was mitogenic, with a dose-dependent (1-1000 nM) proliferative effect and a maximal response at 100 nM. Both Na+/K+ATPase activation and stimulation of proliferation were mediated by binding of Ang II to AT1, as the effects were completely blocked by DuP 753, a specific AT1 antagonist. CGP 42112, an AT2 antagonist, did not affect Ang II actions. The main conclusion of this study is that Ang II exerts its effects on cell proliferation and Na+/K+ATPase in breast cancer epithelial cells, MCF-7, via AT1 activation independently of the Ca(2+) signalling mechanism.     

Angiotensin II stimulation of vascular smooth muscle cells. Secondary signalling mechanisms

Activation of vascular smooth muscle by angiotensin II results in the phospholipase C-mediated generation of two second messengers, inositol trisphosphate (IP3) and diacylglycerol (DG). IP3 is responsible for mobilizing calcium from endoplasmic reticulum whereas DG activates protein kinase C and ultimately Na+/H+ exchange, leading to intracellular alkalinization. The IP3/calcium signal is transient, most likely serving to initiate calcium-mediated events leading to contraction, and is attenuated by activation of protein kinase C. DG formation/protein kinase C activation is sustained and may be enhanced by the concurrent intracellular alkalinization. The delay in induction of the sustained response appears to be related to cellular processing of the angiotensin II-receptor complex. Phospholipase C activity is also modulated by a cholera toxin-sensitive, pertussis toxin-insensitive guanine nucleotide regulatory protein. This guanine nucleotide regulatory protein, movement of the receptor-ligand complex, and the signals generated by the two second messengers, IP3 and DG, interact in a complex manner to cause an integrated response of vascular smooth muscle to angiotensin II stimulation.     

Potent inhibitory effects of type I interferons on human adrenocortical carcinoma cell growth

CONTEXT: Adrenocortical carcinoma (ACC) is a rare tumor with a poor prognosis. Despite efforts to develop new therapeutic regimens for metastatic ACC, surgery remains the mainstay of treatment. Interferons are known to exert tumor-suppressive effects in several types of human cancer. DESIGN: We evaluated the tumor-suppressive effects of type I interferons (IFN)-alpha2b and IFNbeta on the H295 and SW13 human ACC cell lines. RESULTS: As determined by quantitative RT-PCR analysis and immunocytochemistry, H295 and SW13 cells expressed the active type I IFN receptor (IFNAR) mRNA and protein (IFNAR-1 and IFNAR-2c subunits). Both IFNalpha2b and IFNbeta1a significantly inhibited ACC cell growth in a dose-dependent manner, but the effect of IFNbeta1a (IC50 5 IU/ml, maximal inhibition 96% in H295; IC50 18 IU/ml, maximal inhibition 85% in SW13) was significantly more potent, compared with that of IFNalpha2b (IC50 57 IU/ml, maximal inhibition 35% in H295; IC50 221 IU/ml, maximal inhibition 60% in SW13). Whereas in H295 cells both IFNs induced apoptosis and accumulation of the cells in S phase, the antitumor mechanism in SW13 cells involved cell cycle arrest only. Inhibitors of caspase-3, caspase-8, and caspase-9 counteracted the apoptosis-inducing effect by IFNbeta1a in H295 cells. In H295 cells, IFNbeta1a, but not IFNalpha2b, also strongly suppressed the IGF-II mRNA expression, an important growth factor and hallmark in ACC. CONCLUSIONS: IFNbeta1a is much more potent than IFNalpha2b to suppress ACC cell proliferation in vitro by induction of apoptosis and cell cycle arrest. Further studies are required to evaluate the potency of IFNbeta1a to inhibit tumor growth in vivo.