Dehydrogenase and oxoreductase activities of porcine placental 11beta-hydroxysteroid dehydrogenase

Dehydrogenase (cortisol to cortisone) and oxoreductase (cortisone to cortisol) activities of porcine placental 11beta-hydroxysteroid dehydrogenase (11beta-HSD) were measured in tissue fragment cultures on day 75 of gestation. Dehydrogenase activity was over fivefold greater than oxoreductase activity (p < .001). There were positive linear associations (p < .01) between net dehydrogenase activity (dehydrogenase minus oxoreductase) and fetal weight, fetal length, and placental weight. These data indicate a predominance of placental 11beta-HSD dehydrogenase activity at this gestational stage that would insure a net conversion of cortisol to cortisone as it traverses the placenta. The data further suggest that 11beta-HSD activities may provide an optimal glucocorticoid environment that is supportive of enhanced fetal and placental growth.     

Hormonal regulation of the adrenocortical cell

Monolayer cultures of bovine and human adrenocortical cells have been used to study regulation of growth and function. Homogeneous bovine adrenocortical cells exhibit a finite life span of ～60 generations in culture. Full maintenance of differentiated function (steroid hormone synthesis) requires an inducer such as ACTH and antioxidizing conditions. Full induction of differentiated function occurs only when cellular hypertrophy is stimulated by growth factors such as fibroblast growth factor and serum. ACTH and other agents that increase cellular cAMP inhibit replication but do not block growth factor-induced cellular hypertrophy. ACTH and growth factors together result in a hypertrophied, hyperfunctional cell. Replication ensues only when desensitization to the growth inhibitory effects of ACTH occurs. Cultures of the definitive and fetal zones of the human fetal adrenal cortex synthesize the steroids characteristic of the two zones in vivo. ACTH stimulates production of dehydroepiandrosterone (DHA), the major steroid product of the fetal zone, and of cortisol, the characteristic steroid product of the definitive zone. Prolonged ACTH treatment of fetal zone cultures results in a preferential increase in cortisol production so that the pattern of steroid synthesis becomes that of the definitive zone. The preferential increase in cortisol production by fetal zone cultures results from induction of 3β-hydroxysteroid dehydrogenase, Δ^4,5 isomerase activity, which is limiting in fetal zone cells. ACTH thus causes a phenotypic change in fetal zone cells to that of definitive zone cells. In both bovine and human adrenocortical cells, the principal effect of ACTH is to induce full expression of differentiated function. This occurs only under conditions where growth substances and nutrients permit full amplication.     

Investigation of factors influencing 11 beta-hydroxysteroid dehydrogenase (EC 1.1.1.146) activity in midgestational human fetal lung monolayer and explant cultures

We recently suggested that the 11 beta-hydroxysteroid dehydrogenase (11-HSD) activity in midgestational human fetal lung (HFL) cultures comprised at least two enzymes, one oxidative--associated with epithelial cells, the other reductive--related to fibroblast-like cells. In this study, the effects of various hormones on 11-HSD activity were studied by measuring the interconversion of [3H]cortisol and [14C]cortisone. Human chorionic gonadotrophin, placental medium, and low oxygen concentration increased the conversion of cortisone to cortisol while activity in the reverse direction remained unchanged. No effects were seen when adrenocorticotrophin, prolactin, placental lactogen, estrogens, triiodothyronine or oxytocin were added in physiological amounts.     

Feto-maternal production and transfer of cortisol in the rhesus (Macaca mulatta)

Four pregnant rhesus monkeys cnd their fetuses. were infused constantly with ¹⁴C-cortisol and ³H-cortisol. Steady state plasma specific activities for ¹⁴C and ³H-cortisol were obtained after 80 to 90 minutes in both mother and fetus. These data and the rates of infusion of radioactivity were used to calculate the following parameters for both mother and fetus: 1) metabolic clearance rates, 2) production rates, 3) mean adrenal secretory rates, 4) transfer rates from mother to fetus and fetus to mother cnd, 5) the fraction of cortisol in each vascular compartment derived from the maternal and fetal edrenals. Plasma cortisone concentrations, as well as the fraction of cortisone derived from fetal and maternal cortisol were determined. Tetrahydrocortisol and tetrahydrocortisone concentrations were calculated. Mean cortisol secretory rates for the maternal and fetal adrenals were 60.0±11.8 and 1.82±0.42 mg/day. Fifty-eight % of the cortisol in the fetal compartment was of maternal origin. During transfer across the placenta to the fetus, cortisol was largely converted to cortisone. In fetal plasma 76% of the cortisone was of maternal origin. Cortisone concentrations in fetal plasma were higher than those of cortisol.     

Pulmonary phospholipied biosynthesis and the ability of the fetal rabbit lung to reduce cortisone to cortisol during the final ten days of gestation

Incubation of fetal lung tissue with 0.2 μM ¹⁴C-cortisone revealed a 12-fold increase in the rate of reduction of cortisone to cortisol between day 22 and day 30 of gestation in the rabbit. This increase correlated closely with the increase in the rate of incorporation of ¹⁴C-choline into total lung lipids during the same period. In light of these findings it would seem inadequate to attempt to relate the plasma cortisol concentration alone with the rate of lung maturation. In addition, one would need consider both the plasma concentration of cortisone and the activity of 11β-hydroxysteroid dehydrogenase (11β-HSD) in the lung.     

Production of cortisol from cortisone by the isolated,perfused fetal rabbit lung

Perfusion of the isolated 26 day fetal rabbit lung with ³H-cortisone resulted in its conversion to ³H-cortisol and release into the perfusate. Little conversion of ¹⁴C-cortisol to ¹⁴C-cortisone occurred. Quantitative study of homogenized fetal rabbit lung revealed the development of both the cofactor and the enzyme for 11β-hydroxy steroid dehydrogenase activity between 21 and 29 days gestation. These results suggest increasing production of cortisol from cortisone by the fetal rabbit lung as a function of gestational age. This conversion may be of significance with respect to both lung development and parturition, both events being accelerated by cortisol treatment.     

11Beta-hydroxysteroid dehydrogenase type 2 and the regulation of surfactant protein A by dexamethasone metabolites

Glucocorticoid (GC) metabolism by the 11beta-hydroxysteroid dehydrogenase (HSD) system is an important prereceptor regulator of GC action. The HSD enzymes catalyze the interconversion of the endogenous, biologically active GC cortisol and its inactive 11-dehydro metabolite cortisone. The role of the HSD enzymes in the metabolism of synthetic GCs, such as dexamethasone (Dex), is more complex. The human lung is a classic GC-sensitive organ; however, the roles of the HSD enzymes (HSD1 and HSD2) in the human lung are poorly understood. In the present study, we examined the expression of the HSD enzymes in human adult and fetal lung tissues and the human lung epithelial cell line NCI-H441. We observed that human adult and fetal lung tissues, as well as H441 cells, express HSD2 protein and that it is upregulated by Dex (10(-7) M). By contrast, HSD1 protein was undetectable. We also show that the Dex-mediated regulation of surfactant protein A is attenuated by inhibition of HSD2 activity. Furthermore, we demonstrate that unlike the inactive, 11-dehydro metabolite of cortisol (i.e., cortisone), the 11-dehydro metabolite of Dex, 11-dehydro-Dex, competes for binding to the GC receptor (GR) in human lung epithelial cells and retains GR agonist activity. Together, these data suggest that differences exist in the biological activities of the metabolites of cortisol and Dex.     

Effects of cortisol on serially propogated fibroblast cell cultures derived from the rabbit retal lung and skin.

Cortisol affects the growth of serially propogated, fibroblast cell cultures derived from the rabbit fetal lung in a manner which is dependent upon the gestational age of the material used: early in gestation (20 days), the hormone (10(-7)-10(-5) M) stimulates [6-3H]thymidine incorporation into DNA, while in late gestation (28 days), cortisol (10(-7) and 10(-6) M) inhibits this process. Cultures derived from the rabbit fetal skin are inhibited by cortisol (10(-5) M) at both gestational ages. Fibroblasts derived from lung, but not from skin, efficiently convert cortisone to cortisol and this activity increases with advancing gestation. Cortisol does not affect the incorporation of [3H]choline into lecithin by confluent cultures of any of the fibroblast types studied.     

The effect of cortisol on rabbit fetal lung maturation in vitro

Explants of lung tissue from 19-day gestational age fetal rabbits were maintained in organ culture in medium with or without fetal calf serum for 1 to 11 days. Based on the results of biochemical and morphological studies it was apparent that the type II pneumonocyte differentiated in vitro at a time similar to that which occurs with maturation in vivo. The epithelial cells of the presumptive alveoli were undifferentiated at the start of incubation, but within 9 days developed increased amounts of Golgi apparatus and rough endoplasmic reticulum, many microvilli on the luminal surface and numerous lamellar bodies. Secreted lamellar bodies and tubular myelin figures were observed in the lumina of cultured explants. The incorporation of [³H]choline into phosphatidylcholine by lung tissue explants maintained in medium containing 10% fetal calf serum remained relatively constant for 7 days of incubation but thereafter increased two-fold. When explants were maintained in fetal calf serum-containing medium and cortisol (10^?7M) or betamethasone (10^?7M), the incorporation of choline into phosphatidylcholine was two to three times greater than that of explants maintained in serum-containing medium without cortisol. When explants of fetal lung tissue were incubated in the presence of cortisol without fetal calf serum there was no stimulatory effect of cortisol on phosphatidylcholine biosynthesis. Therefore, serum cofactors are necessary for the stimulatory effects of cortisol on fetal lung development. The specific activity of phosphatidate phosphohydrolase (PAPase) increased to very high levels during the culture period. In the presence of serum, cortisol or betamethasone had no effect on the specific activity of phosphatidate phosphohydrolase.     

The effects of cortisol on the primary response of mouse spleen cell cultures to heterologous erythrocytes

Cell viability and the production of direct PFC were studied in mouse spleen cell cultures after cortisol treatment in vivo or in vitro at various times relative to primary stimulation with SRBC in vitro.Cortisol treatment in vivo reduced spleen cell numbers by 88% after 48 hr, but cultures of the remaining cells produced as many PFC in vitro as did cultures of equal numbers of normal spleen cells.In normal spleen cell cultures incubated with cortisol for 4 hr prior to the addition of antigen, peak responses of PFC/culture and PFC/10⁶ cells occurred 24 hr later than in controls and averaged, respectively, 27% and 141% of control values. Minimum viable cell numbers were observed in cortisol-treated cultures after 3 days; thereafter cell numbers gradually increased. These results were not significantly altered when cultures were treated simultaneously with cortisol and antigen.The response was not suppressed if the addition of antigen preceded that of cortisol by more than 4 hr. Suppression was also considerably reduced if fetal calf serum was used when preparing cells for culture.     

Uptake and metabolism of cortisone and cortisol by the fetal rabbit lung

Lungs of rabbit fetuses at 28 days of gestation were incubated with tritium-labeled cortisone (17α,21-dihydroxy-4-pregnene-3,11,20-trione) or Cortisol (11β,17α,21-trihydroxy-4-pregnene-3,20-dione). The fetal lungs metabolized efficiently cortisone yielding cortisol as the major product (64–71% conversion). Cortisol was poorly metabolized, only 10–14% being converted to cortisone and 68–75% of the substrate being recovered unchanged. A small amount of cortisone (5–7% of tissue radioactivity) was also found in the lungs twenty minutes after injection of labeled cortisol to the fetus $\text{in utero}$. Incubation of fetal lungs with labeled cortisone at 37° resulted in specific uptake and binding of radioactivity (predominantly cortisol) to nuclear macromolecules. The amount of cortisol bound to nuclear macromolecules was similar whether the tissue was incubated with cortisol or cortisone. These results demonstrate that the lungs of the rabbit fetus have the capacity to convert the biologically inactive cortisone to the biologically active cortisol, the reverse reaction occurring only to a limited extent.     

Comparison of effects of epidermal and insulin-like growth factors,gastrin releasing peptide and retinoic acid on fetal lung cell growth and maturation in vitro

The role in cell multiplication and maturation of several factors present in the late fetal lung was explored on isolated fetal rat pulmonary fibroblasts and alveolar epithelial type II cells cultivated in serum-free medium. The low degree of reciprocal contamination of each cell population was assessed by immunocytochemistry. Epidermal Growth Factor (EGF) stimulated thymidine incorporation and DNA accumulation in both cell types. In type II cells, it increased labeled-choline incorporation into surfactant phosphatidylcholine (PC), consistently with previous data obtained with lung explant cultures, but not into non-surfactant PC. Insulin-like growth factor (IGF)-I slightly stimulated DNA accumulation in fibroblasts although it did not significantly stimulate thymidine incorporation, contrary to IGF-II which presented a dose-dependent stimulating activity of thymidine incorporation. Neither IGF-I nor IGF-II stimulated type II cell growth. IGFs thus appear to primarily control the growth of lung mesenchyme. In type II cells, they stimulated the most non-surfactant PC biosynthesis. Gastrin releasing peptide (GRP) which was recently reported to promote fetal lung growth in vivo and to stimulate surfactant biosynthesis in lung organ culture revealed as a growth factor for type II cells only, at concentrations below 10 ⁻⁹ M. At concentration 10 ⁻⁸ M, although it did not affect DNA synthesis, GRP tended to increase surfactant and non-surfactant-PC biosynthesis. Retinoic acid inhibited thymidine incorporation into type II cells on a dose-dependent manner but nevertheless enhanced surfactant-PC biosynthesis to a similar extent as EGF. It is suggested that retinoic acid may represent a differentiation or maturation factor for the alveolar epithelium.     

Cell cultures of fetal rat brain : Growth and marker enzyme development

Growth and enzyme development in cell cultures of fetal rat brain were influenced by type of growth medium, cell density, and age of fetal tissue source. Cells grew better in one medium (DMEM), but the other (F12G) enhanced development of choline acetyltransferase activity. One type of growth medium (DMEM) lost efficacy 2 weeks after preparation of complete medium. Cell division rate was density dependent, and choline acetyltransferase development was related to time in culture and cell concentration. Some results suggested division of choline acetyltransferase producing cells. Differences in age of tissue source resulted primarily in differences in growth: cultures of 21 day fetal cells developed more protein per 10⁶ cells inoculated than cultures of cells from younger animals; there was little difference in enzyme activity per culture. Conditions may be controlled such that fetal rat brain cells will grow and express differentiated functions in culture in a predictable manner.     

Depot-specific regulation of the conversion of cortisone to cortisol in human adipose tissue

Objective: Our main objective was to compare the regulation of cortisol production within omental (Om) and abdominal subcutaneous (Abd sc) human adipose tissue. Methods and Procedures: Om and Abd sc adipose tissue were obtained at surgery from subjects with a wide range of BMI. Hydroxysteroid dehydrogenase (HSD) activity (³H‐cortisone and ³H‐cortisol interconversion) and expression were measured before and after organ culture with insulin and/or dexamethasone. Results: Type 1 HSD (HSD1) mRNA and reductase activity were mainly expressed within adipocytes and tightly correlated with adipocyte size within both depots. There was no depot difference in HSD1 expression or reductase activity, while cortisol inactivation and HSD2 mRNA expression (expressed in stromal cells) were higher in Om suggesting higher cortisol turnover in this depot. Culture with insulin decreased HSD reductase activity in both depots. Culture with dexamethasone plus insulin compared to insulin alone increased HSD reductase activity only in the Om depot. This depot‐specific increase in reductase activity could not be explained by an alteration in HSD1 mRNA or protein, which was paradoxically decreased. However, in Om only, hexose‐6‐phosphate dehydrogenase (H6PDH) mRNA levels were increased by culture with dexamethasone plus insulin compared to insulin alone, suggesting that higher nicotinamide adenine dinucleotide phosphate‐oxidase (NADPH) production within the endoplasmic reticulum (ER) contributed to the higher HSD reductase activity. Discussion: We conclude that in the presence of insulin, glucocorticoids cause a depot‐specific increase in the activation of cortisone within Om adipose tissue, and that this mechanism may contribute to adipocyte hypertrophy and visceral obesity.     

Prostaglandin production by type II alveolar epithelial cells.

Prostaglandin production was studied in fetal and adult type II alveolar epithelial cells. Two culture systems were employed, fetal rat lung organotypic cultures consisting of fetal type II cells and monolayer cultures of adult lung type II cells. Dexamethasone, thyroxine, prolactin and insulin, hormones which influence lung development, each reduced the production of prostaglandin E and F alpha by the organotypic cultures. The fetal cultures produced relatively large quantities of prostaglandin E and F alpha and smaller quantities of 6-keto-prostaglandin F1 alpha and thromboxane B2. However, prostaglandin E2 production was predominant. In contrast, the adult type II cells in monolayer culture produced predominantly prostacyclin (6-keto-prostaglandin F1 alpha) along with smaller quantities of prostaglandin E2 and F2 alpha. The type II cells were relatively unresponsive to prostaglandins. Exogenously added prostaglandin E, had no effect on cell growth, and only a minimal effect on cyclic AMP levels in the monolayer cultures.     

Proliferative characteristics of clonal endothelial cell strains

We have utilized clonal strains of bovine fetal aortic endothelial cells to study cellular senescence in a differentiated cell type of physiological significance. Serial subcultivation of nine endothelial clones derived from three fetal calf aortas revealed proliferative life-spans in vitro of 53–125 population doublings (PDs), compared with 60 and 143 PDs for two lines of bovine fetal lung cells and 85 and 147 PDs for two lines of bovine vascular smooth muscle cells. Serial growth curves showed marked reductions associated with endothelial cellular senescence both in cellular growth rate and culture plateau density. Studies of the 24-hour [³H]-thymidine labeling index versus percentage of proliferative life-span completed indicated that clonal endothelial cultures contained a large proportion (greater than 90%) of rapidly cycling cells until about 75% of the life-spans were completed. Senescent endothelial cells showed evidence of large increases in cell area, cell volume, and protein content. In those clones examined, one specialized endothelial function, Factor VIII antigen expression, was retained qualitatively throughout the life-spans.     

The 11 beta-hydroxysteroid dehydrogenase type 2 activity in human placental microsomes is inactivated by zinc and the sulfhydryl modifying reagent N-ethylmaleimide

Proper glucocorticoid exposure in utero is vital to normal fetal organ growth and maturation. The human placental 11 beta-hydroxysteroid dehydrogenase type 2 enzyme (11 beta-HSD2) catalyzes the unidirectional conversion of cortisol to its inert metabolite cortisone, thereby controlling fetal exposure to maternal cortisol. The present study examined the effect of zinc and the relatively specific sulfhydryl modifying reagent N-ethylmaleimide (NEM) on the activity of 11 beta-HSD2 in human placental microsomes. Enzyme activity, reflected by the rate of conversion of cortisol to cortisone, was inactivated by NEM (IC(50)=10 microM), while the activity was markedly increased by the sulfhydryl protecting reagent dithiothreitol (DTT; EC(50)=1 mM). Furthermore, DTT blocked the NEM-induced inhibition of 11 beta-HSD2 activity. Taken together, these results suggested that the sulfhydryl (SH) group(s) of the microsomal 11 beta-HSD2 may be critical for enzyme activity. Zn(2+) also inactivated enzyme activity (IC(50)=2.5 microM), but through a novel mechanism not involving the SH groups. In addition, prior incubation of human placental microsomes with NAD(+) (cofactor) but not cortisol (substrate) resulted in a concentration-dependent increase (EC(50)=8 microM) in 11 beta-HSD2 activity, indicating that binding of NAD(+) to the microsomal 11 beta-HSD2 facilitated the conversion of cortisol to cortisone. Thus, this finding substantiates the previously proposed concept that a compulsorily ordered ternary complex mechanism may operate for 11 beta-HSD2, with NAD(+) binding first, followed by a conformational change allowing cortisol binding with high affinity. Collectively, the present results suggest that cellular mechanisms of SH group modification and intracellular levels of Zn(2+) may play an important role in regulation of placental 11 beta-HSD2 activity.     

Coordination of growth and differentiation in the fetal lung

The male fetal lung begins to synthesize surfactant later in gestation than the female. This delay appears to be caused by androgens. We hypothesized that male fetal lung differentiation is delayed as a consequence of an extended phase of growth which is elicited by androgens. We observed that in vivo fetal lung protein synthesis relative to DNA synthesis peaked earlier in gestation in the female fetal lung and that this event was synchronous with the onset of differentiation. Pregnant rats were treated with dihydrotestosterone (DHT) during pregnancy, and fetal lung growth parameters were measured. Lung wet weight, dry weight, and DNA and protein concentrations were significantly elevated by DHT treatment. Type II cells and fibroblasts were isolated from lungs of DHT-treated fetuses. The number of total cells recovered was increased by 30%; the number of type II cells recovered was increased by 87%; and the number of fibroblasts recovered was increased by 42%. The type II cells which were recovered exhibited increased incorporation of [3H]thymidine into DNA and a reduced ratio of radiolabeled protein to radiolabeled DNA compared to that of cells from control lungs. Further studies were done in vitro with fibroblasts and type II cells isolated from untreated fetal rat lungs. Treatment of the fibroblasts with DHT during culture caused an increase in thymidine incorporation into DNA. This effect was not blocked by simultaneous treatment with cortisol, which normally causes reduced DNA synthesis and induces fibroblast differentiation. Treatment of the type II cells with DHT in culture caused a dose-dependent increase in cell number but a decrease in synthesis of disaturated phosphatidylcholine. These studies provide more direct evidence of the interrelationships between the control of growth and the control of differentiation in the fetal lung. DHT, a signal which delays the onset of expression of differentiation, also induces growth. We conclude that the controls of growth and of differentiation of the fetal lung are reciprocally linked.     

Investigation on possible transformations of cortisol,cortisone and cortisol glucuronide in bovine faecal matter using liquid chromatography–mass spectrometry

Given the close resemblance of the ring A structure of prednisolone and prednisone on the one hand, and of androstadienedione on the other, the transformation of cortisol and cortisone into prednisolone and prednisone in cattle faeces was evaluated. A simple method that does not involve extraction but only the 1:100 dilution of cattle faeces, spiking with 400 ng/mL cortisol, cortisone or cortisol glucuronide and incubation of the suspension, was used. The analyses were performed by HPLC–MS³ to detect the supposed Δ¹ dehydrogenation of the glucocorticoids. The decision limits (CCα) and detection capabilities (CCβ) were 2.0 and 3.0 ng/mL for cortisol, cortisone and prednisolone, 3.0 and 4.0 ng/mL for cortisol glucuronide and 7.0 and 10.0 ng/mL for prednisone, respectively. Intra-day and inter-day coefficients of variation (CV%), were 5.6–6.2 and 5.2–6.6 for cortisol glucuronide, cortisol, cortisone and prednisolone, and 16.0 and 16.2 for prednisone, respectively. The recoveries were in the range 110–143% for all analytes. Regression coefficients (R2) were in the range 0.996–0.999 for all analytes. The results show the hydrolysis of the conjugated form and the dehydrogenation in ring A in diluted faeces. It is therefore predicted that urine contaminated with faeces may be positive for prednisone and prednisolone in the same way as they are positive for boldenone, i.e. as a result of microbiological dehydrogenase activity on cortisol and cortisone.     

The effect of choleragen and epidermal growth factor on proliferation and maturation in vitro of human ectocervical cells

Summary The role of choleragen (CT) and epidermal growth factor (EGF) has been examined in relation to the control of growth and differentiation of adult human cervical epithelial (HCE) cells derived from the ectocervix. Cervical biopsies derived from hysterectomy specimens were trypsin disaggregated and HCE cells were plated at 5×10³/cm² in the presence of 2×10⁴/cm² lethally irradiated Swiss 3T3 fibroblasts. Cultures were grown in Liebovitz medium supplemented with 10% fetal bovine serum and hydrocortisone. Epidermal growth factor at 10 ng/ml and choleragen at 10⁻¹⁰ M were added to cultures either singly or in combination. DNA replication in these cultures was measured autoradiographically after exposing cells to tritiated thymidine for 2 h. Differentiation was assessed histochemically by determining glycogen accumulation using the periodic acid Schiff technique. Choleragen increased colony plating efficiency by at least a factor of two but had no effect on colony size Epidermal growth factor did not increase plating efficiency but did increase colony size. In EGF treated colonies DNA replication occurred throughout the colony compared to CT treated colonies in which replication was restricted to the periphery. In the absence of EGF, population doublings achieved in culture did not exceed 32 and glycogen accumulation was evident in cells early in culture life. Colonies treated with EGF exhibited glycogen accumulation late in culture life and the EGF treated cells achieved at least 50 population doublings in culture. The results are discussed in relation to the role of EGF and choleragen on cell differentiation.