Biologically active chorionic gonadotropin: synthesis by the human fetus.

The kidney, and to a slight extent the liver, of human fetuses were found to synthesize and secrete the alpha subunit common to glycoprotein hormones. Fetal lung and muscle did not synthesize this protein. Since fetal kidney and liver were previously found to synthesize beta chorionic gonadotropin, their ability to synthesize bioactive chorionic gonadotropin was also determined. The newly synthesized hormone bound to mouse Leydig cells and elicited a biological response: namely, the synthesis of testosterone. These results suggest that the human fetus may participate in metabolic homeostasis during its development.

Fetal tissue can synthesize a placental hormone. Evidence for chorionic gonadotropin beta-subunit synthesis by human fetal kidney.

Metabolically active tissues from second trimester human fetuses were examined for their ability to synthesize the placental hormones chorionic gonadotropin and chorionic somatomammotropin. During short-term incubation studies both placenta and fetal kidney were found to synthesize and secrete the beta-subunit of chorionic gonadotropin, whereas its synthesis was not observed in fetal liver, lung or muscle. In addition, chorionic somatomammotropin synthesis and secretion was demonstrated with placental tissue but could not be detected in any of the fetal tissues examined. These observations constitute the first evidence that the genome of a fetal tissue directs the synthesis of what is considered a placental hormone.

A homologous radioimmunoassay for rat corticosteroid-binding globulin.

A rapid, specific, and sensitive RIA was developed for rat corticosteroid-binding globulin (CBG). Rat CBG was purified by affinity chromatography and its precise concentration was determined by amino acid analysis. This rat CBG was injected into rabbits to raise antiserum and was used both as the assay standard and as the tracer after labeling with 125I. Antiserum to CBG was judged specific by immunoelectrophoresis and by the comparison of RIA values with steroid-binding assay values obtained following serum fractionation on ion exchange and sizing resins. The RIA was used to determine CBG levels in pregnant rats (2.65 microM on day 14 falling to 0.95 microM at parturition), their corresponding fetuses (0.24 microM on day 18 and 0.16 microM at parturition), and amniotic fluid (0.051 microM on day 13 rising to 0.21 microM on day 21).

The role of the adrenal in generating the diurnal variation in circulating levels of corticosteroid-binding globulin in the rat.

Corticosteroid-binding globulin (CBG) levels were measured in serum samples collected sequentially from rats into which indwelling catheters had been inserted. A distinct diurnal variation in CBG levels was found, with the highest levels of binding protein at the beginning of the dark period. CBG levels then decreased until a nadir was reached shortly after the beginning of the light period. To examine the role of glucocorticoid in the generation of this pattern, rats were adrenalectomized 10 days before repeating the experiment. Adrenalectomy abolished both the diurnal variation in CBG levels and the high degree of variation in the levels of binding protein between animals. However, adrenalectomy just before the onset of the dark period did not eliminate the expected decrease in CBG levels. To further explore the role of steroid, animals that had been adrenalectomized for 10 days were given saline containing 25 micrograms/ml corticosterone for a period of 24 h. Despite the attainment of normal plasma corticosterone levels, no decrease in CBG levels was observed. When steroid administration was discontinued, however, CBG levels dropped concurrently with the decreasing steroid. These studies show that a diurnal variation in CBG exists and suggest that it is the result of the diurnal variation in glucocorticoid levels.

Steroid-binding proteins in guinea pig milk and plasma.

The levels of two steroid-binding proteins, progesterone-binding globulin (PBG) (1) and corticosteroid-binding globulin (CBG), present in the plasma of pregnant guinea pigs were determined just before and after parturition. Both PBG (3.37 +/- 2.1 microM) and CBG (10.1 +/- 1.7 microM) were present in high levels just before parturition and were found to decrease with a half-life of 2 days after delivery. PBG and CBG were also found in the milk whey of lactating guinea pigs but at much lower levels (26.5 +/- 12 and 375 +/- 18 nM, respectively, on day 1 post partum). The whey content of each protein declined with a half-life of 3 days. The whey steroid-binding proteins were indistinguishable from their plasma counterparts on the basis of ion exchange and gel filtration chromatographies, sucrose gradient centrifugation, susceptibility to heat denaturation, and hormone binding specificity.

Uptake of corticosterone by the perfused rat liver.

The mechanism of hepatic uptake of corticosterone from plasma was investigated in the isolated perfused rat liver using an indicator-dilution method. The hepatic influx rate constant for free corticosterone was determined from measurements of the rate of hepatic uptake of corticosterone from protein-free buffer. The rate of hepatic uptake of corticosterone from pooled normal rat serum was then measured. A general model of hormone transport that does not assume that hormone-protein complexes remain at equilibrium during transit through the hepatic sinusoids was used to ask whether this observed rate of uptake could be accounted for by a pool of free corticosterone that turns over very rapidly. Parameter values used in this analysis included the measured concentrations of albumin and corticosteroid-binding globulin in the serum, literature values for the rate constants describing the interactions of corticosterone with these proteins, and the value of the hepatic influx rate constant for free corticosterone determined in the present study. The rate of hepatic uptake of corticosterone from rat serum that we observed was very similar to the rate of uptake predicted by this model to occur via the pool of free corticosterone.

Increased responsiveness of the dopamine-mediated inhibition of prolactin synthesis after destruction of the medial basal hypothalamus.

Long term destruction of the tuberoinfundibular dopaminergic neurons results in increased responsiveness of the anterior pituitary to the suppression of PRL release by dopamine. In the present study we tested whether long term destruction of these neurons by medial basal hypothalamic lesions would also increase the potency of dopamine in suppressing the release and apparent synthesis of PRL. Anterior pituitaries from ovariectomized control rats and ovariectomized rats lesioned for 2 weeks were incubated for 3 h in medium 199 containing [3H]leucine in the presence or absence of various concentrations of dopamine. Labeled PRL was separated by polyacrylamide gel electrophoresis and quantitated by liquid scintillation spectroscopy. Concentrations of dopamine ranging from 10(-8)-10(-6) M caused a dose-dependent suppression of labeled newly synthesized PRL released into the medium from anterior pituitaries of medial basal hypothalamus-lesioned animals. With pituitaries from the nonlesioned rats, newly synthesized PRL release was progressively inhibited by 10(-7) and 10(-6) M dopamine, while 10(-8) M dopamine actually significantly stimulated PRL release. Total labeled PRL (that released and that remaining in the gland) was equally suppressed by all three concentrations of dopamine in pituitaries from lesioned animals, but only a minor effect was observed at the highest concentration of dopamine with the control pituitaries. Therefore, the potency of dopamine to suppress the release and apparent synthesis (total labeled PRL) of newly synthesized PRL from pituitaries of long term lesioned animals was increased.

Evidence for the direct transfer of corticosteroid-binding globulin from plasma to whey in the guinea pig.

Two steroid-binding proteins, corticosteroid-binding globulin (CBG) and progesterone-binding globulin (PBG), are known to be present in the milk whey of lactating guinea pigs. After injection of radioiodinated CBG into the maternal circulation, labeled CBG was found in the milk whey. The labeled whey CBG was identical to its plasma counterpart on the basis of size (sucrose gradients, Sephacryl S-200 gel filtration, and sodium dodecyl sulfate-gel electrophoresis), charge (DEAE-chromatography and polyacrylamide gel electrophoresis), and immunoprecipitability. In contrast, radioiodinated ovalbumin was not transferred to the milk. These results demonstrate that the CBG present in guinea pig whey results from the direct transfer of CBG from plasma to milk.

Pharmacological evidence that the inhibition of diurnal adrenocorticotropin secretion by corticosteroids is mediated via type I corticosterone-preferring receptors.

These studies were performed to determine pharmacologically the corticosteroid receptor type that mediates the effects of corticosterone (B) on ACTH secretion in adrenalectomized rats. We have compared the effects of treating young male rats at the time of adrenalectomy and throughout the next 5 days with B, dexamethasone (DEX), or aldosterone (ALDO) in doses that elevated plasma levels to concentrations in the range between 0.2-30 nM. Plasma ACTH, corticosteroid-binding globulin (CBG), and thymus weight were measured in the morning or evening, and these steroid-sensitive end points were related to the circulating concentrations of B (total B - CBG-bound B), total DEX, and total ALDO. For the inhibition of ACTH the rank order of potency of the three steroids was B greater than DEX greater than or equal to ALDO in the morning (estimated IC50, 0.7 +/- 0.1, 2.3 +/- 0.5, and 4.9 +/- 1.6 nM for B, DEX, and ALDO, respectively). There was a significant shift to the right in steroid efficacy between morning and evening (estimated IC50 in the evening, 3.9 +/- 0.2 and 9.3 +/- 0.8 nM for B and DEX; ALDO at the concentrations achieved was ineffective). The rightward shift in efficacy may result from the circadian increase in drive to ACTH secretion. The rank order of potency for B and DEX on ACTH and the agreement between the steady state IC50 values achieved for these steroids and the Kd values determined for B and DEX with type I receptors in vitro strongly suggest that feedback control of basal diurnal ACTH by corticosteroids is mediated by association with type I, B-preferring receptors. By contrast, DEX was 3 times more potent than B on CBG (estimated IC50, 1.5 and 4.5 nM, respectively) and tended to be more effective on thymus weight, suggesting that the effects of corticosteroids on these peripheral targets are mediated by association of the steroids with type II glucocorticoid receptors. ALDO coinfused with DEX or B did not alter the inhibitory effects of these on ACTH, suggesting that ALDO does not interfere with these type I, B-preferring receptors in vivo. Because there is little if any evidence for type I corticosteroid receptors in the hypothalamus, these results strongly suggest that the majority of corticosteroid feedback inhibition of basal morning and evening ACTH secretion is mediated transynaptically by the activity of extra-hypothalamic neurons.

Plasma adrenocorticotropin is more sensitive than transcortin production or thymus weight to inhibition by corticosterone in rats.

After adrenalectomy, ACTH, corticosterone-binding globulin (CBG), and thymus wet weight increase in rats as a consequence of the removal of corticosterone (B) and decrease again in response to replacement with glucocorticoids. We have studied the effect of replacing adrenalectomized male rats with a variety of different concentrations of B. Plasma concentrations of ACTH and CBG and thymus wet weights were related to the measured concentration of free ultrafilterable B in plasma. In other experiments, the clearance of [125I]CBG was determined in adrenalectomized rats with and without B replacement, and the time required for the changes in plasma CBG concentrations after removal and/or replacement of B in adrenalectomized rats was determined. Five to 7 days after adrenalectomy and institution of a relatively constant B replacement signal, plasma CBG concentrations were highly correlated with circulating B concentrations (r2 = 0.745; P less than 0.001). The effect of B was on the CBG production rate, since clearance did not change. Because CBG concentrations decrease as total B concentrations increase, there is an amplification of free B concentrations with increasing total B. The relationships of plasma ACTH and CBG and thymus wet weight to circulating free B levels showed that 50% inhibition of ACTH was achieved at a free B concentration of 0.8 +/- 0.05 nM, whereas 50% inhibition of CBG and thymus wet weight were achieved at free B concentrations of 4.6 +/- 0.9 and 4.4 +/- 0.6 nM, respectively. These inhibition values correlate well with the known Kd values for the high affinity type I B receptor (0.5 nM) and for the lower affinity type II glucocorticoid receptor (2.5-5 nM), respectively, suggesting that ACTH secretion, CBG production, and thymus wet weight are regulated by the association of B with these receptor types.

Uptake of cortisol by the perfused rat liver: validity of the free hormone hypothesis applied to cortisol.

The mechanism by which cortisol in plasma enters hepatic cells was investigated using the isolated perfused rat liver. To determine whether hepatic uptake of cortisol from serum can be accounted for entirely by the pool of unbound (free) cortisol, we compared observed uptake rates with the equilibrium-free fraction of cortisol in serum and the rates of dissociation of cortisol from its serum binding proteins (determined using a rapid filtration assay based on transfer of [3H] cortisol to dextran-coated charcoal). More than 95% of the cortisol in both human and rat serum dissociated spontaneously from its binding proteins within 5 sec at 37 C. The fractional unidirectional hepatic uptakes of cortisol from pooled human serum and pooled rat serum were 59.4 +/- 5.4% and 59.5 +/- 1.0% (mean +/- SE), respectively, at the physiological flow rate of 1 ml/min.g liver. The corresponding free cortisol fractions in these sera were 4.53 +/- 0.15% and 8.16 +/- 0.23%, respectively. The fractional unidirectional hepatic uptake of cortisol from protein-free buffer averaged 99.9% (n = 5) at a flow rate of 3 ml/min.g liver. By calculating the appropriate rate constants and applying the Kety-Renkin-Crone equation to the above data, it can be shown that all of the cortisol taken up from serum by the perfused rat liver can be accounted for by the pool of free cortisol, which turns over very rapidly. The physiological significance of this finding is discussed in terms of a general mathematical model of hormone transport that delineates the conditions under which the free hormone hypothesis is and is not valid.

Human fetal adrenal definitive and fetal zone metabolism of pregnenolone and corticosterone: alternate biosynthetic pathways and absence of detectable aldosterone synthesis.

In the rhesus monkey and ovine fetus in utero, aldosterone concentrations do not rise in response to surgical stress, ACTH, or angiotensin-II, all of which are secretagogues for this mineralocorticoid in the adult. To assess the mechanism of this phenomenon in the human fetus, metabolism of pregnenolone and corticosterone by second trimester human fetal adrenal definitive zone and fetal zone tissue was studied. After incubation of fresh tissue with trace amounts of [3H]pregnenolone or [3H]corticosterone, the products of metabolism were separated using high performance liquid chromatography and quantified. The delta 5-3 beta-hydroxysteroids 17-hydroxypregnenolone and dehydroepiandrosterone and their sulfates comprised 85-90% of metabolized pregnenolone. In the fetal zone, cortisol was the predominant secreted delta 4-3-ketosteroid, accounting for 6-8% of the metabolized pregnenolone. In the definitive zone, progesterone and corticosterone were the predominant secreted delta 4-3-ketosteroids, each accounting for about 2% of the metabolized pregnenolone. 11-Dehydrocorticosterone and sulfates were the only metabolites detected after incubation of fetal adrenal tissue with corticosterone. 11-Dehydrocorticosterone accounted for more than 80% of the metabolized corticosterone in the definitive zone and 50% in the fetal zone. Incubations with secretagogues or antioxidants (10 nmol/L ACTH, 10 nmol/L angiotensin-II, 21 mmol/L potassium, 100 mmol/L dimethylsulfoxide, 5 mumol/L metyrapone, or 100 mumol/L butylated hydroxyanisole) did not change the pattern or extent of precursor metabolism. No aldosterone, 18-hydroxycorticosterone, or 18-hydroxydeoxycorticosterone was detected in baseline or stimulated incubations of human fetal tissue. In contrast, adult human zona glomerulosa metabolized corticosterone to aldosterone, 18-hydroxycorticosterone, and 11-dehydrocorticosterone under similar conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial characterization of a novel growth factor from the blood of women with preeclampsia.

Sera obtained before delivery from women with preeclampsia contain greater mitogenic activity than sera drawn from the same women 24-48 h after parturition or sera from normal parturients. These studies describe the initial characterization of the blood-borne mitogenic factor(s) from preeclamptic women which we have named ELMER (Endogenous Ligand conferring MitogEnic Response). ELMER appears to be a unique mitogen with characteristics that are not identical to those of other known growth factors. ELMER is present in serum as an acid- and heat-labile protein, approximately 160,000 daltons in size, which is a potent mitogen for human fibroblasts but not for human endothelial cells. Its presence in plasma suggests that it is a circulating factor rather than a product of blood coagulation ex vivo. We believe that ELMER represents a potential serum marker of preeclampsia and that it may play roles in the vasospasm and proliferative vascular lesion, termed atherosis, frequently associated with the preeclamptic syndrome.

Cellular localization of chorionic gonadotropin in human fetal kidney and liver.

Earlier, we reported that second trimester human fetal kidney and, to a much lesser extent, human fetal liver were capable of synthesizing and secreting the beta-subunit of hCG. Recently, we also have shown that these tissues, likewise, synthesize and secrete the alpha-subunit of hCG. The hCG produced is biologically active. To determine the cellular localization of these peptides, immunocytochemical studies were performed on human fetal tissues using antibodies against beta hCG, alpha hCG, and the intact hormone. Placental syncytiotrophoblast served as an immunopositive control. In the human fetal kidney, the ascending (thick) limb of the loop of Henle, distal convoluted tubule, and occasional cells in the collecting ducts were distinctly immunopositive for both beta hCG and the alpha-subunit. Small amounts of light positive staining occurred in only a few hepatocytes. Placental syncytiotrophoblast was routinely positive for both subunits, but fetal lung and striated muscle were negative. These immunocytochemical results indicate that immunoreactive beta hCG as well as the alpha-subunit are present in placental syncytiotrophoblast, in the distal renal nephron, and in a limited population of hepatocytes. The qualitative number and intensity of immunopositive cells closely correlate with the quantitative amounts of their hCG subunit synthesis. Taken together with our previous biosynthetic data, the immunocytochemical localization reported here indicates the probable cellular sites of alpha- and beta hCG synthesis in these tissues. The presence of comparable alpha- and beta-subunit staining in identical cell populations suggests that both hCG subunits and, therefore, perhaps intact hCG are produced at these same cellular sites during fetal life.

Immunocytochemical localization of corticosteroid-binding globulin in rat tissues.

Previous studies utilizing steroid-binding assays have suggested that corticosteroid-binding globulin (CBG)-like glucocorticoid binding sites are present in various tissues of the rat. It is not known, however, whether such binding reflects the intracellular presence of CBG derived from serum or a special class (type III) of receptors. In order to elucidate this problem, immunocytochemical localization of rat CBG was carried out using a specific antiserum prepared against rat serum CBG and the peroxidase-antiperoxidase technique. Positive staining was found in certain cells of the liver, the distal and/or convoluted tubules of the kidney, the uterus, the follicular cells of the thyroid, and some cells of the anterior pituitary. Other tissues including heart, muscle, thymus, hypothalamus, supraoptic and paraventricular nuclei, and diaphragm were negative. The presence of immunoreactive CBG in specific cells of some glucocorticoid-responsive tissues and not others raises interesting questions concerning the transport of glucocorticoids and their mechanism of action.

Development of a hybrid artificial pancreas with a dense polyurethane membrane.

We have synthesized a series of strong, elastomeric polyurethaneureas and have used them to fabricate non-porous film and hollow fiber membranes. The solvent cast membranes are non cytotoxic, angiogenic, and permeable to gases, nutrients, secretagogues, and cell products via purely concentration driven transport. Permeability to water, glucose, and protein increases monotonically with membrane water absorption above a threshold value. Water absorption increases with soft segment hydrophilicity, soft segment molecular weight, and soft segment volume fraction of the (dry) segmented polyurethanes. Cell lines (RAJI and MOPC-31C) and primary cells (porcine islets) contained within our membranes have been maintained in culture for up to 6 months with nutrients supplied only by the external media. Cells within membrane devices were protected from immune rejection when implanted into murine hosts. Simple, compact devices containing porcine islets restored normoglycemia and near normal response to glucose tolerance tests in diabetic mice for at least 2 months. Explants had a high degree of vascularization adjacent to the membrane, with little or no fibrous tissue. These properties, and the material's ability to support cell function and protect xenogeneic cells from immunologic rejection, suggest that it would be useful in the construction of hybrid artificial organs and in in vitro cell culture.

The role of tyrosine in the binding of steroid by progesterone binding globulin from the guinea pig.

Treatment of progesterone binding globulin (PBG) with tetranitromethane (TNM) resulted in a loss of steroid binding activity (inactivation) which was dependent on both time and concentration of reagent. Scatchard analysis of binding revealed that inactivation was due to a decrease in binding site number with no effect upon the affinity of PBG for steroid. Incorporation studies demonstrated that the loss of binding activity correlated with the incorporation of 1.3 nitro groups per molecule of PBG. The involvement of the steroid binding site in the reaction was shown by the ability of progesterone, but not cortisol, to protect against inactivation. Treatment with N-acetylimidazole did not inactivate PBG nor did the conversion of nitrotyrosyl residues to amino-tyrosines regenerate binding activity, suggesting that the pheolic hydroxyl is not involved in steroid binding. These studies suggest that inactivation was due to the incorporation of a bulky group into the aromatic ring of a tyrosine present at the steroid binding site thus blocking its ability to participate in hydrophobic interactions with the ligand.

Corticotropin-releasing factor can stimulate gonadotropin secretion by human fetal pituitaries in superfusion.

In previous studies, corticotropin-releasing factor was found to elicit a rise in circulating adrenocorticotropic hormone in human subjects and laboratory animals, but no stimulatory effect of corticotropin-releasing factor on other pituitary hormones was detected. Since stress may be associated with luteinizing hormone changes as well as with those of corticotropin-releasing factor and adrenocorticotropic hormone, we quantified gonadotropin responses to corticotropin-releasing factor and arginine vasopressin in 11 human fetal pituitaries with use of both superfusions and static incubations. Exposure to corticotropin-releasing factor brought about a significant increase in adrenocorticotropic hormone and gonadotropin concentrations in the effluent media by means of the superfusion system. Similar concentrations of corticotropin-releasing factor significantly increased adrenocorticotropic hormone secretion into the medium by dispersed fetal pituitary cells cultured on an extracellular matrix but failed to increase luteinizing hormone and follicle-stimulating hormone secretion. Exposure to 3 mmol/l 8-bromo-cyclic adenosine monophosphate caused an increase in all three peptides, both in superfusion and static incubations. Dose-response studies showed that the effect on gonadotropin secretion occurred at concentrations of 8-bromo-cyclic adenosine monophosphate two orders of magnitude lower than those affecting adrenocorticotropic hormone secretion. The purity of corticotropin-releasing factor and arginine vasopressin used in these studies was confirmed by high-performance liquid chromatography. These in vitro results are consistent with a paracrine effect of corticotropes acting on gonadotropes. The combination of static incubation and superfusion has proved useful in elucidating the effects of different secretagogues on pituitary cells.