Addition Of Hydrocortisone Research Articles

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Dr Al-Qattan makes an interesting point of performing a study on the injection of triamcinolone and collagenase in combination. I think this might be a worthwhile undertaking. My group at the University of Kansas performed an in vitro study in which collagen was incubated for 48 hours with nothing, triamcinolone alone, hydrocortisone alone, collagenase alone, triamcinolone and collagenase, and collagenase and hydrocortisone. After 48 hours the yield of salt-soluble collagen was measured. With nothing added to the collagen, there was no yield of salt-soluble collagen, as was the case when triamcinolone and hydrocortisone were added alone. But when collagenase was added a definite yield of salt-soluble collagen was obtained; that yield was doubled with the addition of triamcinolone, whereas the addition of hydro-cortisone to collagenase yielded acid-soluble collagen which is not soluble in the body. 1 Ketchum LD Robinson DW Masters FW The degradation of mature collagen: a laboratory study. Plast Reconstr Surg. 1967; 40: 89-91 Crossref PubMed Scopus (36) Google Scholar Therefore, on the basis of that in vitro study, it does theoretically make sense to combine triamcinolone with collagenase in the injection of nodules of Dupuytren's disease.

Optimization of rat hepatocyte culture in citrated human plasma.

Maintenance of liver-specific functions in hepatocyte cultures during plasma exposure is critical for the clinical application of bioartificial liver assist systems. Sodium citrate is a common anticoagulant but has been shown to be cytotoxic to hepatocytes. We have tested the effect of various supplements on the viability and function of adult primary rat hepatocytes exposed to citrated plasma. Freshly isolated rat hepatocytes were cultured in the collagen gel sandwich configuration in culture medium for 6 days followed by exposure to citrated human plasma with various supplements for 1 week. Controls were left in culture medium throughout. Viability and synthetic functions were evaluated. Hepatocytes exposed to unsupplemented citrated plasma lost significant viability and function within the first 2 days. Cells cultured in plasma supplemented with a fivefold concentrate of standard hepatocyte culture medium maintained urea (1. 2-2.1 micromol/day/10(6) cells) and albumin (51-62 microg/day/10(6) cells) synthesis rates equal to or higher than those of controls. Among the various components of the concentrated medium supplement, calcium chloride (1.8 mM), magnesium sulfate (0.8 mM), amino acids (fourfold Basal Medium Eagle amino acids including 4 mM glutamine), and glucagon (14 ng/ml) were found to be essential in maintaining urea synthesis. Maintenance of a high albumin synthesis rate also required the addition of hydrocortisone (7.5 microg/ml) and insulin (0.5 U/ml). Appropriate metabolic and hormonal supplementation of citrated human plasma prevents its cytotoxic effects and may be used in conjunction with in vivo use of bioartificial liver assist systems.

The host-parasite relationship between the rainbow trout (Oncorhynchus mykiss) and the ectoparasite Argulus foliaceus (Crustacea: Branchiura): epithelial mucous cell response, cortisol and factors which may influence parasite establishment.

The effects of short-term infection with the branchurian crustacean ectoparasite Argulus foliaceus, and the fish stress hormone cortisol (which is reported to stimulate mucus discharge), were studied on the mucous cell population of the head skin of rainbow trout (Oncorhynchus mykiss). Argulus infection did not raise plasma cortisol significantly and had no effect on the number of epidermal mucous cells in the head skin. Cortisol was administered twice to groups of trout via the food, significantly elevating circulating plasma cortisol at 24 h post feeding without affecting numbers of mucous cells, and increasing the numbers of vesicles in the upper cells of the epidermis. Subsequent infection with the parasite (6 Argulus/fish) did not affect either plasma cortisol or total numbers of mucous cells at 48 h post-infection with the parasite, but led to a significantly lower parasite infestation per fish in the cortisol-administered groups. A 24 h culture system was used to expose pieces of trout skin to 50 ng/ml cortisol in vitro to investigate whether cortisol alone would stimulate reductions in mucous cell numbers. These were unaffected by the addition of cortisol. The in vivo and in vitro results are discussed in relation to the current understanding of crustacean host-parasite interactions.

Increase in prolactin receptor (PRL-R) mRNA level in the mammary gland after hormonal induction of lactation in virgin ewes☆

In order to examine the hormonal regulation of the prolactin-receptor (PRL-R) gene expression during mammary gland development, ewes were treated to induce lactation via an estrogen-progesterone-hydrocortisone and ovine growth hormone treatment. In situ hybridization analysis was used and revealed that sex steroids increased PRL-R mRNA levels in the mammary gland. Using RNase protection assay we showed that the estradiol + progesterone treatment increased both the levels of the long and the short forms of PRL-R mRNA. Addition of hydrocortisone increased the level of α S1-casein transcripts and the level of the ratio of the long to the short form of the PRL-R mRNA. This ratio can be further enhanced by addition of ovine growth hormone to the latter treatment. This suggests a role of hydrocortisone and ovine growth hormone in the alternative splicing that leads to the preferential expression of the long form of the PRL-R mRNA. In conclusion, the present experiments suggest that estrogen, progesterone and hydrocortisone are the major regulators of the PRL-R gene expression during pregnancy and prepare the mammary gland for its differentiation.

Neuropilin-1 Is Expressed on Bone Marrow Stromal Cells: A Novel Interaction With Hematopoietic Cells?

In adult bone marrow, hematopoietic stem cells are found in close association with distinctive stromal cell elements. This association is necessary for maintenance of hematopoiesis, but the precise mechanisms underlying the cross-talk between stromal cells and hematopoietic stem cells are poorly understood. In this study, we used a bone marrow stromal cell line (MS-5) that is able to support human long-term hematopoiesis. This hematopoietic-promoting activity cannot be related to expression of known cytokines and is abolished by addition of hydrocortisone. Using a gene trap strategy that selects genes encoding transmembrane or secreted proteins expressed by MS-5 cells, we obtained several insertions that produced fusion proteins. In one clone, fusion protein activity was downregulated in the presence of hydrocortisone, and we show that insertion of the trap vector has occurred into the neuropilin-1 gene. Neuropilin-1 is expressed in MS-5 cells, in other hematopoietic-supporting cell lines, and in primary stromal cells but not in primitive hematopoietic cells. We show that neuropilin-1 acts as a functional cell-surface receptor in MS-5 cells. Two neuropilin-1 ligands, semaphorin III and VEGF 165, can bind to these cells, and the addition of VEGF 165 to MS-5 cells increases expression of 2 cytokines known to regulate early hematopoiesis, Tpo and Flt3-L. Finally, we show that stromal cells and immature hematopoietic cells express different neuropilin-1 ligands. We propose that neuropilin-1 may act as a novel receptor on stromal cells by mediating interactions between stroma and primitive hematopoietic cells.

Localization of nitric oxide synthases and nitric oxide production in the rat mammary gland.

We investigated nitric oxide (NO) production and the presence of nitric oxide synthase (NOS) in the mammary gland by use of an organ culture system of rat mammary glands. Mammary glands were excised from the inguinal parts of female Wistar-MS rats primed by implantation with pellets of 17beta-estradiol and progesterone and were diced into approximately 3-mm cubes. Three of these cubes were cultured with 2 ml of 10% FCS/DMEM plus carboxy-PTIO (an NO scavenger, 100 microM) in the presence or absence of LPS (0.5 microgram/ml) for 2 days. The amount of NO produced spontaneously by the cultured mammary glands was relatively minute at the end of the 2-day culture period, and the NO production was significantly enhanced by the presence of LPS. This enhancement of NO production was completely eliminated by addition of hydrocortisone (3 microM), an inhibitor of inducible NOS (iNOS), to the incubation medium. Immunoblot analyses with specific antisera against NOS isoforms such as iNOS, endothelial NOS (eNOS), and brain NOS (bNOS) showed immunoreactive bands of iNOS (122 +/- 2 kD) and eNOS (152 +/- 3 kD) in extracts prepared from the mammary glands in the culture without LPS. The immunoreactive band of iNOS was highly intense after the treatment of mammary glands with LPS, whereas the corresponding eNOS immunoreactive band was faded. The immunohistochemical study of anti-iNOS antiserum on frozen sections of the cultured mammary glands showed that an immunoreactive substance with the antiserum was localized to the basal layer (composed of myoepithelial cells of alveoli and lactiferous ducts) of the mammary epithelia and to the endothelium of blood vessels that penetrated into the interstitium of the mammary glands. Histochemical staining for NADPH-diaphorase activity, which is identical to NOS, showed localization similar to that of iNOS in the mammary glands. Similar observations were noted in the immunohistochemistry of eNOS. In contrast, the immunoreactive signal with the bNOS antiserum was barely detected in the epithelial parts of alveoli and lactiferous ducts of the mammary glands. These observations demonstrate that three isoforms of NOS are present not only in the endothelium of blood vessels but also in the parenchymal cells (the glandular epithelium) of the rat mammary gland, such as epithelial cells and myoepithelial cells, and suggest that NO may have functional roles in the physiology of the mammary glands.

Hydrocortisone Regulates the Dynamics of Plasminogen Activator and Plasminogen Activator Inhibitor Expression in Cultured Murine Keratinocytes

The plasminogen activators tPA and uPA, and their inhibitors, PAI-1 and PAI-2, have been associated with epithelial homeostasis and wound healing. In these studies, we investigate the effect of the steroid hormone hydrocortisone, a commonly used therapeutic modality for skin, on PAs/PAIs in serum- and plasminogen-free primary cultures of murine keratinocytes. SDS–PAGE fibrin zymography showed that addition of 1 μM hydrocortisone to cultures significantly reduced tPA fibrinolytic activity in both cell extracts and conditioned medium. uPA activity in conditioned medium was similarly inhibited. Cells were also cultured in the presence of dibutyryl cyclic AMP (dbcAMP). dbcAMP (5 mM) alone enhanced uPA and tPA fibrinolytic activity in conditioned medium, but this increase was diminished in the presence of 1 μM hydrocortisone. Immunoblots revealed a three- to fivefold induction of free PAI-1 by hydrocortisone which was partially blocked by dbcAMP. Northern blots showed that PAI-1 mRNA increased threefold 2 h after addition of hydrocortisone and remained elevated at least 8 h. In contrast, uPA and tPA mRNA were unchanged over the same time course. uPA, tPA, and PAI-1 mRNA increased in the presence of dbcAMP; levels remained elevated at least 8 h. HC suppressed the induction of uPA and tPA by dbcAMP. Studies directed at identifying plasminogen mRNA showed that in this culture system, keratinocytes produce no plasminogen mRNA either in the presence or in the absence of hydrocortisone or dbcAMP. Collectively, these results show that keratinocyte plasminogen activator activity is suppressed by hydrocortisone as a function of increased PAI-1 combined with an attenuation of PA induction by agents that increase intracellular cAMP. These results provide additional information to further define the mechanism by which glucocorticoids inhibit wound healing.

Effects of cortisol on the expression of interleukin-6 and interleukin-1 beta in human osteoblast-like cells

High levels of glucocorticoids are believed to alter bone remodeling by decreasing bone formation and increasing bone resorption. It has been suggested that different cytokines, like interleukin-6 (IL-6) and interleukin-1 (IL-1), are involved in bone resorption by activating immature osteoclasts, and some studies indicate that IL-6 promotes bone formation by a mitogenic effect on osteoblasts. The aim of the present investigation was to study whether cortisol regulates the expression of IL-6 and IL-1 beta in human osteoblast-like cells. A high dose of cortisol (10(-7)M) decreased, as expected, the C-terminal propeptide of type I collagen released into the culture medium. The IL-6 mRNA levels and IL-6 protein released into the culture medium were also decreased by cortisol in a dose-dependent manner. The maximum effect was seen at 1 microM cortisol (mRNA 23.1 +/- 7.9% of control culture; protein 28.2 +/- 8.3% of control culture). The decrease in IL-6 mRNA levels was apparent 4 h after the addition of cortisol and was still present 20 h later. The decrease in IL-6 protein released into the culture medium was seen 20 h later than the decrease in IL-6 mRNA levels. The production of IL-1 beta protein released into the culture medium was decreased in a dose-dependent manner after the addition of cortisol with a maximum effect at 1 microM. The effect of cortisol on IL-1 beta protein released into the culture medium was seen 16 h after the addition of cortisol. To summarize, cortisol decreases the expression of IL-6 as well as IL-1 beta in human osteoblast-like cells.

Cortisol increases growth hormone-receptor expression in human osteoblast-like cells.

It is well known that high levels of glucocorticoids cause osteoporosis and that physiologic levels of growth hormone (GH) are required for normal bone remodeling. It has been suggested that glucocorticoids regulate GH-responses via the regulation of GH-receptor expression. The aim of the present study was to investigate whether cortisol plays a role in the regulation of GH-receptor expression in cultured human osteoblasts. The effect of serum starvation and cortisol on GH-receptor expression was tested in human osteoblast (hOB)-like cells. Serum starvation for 24 h resulted in an increase in GH-receptor mRNA levels (90 +/- 1% over control culture). Cortisol increased GH-receptor mRNA levels in a dose-dependent manner with a maximal effect at 10(-6)M. The stimulating effect of cortisol on GH-receptor mRNA levels was time-dependent, reaching a peak 12 h after the addition of cortisol (126 +/- 29% over control culture) and remaining up to 12 h later. The increase in GH-receptor mRNA levels was accompanied by an increase in 125I-GH binding which reached a maximum at 24 h (196 +/- 87% over control culture). In conclusion, glucocorticoids increase GH-receptor expression in hOB-like cells. Further studies are needed to clarify whether glucocorticoid-induced regulation of the GH-receptor is important in human bone physiology.

Cortisol metabolism and its inhibition by glycyrrhetinic acid in the isolated perfused human placental lobule

We have previously reported the placental metabolism of prednisolone to prednisone, 20α- and β-dihydroprednisone and 20β-dihydroprednisolone. In this study, the disposition of cortisol was investigated in vitro in the dual perfused, isolated human placental lobule after the addition of cortisol (1.2 μmol, n = 3 and 12 μmol, n = 4) to the maternal compartment. Analysis of 5 h maternal and fetal perfusate samples by high performance liquid chromatography-electrospray-tandem mass spectrometry (HPLC-ESI-MS/MS) revealed that cortisol was mainly metabolized to cortisone, but a significant production of 20α-dihydrocortisone, 20β-dihydrocortisone, 20α-dihydrocortisol and 20β-dihydrocortisol was also detected. Saturability of metabolism but not transfer was demonstrated. Metabolism was eliminated by co-perfusion with the potent 11β-hydroxysteroid dehydrogenase (11β-HSD) enzyme inhibitor 18β-glycyrrhetinic acid (GA). The disposition of GA was analysed using HPLC-atmospheric pressure chemical ionisation-MS/MS (HPLC-APCI-MS/MS). GA was found to transfer from the maternal to the fetal circulations without detectable metabolism during 6 h of perfusion.

Insulin and prolactin synergize to induce translation of human serum albumin in the mammary gland of transgenic mice.

A dramatic uncoupling of the expression of chimaeric beta-lactoglobulin (BLG)/human serum albumin (HSA) gene constructs at the RNA and protein levels was observed in cultured mammary explants of virgin transgenic mice. Upon explantation, both HSA RNA and protein were expressed at high levels. However, when the explants were grown in hormone-free medium. HSA RNA continued to accumulate, whereas the synthesis of the corresponding protein was dependent on the presence of insulin and prolactin with a minor contribution of hydrocortisone. The untranslated HSA RNA was indistinguishable from its translatable counterpart in its mobility on agarose gels, was transported normally from the nucleus to the cytoplasm and was translated efficiently in rabbit reticulocyte lysate. In the presence of cycloheximide, HSA RNA rapidly disappeared suggesting a dependency on ongoing protein synthesis. Its estimated half-life of 5-6 h in hormone-free medium increased significantly in the presence of insulin, hydrocortisone and prolactin and was comparable to that of beta-casein RNA. The uncoupling of the expression of the BLG/HSA transgenes at the RNA and protein levels was also confirmed by in situ hybridization and immunohystochemistry on sections from virgin mammary explants. HSA synthesis was initiated within 13 h of the addition of insulin and prolactin in explants that had accumulated untranslated HSA RNA and was fourfold higher than that observed with insulin alone. Addition of hydrocortisone contributed to an additional 20% in HSA synthesis. We believe this is the first demonstration of translational control of exogenous milk protein gene expression in the mammary gland of transgenic animals.

Effect of hydrocortisone succinate on growth of Chlamydia pneumoniae in vitro.

We examined the effect of hydrocortisone succinate on the growth of three isolates of Chlamydia pneumoniae in vitro. There was a significant increase in the number of inclusions seen in two of the C. pneumoniae strains in the presence of hydrocortisone. There was no significant increase in the number of inclusions with various concentrations of hydrocortisone over time. The addition of hydrocortisone did not affect the in vitro activities of azithromycin, erythromycin, and doxycycline against C. pneumoniae.

Long-term productive episomal hepatitis B virus replication in primary cultures of adult human hepatocytes infected in vitro.

We have previously increased the efficiency of hepatitis B virus (HBV) infection of human hepatocytes in vitro by using polyethylene glycol. After further documenting by neutralization experiments, this in vitro infection, we used this model to define new culture conditions that would maintain stable episomal replication for several weeks. We found that in the presence of 10% porcine serum and 2% dimethyl sulphoxide (DMSO), high-density cultures survived more than 3 months, while addition of hydrocortisone instead of DMSO resulted in survival of less than 1 month. HBV episomal replication was maintained without any evidence of viral integration into the host genome. The maintenance of HBV replication was demonstrated by: first, stability of the covalently-closed-circular DNA in the nucleus and relaxed circular and single-stranded replicative intermediates in the cytoplasm; second, detection of two major transcripts of 3.5 and 2.1-2.4 kb corresponding to the pregenomic and surface genes respectively; and third, continuous secretion of mature viral particles in the supernatant of infected cells. We showed that under these culture conditions, hepatocytes were blocked in the G1 phase of the cell cycle and did not spontaneously proliferate. Upon hepatocyte growth factor (HGF) stimulation, however, the ability of hepatocytes to divide was demonstrated and was compared in infected and non-infected cells. No change in proliferative capacity and no variation in c-myc and c-jun levels could be found. Hepatocyte survival was not modified in infected cells, confirming that HBV is not cytopathic for normal human hepatocytes. These new culture conditions represent substantial progress in the study of HBV-host cell interactions.

Mesenchymal regulation of differentiation of intestinal epithelial cells.

Intestinal epithelial differentiation requires the presence of mesenchyme, but the exact nature of this epithelial-mesenchymal interaction is unknown. The role of humoral and extracellular matrix factors in determining intestinal epithelial differentiation was examined by exposing a nontransformed, immature, rat small-intestinal cell line (IEC-6) to intestinal fibroblast-conditioned media or by growing the cells on matrix deposited by intestinal fibroblasts. Differentiation was assessed morphologically and by induction of the disaccharidase sucrase, which is a marker of intestinal epithelial maturation. When exposed to fetal intestinal fibroblast-conditioned medium or grown on extracellular matrix prepared from fetal intestinal fibroblasts, IEC-6 cells showed a significant increase in sucrase activity. The addition of cortisol to IEC-6 cells cultured on intestinal matrix also led to increased sucrase expression, as measured by enzyme activity and protein and messenger RNA levels. Ultrastructurally, cells cultured on intestinal fibroblast matrix in the presence of cortisol displayed a more differentiated phenotype. In contrast, there was no induction of sucrase activity in cells exposed to lung-conditioned media or grown on lung fibroblast matrix. These studies suggest that both humoral and matrix factors from intestinal mesenchyme are involved in intestinal epithelial differentiation and that these factors appear to be organ specific.

Development of Anticandidal Delivery Systems: (I) Anticandidal Activities of Antifungal Agents and Synergistic Combination with Other Drugs

AbstractSusceptibility testing with antifungal agents, e. g., minimum inhibitory concentration (MIC) determination, is performed to obtain reliable data that permit selection of the most suitable agents for treatment of an infective condition. To determine the drugs that provide maximum effectiveness against oral candidiasis, the MICs of various antifungal agents were determined. Also, synergism between two chosen antifungal agents was evaluated, and the effect of benzocaine, an anesthetic, and hydrocortisone, an antiinflammatory agent, on their MICs was examined. It was observed that among all the drugs tested, clotrimazole was the most promising candidate for use as an oral local antifungal. The combination of clotrimazole and chlorhexidine resulted in a decrease in the MIC. While the addition of hydrocortisone to this combination resulted in a slight increase in the MIC, the inclusion of benzocaine resulted in a substantial decrease in the MIC of the antifungal agent combination.

Hepatocyte growth factor stimulates extensive development of branching duct-like structures by cloned mammary gland epithelial cells.

Although epithelial-mesenchymal (stromal) interactions are thought to play an important role in embryonic and postnatal development of the mammary gland, the underlying mechanisms are still poorly understood. To address this issue, we assessed the effect of fibroblast-derived diffusible factors on the growth and morphogenetic properties of a clonally derived subpopulation (clone TAC-2) of normal murine mammary gland (NMuMG) epithelial cells embedded in collagen gels. Under control conditions, TAC-2 mammary gland epithelial cells suspended within collagen gels formed either irregularly shaped cell aggregates or short branching cord-like structures. Addition of conditioned medium from Swiss 3T3 or MRC-5 fibroblasts dramatically stimulated cord formation by TAC-2 cells, resulting in the development of an extensive, highly arborized system of duct-like structures, which in appropriate sections were seen to contain a central lumen. The effect of fibroblast conditioned medium was completely abrogated by antibodies against hepatocyte growth factor (also known as scatter factor), a fibroblast-derived polypeptide that we have previously shown induces tubulogenesis by Madin-Darby canine kidney epithelial cells. Addition of exogenous recombinant human hepatocyte growth factor to collagen gel cultures of TAC-2 cells mimicked the tubulogenic activity of fibroblast conditioned medium by stimulating formation of branching duct-like structures in a dose-dependent manner, with a maximal 77-fold increase in cord length at 20 ng/ml. The effect of either fibroblast conditioned medium or hepatocyte growth factor was markedly potentiated by the simultaneous addition of hydrocortisone (1 microgram/ml), which also enhanced lumen formation. These results demonstrate that hepatocyte growth factor promotes the formation of branching duct-like structures by mammary gland epithelial cells in vitro, and suggest that it may act as a mediator of the inducing effect of mesenchyme (or stroma) on mammary gland development.

Hydrocortisone Differentially Affects the Ability of Murine Stromal Cells and Human Marrow-Derived Adherent Cells to Promote the Differentiation of CD34++/CD38– Long-Term Culture-Initiating Cells

Very primitive human hematopoietic progenitor cells are identified indirectly by their ability to give rise to clonogenic progenitors in the presence of either human or murine stromal cells. These long-term culture-initiating cell (LTC-IC) assays are usually performed in the presence of hydrocortisone based on the initial observation that hydrocortisone was required for prolonged hematopoiesis in standard long-term bone marrow cultures. In this report, we investigated the role of hydrocortisone in LTC-IC assays initiated with CD34++/CD38- cells seeded onto either human bone marrow LTC-derived adherent cells or a murine marrow-derived stromal cell line, MS-5. It was found that weekly addition of hydrocortisone to the cultures reduced the frequency of LTC-IC (from 1/5 to 1/20) calculated from limiting dilution experiments and also reduced fivefold to 10-fold the number of their progeny clonogenic cells detected after 4 to 5 weeks. In contrast, the frequency and differentiative potential of CD34++/CD38- grown in the presence of human marrow feeders was unaltered by the addition of glucocorticoids. Data are consistent with the hypothesis that hydrocortisone inhibited LTC-IC differentiation by downregulating the expression of a synergistic factor produced by MS-5 cells. (1) In the absence of hydrocortisone, the number of clonogenic progenitors generated by LTC-IC was much higher in cultures seeded on MS-5 than in cultures seeded on human marrow adherent cells, which was also true when cytokines were added to the cocultures. However, based on the phenotype of the colonies, progenitors produced in MS-5 cocultures were more mature than those generated on human marrow adherent cells. (2) Hydrocortisone counteracted the stimulatory effect of recombinant human cytokines (interleukin-3, interleukin-6, and steel factor) in assays performed on MS-5 but not on human marrow feeders. (3) Hydrocortisone led to a 50% decrease in the numbers of colony-forming units-granulocyte-macrophage found in methycellulose colony assays of CD34++/CD38- cells performed in the presence of MS-5 cells. Taken together, our results indicate that hydrocortisone acts differently on a murine stromal cell line and on marrow-derived human stromal cells and may suppress the expression by MS-5 cells of an activity selectively promoting amplification of clonogenic cells derived from primitive LTC-IC.

Transforming growth factor-beta stimulates, and glucocorticoids and epidermal growth factor inhibit brain natriuretic peptide secretion from cultured human amnion cells.

We previously reported the massive secretion of brain natriuretic peptide (BNP) from human amnion cells and suggested the possible role of BNP in the maintenance of human pregnancy. In this study, to elucidate the regulatory mechanism of BNP secretion from amnion cells, we measured the BNP level in the culture medium of amnion cells by RIA after incubation in the presence of various substances. Among the agents examined, cortisol (1 x 10(-7) to 1 x 10(-6) mol/L), dexamethasone (1 x 10(-8) to 1 x 10(-6) mol/L), and epidermal growth factor (EGF; 2 x 10(-11) to 2 x 10(-8) mol/L) inhibited BNP secretion from the cultured amnion cells in a dose-dependent manner. By contrast, transforming growth factor-beta (TGF beta; 4 x 10(-11) to 4 x 10(-9) mol/L) caused a 3- to 5-fold increase in BNP secretion. TGF beta-augmented BNP secretion was abolished by the addition of cortisol or EGF to the culture medium. Moreover, in this study, we revealed the presence of bioactive TGF beta in human amniotic fluid (approximately 4 x 10(-10) mol/L). The present finding of tight regulation of BNP secretion from amnion cells by cortisol, EGF and TGF beta, all at the concentrations physiologically present in human amniotic fluid, implies a physiological role of BNP secretion from amnion cells in the pregnant uterus.

Modulation of arginine vasopressin secretion from cultured ovine hypothalamic cells by glucocorticoids and opioid peptides.

In sheep, arginine vasopressin (AVP) appears to be a more potent ACTH-releasing factor than ovine corticotrophin-releasing hormone. In order to investigate the neuroendocrine regulation of AVP secretion we have developed a novel system for maintaining fetal ovine hypothalamic neurones in serum-free culture. Hypothalamic neurones derived from fetal sheep at day 70 gestation (term = 145 days) secreted AVP under basal conditions and in response to repeated potassium-induced depolarizations, for up to 35 days in vitro. AVP secretion was time- and calcium-dependent. AVP secreted from ovine hypothalamic cells co-eluted with synthetic AVP on a Sephadex chromatography column and diluted in parallel with AVP standard in the radioimmunoassay. The addition of cortisol (150 nM) to medium bathing ovine hypothalamic cells significantly inhibited basal, and potassium-induced AVP secretion without altering the AVP content of the cell cultures. Furthermore, the opioid peptide [D-Pro10]Dynorphin(1-11) which acts via the kappa opioid receptor, significantly inhibited basal and potassium-stimulated AVP secretion, an effect which was abolished when cells were cultured in the presence of cortisol. These data show that hypothalamic AVP is a site for negative feedback regulation within the ovine hypothalamic-pituitary-adrenal axis. Furthermore, these data suggest that the kappa opioid system inhibits AVP secretion from ovine hypothalamic neurones, a response which is modulated by glucocorticoids.

Regulation of surfactant protein D expression by glucocorticoids in vitro and in vivo.

SP-D is a recently described lung-associated protein that is produced by alveolar type II cells and may function in pulmonary host defenses. Since little is known regarding the hormonal regulation of SP-D, and since the other surfactant proteins (SP-A, SP-B, and SP-C) are known to be regulated by glucocorticoids, we sought to determine the effects of glucocorticoids on SP-D mRNA and protein expression, both in vitro and in vivo, in the fetal rat lung. In vitro experiments were performed on lung explants from fetuses on gestational day 15 or 18. Explants were cultured in serum-free conditions with or without hydrocortisone for 3 days. SP-D mRNA expression was evaluated by Northern blot analysis. SP-D protein expression was analyzed using a polyclonal antibody against SP-D and standard immunohistochemical techniques. The expression of SP-D mRNA increased in fetal day 15 explants but remained unchanged in fetal day 18 explants cultured without the addition of hydrocortisone, compared with in vivo controls. The addition of hydrocortisone resulted in increases in SP-D mRNA expression at both gestational ages. This pattern of SP-D mRNA expression was compared with the expression of the other surfactant proteins and found to be most similar to that of SP-B. In vivo experiments were performed using maternal administration of dexamethasone (1 mg/kg) or an equal volume of saline on fetal days 15, 16, and 17 or on fetal day 17 with sacrifice on fetal day 18.(ABSTRACT TRUNCATED AT 250 WORDS)