Search

We studied ligand binding to adrenergic receptors in various brain regions of adult ovariectomized rats after estrogen treatment or induction of sexual behaviours after treatment with estrogen and progesterone. Rats, ovariectomized for 2 weeks, were given estradiol benzoate (10 μg/kg) s.c on day 0. On day 2 they were injected with progesterone (0.5 mg/rat) at 7.45 a.m. The rats were killed shortly before 1.00 p.m., at a time when the serum LH levels were still markedly reduced. However, this steroid regimen was 100 % effective in inducing sexual behaviour. Effects of α- and β-adrenergic receptors were evaluated by the use of the radioligands, 3 H-dihydroalprenolol, an unselective β-adrenergic blocking agent, 3 H-clonidine, an α 2 -adrenergic agonist, and 3 H-prazosin, an α 1 -adrenergic antagonist. Progesterone was shown to induce a low affinity binding site in the cerebral cortex for 3 H-dihydroalprenolol, while no effects on 3 H-dihydroalprenolol binding characteristics could be demonstrated following estrogen alone or estrogen-progesteron treatment in the amygdaloid-entorhinal area and in the whole hypothalamus including the preoptic region. The binding characteristics of 3 H-clonidine was changed by estrogen alone or estrogen-progesterone treatment in a different manner. Within the amygdaloid-entorhinal cortex, estrogen-progesterone treatment produces a lowering of the affinity of the low affinity binding site, and in the whole hypothalamus including the preoptic area, estrogen-progesterone treatment produces a slight increase in the number of high affinity 3 H-clonidine binding sites. With regard to 3 H-prazosin binding characteristics combined treatment with estrogen and progesterone produced a lowering of the affinity of the low affinity component within the hypothalamus leaving the 3 H-prazosin binding sites in the cerebral cortex unaffected. The most marked changes were found in amygdaloid-entorhinal cortex, where the K D value of the high affinity component was increased by 50 %, while the B max and K D values of the low affinity component were reduced. These results demonstrate that the α- and β-adrenergic receptors are sensitive to combined treatment with estrogen and progesterone resulting in the induction of sexual behaviour. The changes are ligand and region specific and demonstrate that progesterone in estrogen-primed ovariectomized rats can produce preferential changes in the low affinity component of β-adrenergic and α 1 -adrenergic receptors in the cerebral cortex and hypothalamus, respectively and changes in both the high and low affinity component of α 1 receptors of the amygdaloid-entorhinal cortex. The findings give futher evidence that estrogen treatment alone can produce changes in both the high and low affinity component of α 2 -adrenergic receptors in the amygdaloid-entorhinal cortex and in the hypothalamus. It seems possible that estrogen and progesterone induce their effects on α- and β-adrenergic receptors via actions on estrogen and progesterone receptors present within the brain. Activation of the steroid receptors may in turn produce changes in genetic transcription which can effect various aspects of the biochemical signals regulating the catecholamine receptors. It is presently unknown if these changes in the characteristics of catecholamine receptors can be related to the ability of progesterone to induce sexual behaviour in the present experiments or in general if they represent a major change in the neurotransmission of the respective synapses.

Results of studies of hypophysectomized rats suggest that growth hormone serves as a final common mediator through which gonadal steroids and other modifiers of pituitary function alter the expression of gender-specific liver genes such as the sexually dimorphic pair of cytochrome P-450 isozymes, male-specific P-450h and female-specific P-450i. We tested the effects of growth hormone in a system for primary monolayer culture of adult rat hepatocytes on a laminin-rich extracellular matrix (matrigel), which permits sustained expression of both constitutive and inducible liver genes in a chemically defined medium. Cultures of freshly isolated hepatocytes prepared from untreated male rats and samples of the intact donor liver contained readily detectable quantities of immunoreactive P-450h protein (measured on immunoblots of cell microsomes) and P-450h mRNA (measured on Northern blots of cellular RNA). Neither P-450i immunoreactive protein nor P-450i mRNA were present. Addition of physiologic concentrations of human or bovine growth hormone, but not of prolactin, to culture medium lacking insulin or other hormones resulted in prompt induction of P-450i immunoreactive protein and P-450i mRNA. Induction of P-450i mRNA in male hepatocyte cultures was dependent on the concentration of growth hormone, required as little as 24 hr of exposure, and was markedly attenuated in cultures maintained on type I collagen rather than on matrigel. Growth hormone treatment also induced the level of mRNA for insulin-like growth factor I, whereas the amount of mRNA for the male-specific urinary protein alpha 2 mu-globulin was unaffected. Cultures of hepatocytes derived from untreated adult female rats retained high levels of P-450i mRNA but only if the culture medium contained growth hormone. None of the tested treatments with estrogens, androgens, glucocorticoids, or growth hormone induced P-450h mRNA or P-450h immunoreactive protein in cultures of female hepatocytes. We conclude that the somatogenic effects of growth hormone acting alone and directly on the hepatocyte in culture are sufficient to "feminize" the cytochrome P-450 phenotype. The present culture system offers a way to explore the molecular basis for hormonal control of liver gene expression.

The induction of prolactin (Prl) liver receptors in the male rat by subfractions of human growth hormone (hGH) was studied. A heterogeneous preparation of hGH, devoid of immunoassayable Prl activity was fractionated by preparative isoelectric focusing (IEF). Induction of Prl receptors and the metabolism of 4-(4-14C)-andostene-3, 17-dione (A4) were studied in the liver microsomal fraction following continuous adminstration of the subfractions at a rate of 2.5 ug/h for one week. Induction of Prl receptors was noted with several subfractions which differed in their immunoreactivity. There was a relation between the Prl receptor induction and the A4 metabolism. Serum levels of testosterone (T) 5α-dihydrotestosterone (DHT) and of triglycerides (TG) appear to be unrelated to Prl receptor induction.

Hepatocytes from adult rats were isolated and cultivated as primary monolayers for 3 days in a medium containing only 1% homologous serum. The metabolism of 4-androstene-3,17-dione was followed in hepatocytes from male, female and hypophysectomized animals. It was found that immediately after preparation, 5alpha-reductase and 16alpha-hydroxylase activities were present in the cells in amounts comparable to those found in microsomal preparations from liver homogenates. After 3 days in culture, these enzyme activities had decreased to about half the values measured on day 0. During this time the sexual differences in steroid metabolism in the cells were stable, i.e. there was no detectable induction of 16alpha-hydroxylase in cells from female animals, and the relative sex difference in 5alpha-reductase activity persisted.

The localization of various neuropeptides is described in the gut and in the hypothalamus in the rat. Evidence is given for the presence of material resembling corticotropin-like intermediate peptide in arcuate and periarcuate neurons, projecting to various hypothalamic nuclei, limbic areas and the thalamus. beta-Endorphin and glucagon decrease dopamine turnover in the median eminence, while secretin increases dopamine turnover and vasoactive intestinal polypeptide (VIP) has no effect. beta-Endorphin, VIP, secretin, and glucagon all produce discrete changes in norepinephrine turnover in various hypothalamic nuclei. Mainly increases of norepinephrine turnover were observed. These catecholamine turnover changes appear to cause changes in the secretion of prolactin and growth hormone. The results therefore indicate that gut hormones and opioid peptides may act directly on the hypothalamus on specific types of receptors to participate in the control of hypothalamic functions such as control of hormone secretion from the anterior pituitary and of food intake. It seems possible that gastrointestinal peptides released from the gastrointestinal tract into the circulation under certain circumstances could reach the hypothalamus and modulate its activity via the above-mentioned mechanisms. It may therefore be speculated that disturbances in gastrointestinal functions could lead to pathological changes in food intake via modulation of hypothalamic activity.

Quantitation of estrogen receptor has been performed in cytosol prepared from 75 specimens of breast cancer tissue from patients who had not received hormonal therapy. The study was performed in order to compare an immunoassay (Abbott Laboratories, North Chicago, IL) with our currently used method for estrogen receptor analysis based on isoelectric focusing of [3H]estradiol-receptor complex in polyacrylamide gels. Using linear regression analysis, a regression coefficient (slope) of 1.30 and a correlation coefficient of 0.75 were calculated. The differences in results between the two methods are probably partly explained by the fact that the ligand-based method only measures unoccupied receptor, whereas the immunoassay detects the total amount of receptor, resulting in generally slightly higher concentrations with the latter method. However, in five of 75 specimens the ligand-based method gave a considerably higher concentration of estrogen receptor. This was most probably explained by partial proteolysis resulting in the formation of receptor fragment(s), which was undetectable with the immunoassay but detectable with the ligand-based method. These observations underline the importance of careful handling of specimens during the whole immunoassay procedure.

Measurement of estrogen receptor content in human breast cancer has become a valuable tool to predict the response of a tumor to endocrine manipulations. The aim of this study was to see whether steroid receptor assay could also be used to predict the value of endocrine therapy in human prostatic carcinoma. Biopsies from 25 primary tumors were analyzed with regard to quantity of methyltrienolone (a synthetic androgen) binding to the receptor 20 specimens were receptor-positive and 5 were receptor-negative. The correlation between receptor content and response to endocrine treatment was approximately approximately equal to 80%. The steroid receptor profiles of 5 lymph node metastases were also analyzed. 4/5 contained methyltrienolone receptors and 2/5 contained progestin receptors. 3/5 were glucocorticoid receptor-positive while all specimens were estrogen receptor-negative.

Specific lesions in the periventricular area of the hypothalamus in male rats lead to a partial feminization of the liver steroid metabolism and a simultaneous reduction of the somatostatin level in the median eminence. Administration of an antiserum against somatostatin causes a similar degree of feminization of liver metabolism in male rats. Thus somatostatin could be the neuroendocrine regulator of the sexually differentiated metabolism of steroids in rat liver. A possible influence from the amygdaloid complex in regulating hepatic steroid metabolism is also indicated since large lesions in the amygdala cause a slight feminization of hepatic steroid metabolism in male rats. The female pattern of hepatic steroid metabolism is induced following frequent administration of hGH. The feminizing effect of hGH on hepatic steroid metabolism does not require the presence of gonads, adrenals, or thyroid. The somatogenic property of hGH seems to be responsible for the feminizing effect since purified rGH alone gives a complete feminization of hepatic steroid metabolism in hypophysectomized animals. rGH purified from male or female pituitary glands is equally efficient in feminizing hepatic steroid metabolism. Furthermore, male or female rGH have the same apparent molecular weight and isoelectric point. The mechanism whereby GH regulates hepatic steroid metabolism could be related to the sexually differentiated secretory profile of GH in the rat. A continuous presence of GH in the circulation seems to be a prerequisite for a female pattern of hepatic steroid metabolism. By analogy, it may be suggested that the high-peak, low-trough secretory pattern of GH characteristic of male rats causes a masculine type of liver steroid metabolism. Gonadal hormones affect both the secretory profile of GH and hepatic steroid metabolism. It is most probable that gonadal hormones affect liver steroid metabolism via modulations of the GH secretory profile. Possibly, estrogen exerts its effect directly on the pituitary by stimulating GH secretion. Androgen most probably has its primary site of action in the anterior hypothalamus or in extrahypothalamic areas. A plausible mechanism is that androgens stimulate the hypothalamic GH-inhibitory center. An overall view of the present hypothesis concerning hypothalamopituitary regulation of the sexually differentiated hepatic steroid metabolism in the rat is presented in Figure 2.

Cytochrome P-45011 beta has been solubilized and partially purified from bovine adrenal cortex mitochondria using chromatography on Octyl-Sepharose CL-4B or DEAE-Sepharose CL-6B. The partially purified P-450 preparations were about 90% pure as judged by SDS-polyacrylamide gel electrophoresis. In the presence of purified preparations of adrenodoxin reductase and adrenodoxin, the partially purified P-450 preparations catalyzed NADPH-supported 11 beta-hydroxylation of unconjugated and sulphoconjugated deoxycorticosterone. In presence of Triton X-100 the partially purified cytochrome P-45011 beta had a Stoke's radius of 4.5 nm, a sedimentation coefficient of 3.1 S and a partial specific volume of about 0.85 cm3/g. These results indicate that the cytochrome P-45011 beta-Triton X-100 complex has a molecular weight of about 100 000 and that P-45011 beta bound about 1.1 g of Triton X-100 per g of protein. The P-45011 beta-Triton X-100 complex was catalytically active in hydroxylation reactions supported by NADPH or the hydroxylating agent ortho-nitroiodosobenzene, suggesting that the monomer of cytochrome P-45011 beta is an active form of the protein.

Subacute exposure of rats to varying concentrations of toluene vapours leads to increases of amine levels and turnover in several types of hypothalamic noradrenaline nerve terminal systems. Furthermore, the exposure of toluene in concentrations ranging from 80-3000 ppm and benzene (1500 ppm) produced differential effects on dopamine and noradrenaline turnover within various parts of the hypothalamus. These regions are involved in neuroendocrine regulation and disturbances in the secretion of prolactin were demonstrated after exposure to toluene.

The exposure of newborn infants to the plasticizer di-(2-ethylhexyl) phthalate (DEHP) and its primary metabolite mono-(2-ethylhexyl) phthalate (MEHP) was studied during exchange transfusions by measuring their contents in the infused blood. Plasma concentrations of DEHP and MEHP in the blood withdrawn from the infants during the transfusions also were determined. The amounts of DEHP and MEHP inadvertently infused varied from 1.7 to 4.2 and 0.2 to 0.7 mg per kg body weight, respectively. Immediately after the transfusions, the plasma levels of DEHP in the individual infants varied between 3.4 and 11.1 micrograms per ml. MEHP in the corresponding samples ranged from 2.4 to 15.1 micrograms per ml. Judging from plasma concentrations of DEHP and MEHP during and after transfusion, there was no gradual accumulation of these substances in the plasma during the course of the transfusion. In the two infants who underwent a second exchange transfusion, significant levels of phthalates were found at 16 and 23 hours, respectively, after the first transfusion. Plasma concentrations of DEHP in these infants declined at a faster rate than those of MEHP, thus pointing to the importance of examining the pharmacokinetics of this potentially toxic metabolite.

Limited proteolysis of the glucocorticoid receptor has proven to be a valuable tool for a functional analysis of the receptor protein. With the help of these analyses, it has been possible to describe three functional domains of the receptor protein. The native glucocorticoid-receptor complex contains a steroid-binding domain (A), a DNA-binding domain (B) and an immunoactive domain (C). This form of the glucocorticoid receptor has a Stokes radius of 6.1 nm and a molecular weight of 94 K when purified. Two steroid-binding proteolytic receptor fragments can be found. The larger one has a Stokes radius of 3.3 - 3.6 nm and a molecular weight of 39 K and contains both the steroid- and DNA-binding sites (A + B). The smaller steroid-binding receptor fragment, with a Stokes radius of 1.9 nm and a molecular weight of 27 K, contains only the steroid-binding domain (A). Analysis of the proteolytic fragments of the glucocorticoid receptor using the specific anti-receptor antibodies revealed the occurrence of a fragment with Stokes radius 2.6 nm following limited proteolysis of the receptor by alpha-chymotrypsin. This fragment contains neither the steroid-binding nor the DNA-binding domains but consists only of the immunoactive domain (C). Further proteolysis of this fragment results in an even smaller form with Stokes radius 1.4 nm. The apparent identity of the larger of the two proteolytic forms of the glucocorticoid receptor (the 3.3 - 3.6 nm form) with the receptor isolated from certain corticosteroid-resistant cells, together with the lack of the immunoreactive domain in these cells appears to indicate an important function of this domain with regard to the biological activity of the receptor.

Rat liver somatogenic receptors have been characterized by gel permeation chromatography, sucrose density gradients in H2O and D2O, and affinity cross-linking using 125I-bovine growth hormone (bGH) as a specific somatogenic receptor ligand. Cross-linking of 125I-bovine growth hormone to a Triton X-100-treated low density fraction isolated from livers of late pregnant rats followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis under reducing conditions showed three major binders with Mr 95,000, 86,000, and 43,000 and a minor binder of Mr 55,000, after correction for bound ligand assuming a 1:1 binding ratio of ligand-receptor. The Mr 86,000, 55,000, and 43,000 species were recovered in the detergent-soluble supernatant after high-speed centrifugation, whereas the Mr 95,000 species remained Triton X-100 insoluble. Detergent-soluble 125I-bGH-receptor complexes were further analyzed by sedimentation into sucrose density gradients. The sedimentation coefficient was S20,w = 5.2 S and the partial specific volume v = 0.72 ml/g. Gel permeation chromatography on a Sepharose S-400 column indicated a Stokes radius of 61 A for the 125I-bGH-receptor-Triton X-100 complex. Based on these figures, the molecular weight of the complex was calculated as 131,100. The molecular weight of the ligand-free receptor-Triton X-100 complex was calculated as Mr 109,100. Affinity cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the 61 A peak from Sephacryl S-400 chromatography (cf. above) showed two binding entities, one major and one minor with Mr values 86,000 and 43,000, respectively, in the absence of reductant. When electrophoresis was run in the presence of reductant the Mr 43,000 species was the major binding entity. Furthermore, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (first dimension, nonreducing and second dimension, reducing) showed that a disulfide-linked binder at Mr 43,000 is contained within the Mr 86,000 species. As with pregnant rats, female and male rats both showed 125I-bovine growth hormone binders of Mr 95,000, 84,000, 55,000, 43,000, and additionally an Mr 35,000 binder.

This brief report deals with some recent observations relating to the assoclation of the Mr approximately 90,000 heat shock protein (hsp90) with the glucocorticoid receptor. In its nonactivated state, stabilized by sodium molybdate, the glucocorticoid receptor exists as a 9S heteromeric complex containing a single Mr approximately 94,000 steroid-binding unit and a dimer of hsp90. Monospecific antibodies raised against the purified rat glucocorticoid receptor-associated hsp90 interact with the molybdate-stabilized receptor. They also immunoprecipitate the Mr approximately 27,000 steroid-binding fragment of the receptor generated by trypsin treatment. Thus, hsp90 interacts with the ligand-binding domain of the glucocorticoid receptor. Furthermore, dissociation of the glucocorticoid receptor-hsp90 complex results in a major reduction of the affinity of the Mr approximately 94,000 receptor entity for its ligand. The heteromeric 9S complex does not bind to DNA. When it is activated to a DNA-binding state, the hsp90 dissociates from the ligand-binding protein. In vitro, activation of the cytosolic rat glucocorticoid receptor to a DNA-binding state is inducible by the binding of ligand. Taken together, our observations indicate the existence of important connections between the association of hsp90 and the functions of ligand- and DNA-binding of the glucocorticoid receptor.

The [3H]estramustine-binding macromolecule in human prostate was partially characterized using a number of chromatographic procedures. Although human estramustine-binding protein (HEMBP) had a marked tendency to aggregate in several systems, a molecular weight of about 54,000 was determined by gel filtration on Sephacryl S-200 Superfine and high-performance liquid chromatography. A sedimentation-coefficient of about 3.6S was obtained for HEMBP when analyzed by sucrose density gradient centrifugation. Isoelectric focusing in polyacrylamide gels indicated an isoelectric point of 4.7 to 4.8, which was in agreement with the elution position of HEMBP following chromatofocusing on Polybuffer Exchanger 94. Furthermore, HEMBP was eluted from diethylaminoethyl-Sepharose with 0.23 M KCl, was retained by concanavalin A-Sepharose (indicating that HEMBP is a glycoprotein), but did not interact with Affi-Gel Blue. Special efforts were concentrated on establishing that HEMBP was a species distinct from human serum albumin. Separation between the [3H]estramustine-labeled HEMBP and the [3H]estramustine-human serum albumin complex was obtained both on sucrose density gradients by chromatography on Affi-Gel Blue, by chromatofocusing, by gel filtration, by isoelectric focusing, and on concanavalin A-Sepharose by affinity chromatography. Twenty-two of 27 human benign hyperplastic prostate cytosol samples were found to contain protein immunochemically similar to estramustine-binding protein (EMBP) purified from rat ventral prostate as determined by the EMBP radioimmunoassay method. Concentrations from 0.2 to 139.6 ng EMBP per mg of total cytosolic protein (mean, 19.3) were determined. Furthermore, four of seven prostatic cancer specimens as well as two of two normal prostatic specimens were also found to contain rat EMBP-immunoreactive material. The unequivocal demonstration of the presence of a HEMBP is of great potential interest in consideration of estramustine phosphate (Estracyt) therapy against prostatic carcinoma. It is not inconceivable that the concentration of HEMBP in the carcinomatous tissue will be of significance in determining the drug uptake in the malignant tissue.

We have investigated the physiochemical characteristics of trypsin-treated, molybdate-stabilized glucocorticoid-receptor complexes from rat liver in the presence of 10 mM sodium molybdate by high performance ion-exchange chromatography, high performance size-exclusion chromatography, and sedimentation analysis. Trypsin treatment was performed under conditions previously reported to degrade the monomeric Mr approximately 94,000 steroid-binding protein to an Mr approximately 27,000 ligand-binding entity (Wrange, O., and Gustafsson, J.-A. (1978) J. Biol. Chem. 253, 856-865). Also in the presence of molybdate, an Mr approximately 27,000 steroid-binding fragment was obtained by limited trypsinization. However, no major differences in the tested physicochemical parameters were seen when trypsin-treated glucocorticoid-receptor complexes were compared with crude cytosolic complexes. Furthermore, the Mr approximately 27,000 steroid-binding fragment generated in the presence of molybdate could be immunoprecipitated by antibodies specific for the glucocorticoid receptor-associated Mr approximately 90,000 heat shock protein. These results provide direct evidence for an interaction of the Mr approximately 90,000 heat shock protein with the steroid-binding domain of the glucocorticoid receptor, known to correspond to the C-terminal third of the receptor protein.

The structure of the membrane-bound and Triton X-100-solubilized female rat liver prolactin receptor has been studied by affinity cross-linking/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and sucrose/H2O and sucrose/D2O density gradient centrifugation. Hydrodynamic characterization revealed that the 125I-human growth hormone receptor-detergent complex represents a molecular species with a Stokes radius of 61 A, a sedimentation coefficient of 5.0 s, and a calculated molecular weight of 158,000. The molecular weight of the receptor was calculated to be 92,000. Three lactogenic hormone-binding species with Mr values of 87,000, 40,000, and 35,000, respectively, were repeatedly found when detergent-solubilized preparations were analyzed using an affinity cross-linking technique. Estrogen treatment of female rats increased the intensity of these bands. Occasionally, an Mr 165,000 hormone-binding species was also found. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis studies (first dimension, nonreducing; second dimension, reducing) demonstrated that disulfide- and nondisulfide-linked hormone-binding species with Mr values of 40,000 and 35,000 are contained within the Mr 87,000 species. It is concluded that the Triton X-100-solubilized female rat liver prolactin receptor has a molecular weight of about 90,000. This molecular species contains Mr 40,000 and Mr 35,000 hormone-binding subunits. It cannot be determined whether these subunits are combined with each other or with as yet undetected subunit(s) to make up the Mr 90,000 species, or whether each one of these subunits is a proteolytic fragment of the Mr 90,000 species.

The sulfate-specific hydroxylase system in liver microsomes from rats has been investigated with respect to its substrate specificity. Eighteen different C18, C19, C21, and C27 steroid sulfates and the coresponding free steroids have been incubated with microsomal preparations from male and female rats. The sulfate-specific system was only present in preparations from female rats and primarily catalyzed hydroxylation in position 15beta but also in position 7beta. In contrast to this, male liver microsomes were more efficient than female liver microsomes in hydroxylating free steroids; these were hydroxylated in positions 2alpha,2beta,6alpha,6beta,7alpha,7beta,16alpha, and 18. The sulfate-specific hydroxylase system in female liver microsomes was found to have rigid requirements c concerning the structure of ring D in the substrate molecule; only 17beta-sulfates (C18 and C19 steroids) and 21-sulfates (C21 steroids) were hydroxylated. Less rigid criteria, however, exist concerning the structure of ring A. The following K-m values were determined for microsomal 15beta-hydroxylation: 5alpha-androstane-3alpha,17beta-diol disulfate, 17.2 muM; 5beta-androstane-3alpha,17beta-diol disulfate, 16muM;5alpha-androstane-3alpha,17beta-diol 17-sulfate, 26 muM; and estradiol 17-sulfate, 181 muM. Some of the regulatory mechanism controlling the activity of the sex-specific 15beta-hydroxylase system also have been studied and compared to the mechanism controlling the activities of the less specific 2alpha-, 7alpha-, and 18-hydroxylase systems active on 5alpha-[4-14C]androstane-3alpha,17beta-diol. Biliary drainage did not affect the 15beta-hydroxylase activity, whereas the 2alpha- and 7alpha-hydroxylase activities decreased..

Hypophysectomy of male animals has little effect on the hepatic 4-androstene-3,17-dione (androstenedione) metabolism except at the kinetic level where changes in the apparent Km of the 16 alpha- and 7 alpha-hydroxylases are seen. On the other hand, hypophysectomy of female animals leads to a "masculinization" of hepatic androstenedione metabolism, following the changes seen in Vmax of the respective enzymes probably due to the removal of the source of "feminizing factor" thought to maintain the female-type metabolism in the liver. There seems to be a temporal dissociation of the effects on the various enzymes indicating different cellular control mechanisms for these enzymes. Oestrogen treatment of male rats causes "feminization" of the hepatic androstenedione metabolism. The time study shows an initial increase in 17-hydroxysteroid oxidoreductase, 6 beta- and 16 alpha-hydroxylase activities followed by a decrease to the levels of females. This biphasic effect is possibly due to an initial direct effect of the oestrogen on the liver followed by an indirect effect via the hypothalamo-pituitary system. The changes in enzyme activity noted are related to changes in Vmax of the respective enzymes although changes in apparent Km are also seen.

In vivo studies were carried out on three bile fistula patients to further elucidate the side chain oxidation pathways from C-27 bile acids to cholic acid in man. Two patients each received (25-R)- and (25-S)-3 alpha, 7 alpha,-12 alpha-trihydroxy-5 beta-[7 beta-3H]cholestanoic acid (THCA) on consecutive days and three patients wee administered 3 alpha, 7 alpha, 12 alhpa, 24 xi-tetrahydroxy-5 beta-[7 beta-3H]cholestanoic acid (varanic acid). The varanic acid was biosynthetically prepared with rat liver microsomes and was probably the 24 alpha isomer. The patients efficiently (84 to 97%) converted both (R)- and (S)-THCA to cholic acid. There was no apparent significant difference in the ability of either (R)- or (S)-THCA to form cholic acid. Varanic acid was poorly converted (20 to 27%) to cholic acid in all three patients. From 49 to 75% of the administered 3H activity was recovered in the bile as other labeled products. The bulk (30 to 35%) of this 3H activity was identified by thin layer chromatography as varanic acid. The rate of conversion of (R)-THCA, (S)-THCA, and varanic acid was extremely rapid in all three patients with a t 1/2 of 35 to 74 min. The findings suggest that (a) the stereospecific configuration at C-25 of THCA has no significant effect on the efficiency of side chain oxidation to cholic acid; and (b) side chain cleavage pathways may exist which do not pass through varanic acid, or the oxidation of varanic acid in man is highly stereospecific with respect to the hydroxyl group at C-24. To prove the latter, it will be necessary to compare the metabolism of the 24 alpha and 24 beta isomers of varanic acid.

Germfree (GF) rats and antibiotic-treated rats with cannulated bile ducts (ABC) were given single oral doses of 2-(S-(N-[2H3]acetyl)cysteine)-N-isopropyl[1-14C]acetanilide. The GF rats excreted the dose in about equal quantities in the urine and feces; and the ABC rats excreted the dose in about equal quantities in the urine, feces, and bile. The mercapturate (60-75% of the dose in both cases) was isolated and the amount of exchange of N-acetyl deuterium to N-acetyl hydrogen was determined for all samples by mass spectrometry. The percentages of exchange were: ABC rats, bile 6.5%, urine 13%, feces 0.0%; GF rats, urine 15%, feces 4.7%. The remainder of the doses was present as the mercapturic acid sulfoxide. ABC rats dosed with 2-(S-[3,3-3H]cysteine)-N-isopropyl[1-14C]acetanilide (14C/3H = 0.50) excreted 42% of the dose in the urine and bile as the mercapturic acid that had a 14C/3H ratio the same as the original cysteine conjugate. The results of these studies show that a mercapturic acid and a cysteine conjugate can be absorbed from the gastrointestinal tract and resecreted in the bile or excreted by the kidney without having undergone metabolism other than acetylation of the cysteine nitrogen atom and oxidation of the sulfur to a sulfoxide. ABC rats were also dosed with 2-methylthio-N-isopropyl[1-14C]acetanilide, a suspected metabolic intermediate in the metabolism of propachlor (2-chloro-N-isopropylacetanilide). The dose was excreted in about equal quantities in the bile (48%) and urine (46%) as metabolites with a methylsulfonyl group in the 2-position. All are known metabolites of propachlor in conventional rats.

Rabbit antibodies raised against the major isozymes of cytochrome P-450 isolated from hepatic microsomes of beta-naphthoflavone- (BNF) and phenobarbital-treated rats (cytochrome P-450 BNF-B2 and cytochrome P-450 PB-B2, respectively) and against rat liver NADPH-cytochrome P-450 reductase were used to localize these enzymes immunohistochemically in the rat ventral prostate. Using the unlabeled antibody peroxidase-antiperoxidase technique, NADPH-cytochrome P-450 reductase was detected exclusively in the epithelial cells of the gland to the same magnitude in untreated, phenobarbital-, and BNF-treated rats. Cytochrome P-450 BNF-B2-like immunoreactivity was exclusively present in the glandular epithelium in BNF-treated rats, whereas staining could not be visualized in untreated or in phenobarbital-treated rats. The staining for NADPH-cytochrome P-450 reductase was more uniformly distributed within the epithelium than was the cytochrome P-450 BNF-B2-like immunoreactivity. Cytochrome P-450 PB-B2-like immunoreactivity was not found, regardless of animal pretreatment. These findings support our previous results (Haaparanta, T., Halpert, J., Glaumann, H., and Gustafsson, J-A., Cancer Res. 43: 5131-5137, 1983) demonstrating the presence of constitutive NADPH-cytochrome P-450 reductase in the prostate and that an isozyme of cytochrome P-450 is highly inducible by BNF in this gland. The significance of these findings are discussed in view of the essentially unknown etiology of human prostatic cancer.

Male rats were exposed to severe 14 day immobilization stress. Body weight, body temperature, food and water intake, behavioral parameters, and serum corticosterone levels were measured during and after the stress period. On the 7th day after cessation of stress the experimental animals together with the control rats were taken to immunocytochemical analysis involving morphometry and microdensitometry of tyrosine hydroxylase (TH), 5-hydroxytryptamine (5-HT), various neuropeptide, and glucocorticoid receptor (GR) immunoreactivities (IRs) in a large number of regions of the central nervous system. In addition, adrenocorticotropic hormone (ACTH) IR was analyzed in the pituitary gland. Seven days following cessation of the chronic stress food intake, total locomotion and forward locomotion had been restored to normal. Serum corticosterone levels appeared to remain increased even 6 days following cessation of the chronic immobilization stress, probably caused by increased release of ACTH. Paraventricular corticotropin releasing hormone (CRF) IR was negatively correlated with the pituitary ACTH IR, indicating that the increase in ACTH release was produced by an increased release of CRF from the hypothalamus. The major immunocytochemical change observed 7 days after cessation of stress was a disappearance of 5-HT IR in the 5-HT cell groups B 1, B 2, B 3, and B 7. 5-HT IR in nerve terminals was only affected in the dorsal horn, where 5-HT IR was increased in the substantia gelatinosa. GR IR was found to be significantly increaed in monoaminergic cell groups: serotoninergic B 7, dopaminergic A 12, and noradrenergic A 1, A 2, and A 6. A trend for a reduction of TH IR was observed in nigral DA cells associated with significant reductions in TH IR in striatal DA nerve terminals. Finally, increases in 5-HT and substance P (SP) IR were found in the nerve terminals of the substantia gelatinosa of the cervical spinal cord in the stress group. In the present experimental model evidence has been obtained for a maintained activation of the hypothalamic-pituitary-adrenal axis as evaluated 7 days after cessation of severe chronic immobilization stress. The reduction of 5-HT IR in various 5-HT cell groups indicates a reduction of 5-HT synthesis, which may also be associated with reduced 5-HT release from the nerve terminals, since no depletion was observed in terminal regions and in one case an increase in 5-HT IR was noted (substantia gelatinosa). The increase in GR IR, demonstrated in the NA and 5-HT cell groups in the presence of a maintained hypersecretion of corticosterone may represent signs of an upregulation of GR synthesis and/or increased translocation, which take place in the presence of maintained hypersecretion of corticosterone. Thus, 5-HT and NA neurons may respond more effectively to circulating glucocorticoids after severe chronic stress. In this way glucocorticoids may protect against stress-induced exhaustion of neurons leading to impairment of transmission. Studies on TH IR suggest a deficit in the DA transmission line of the nigrostriatal DA neurons, but of no other CA neurons studied. Such effects may contribute to behavioral suppression. Finally, the stress-induced increases in 5-HT and SP IR in the substantia gelatinosa may in part underlie the phenomenon of stress-induced analgesia.

Hormonal therapy is the dominating form of treatment for prostatic carcinoma. The majority of cases (80%) are well controlled for varying times with this regimen. However, thus far there have been no adequate methods to predict in which cases hormonal therapy is of less benefit. Measurement of cancer tissue content of intracellular hormone receptors constitutes progress toward a more individualized therapy in prostatic carcinoma. In this study biopsies from 16 cancer patients were taken before therapy was given, and the specimens were analyzed with regard to content of specific methyltrienolone-binding sites. A correlation has been made between receptor content and clinical response to hormonal therapy in each case. Twelve specimens contained measurable amounts of steroid receptors. Of these, one patient died during irradiation therapy before onset of hormonal treatment. However, of the remaining 11 patients, 9 responded well to hormones (9/11 approximately 82%). The two receptor-positive nonresponders had the lowest measurable receptor levels in the series. Four specimens contained no detectable amounts of receptors. Three of these patients showed no response to therapy (3/4 = 75%) but one was "false negative." Our data indicate that steroid receptor analysis may become a valuable diagnostic tool in individualizing the therapy for prostatic cancer.

Treatment with beta-naphthoflavone (BNF) was found to induce 7-ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities about 500-fold in the microsomal fraction of the rat ventral prostate but had no effect on aminopyrine N-demethylase or reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase activities. Phenobarbital (PB) treatment did not alter any of these enzyme activities. Antibodies raised in rabbits against rat liver cytochrome P-450 reductase (P-450 reductase) and against P-450 BNF-B2 and P-450 PB-B2, the major forms of P-450 isolated from liver microsomes of BNF- and PB-treated rats, respectively, were used to characterize the P-450-dependent monooxygenase system in the rat ventral prostate. Anti-P-450 reductase immunoglobulin G inhibited reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase activity in prostatic microsomes, and anti-P-450 BNF-B2 but not anti-P-450 PB-B2 immunoglobulin G inhibited the BNF-induced prostatic microsomal 7-ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities. A highly sensitive immunoblotting method was used to quantitate P-450 BNF-B2, P-450 PB-B2, and P-450 reductase in prostatic microsomes. Using this technique, prostatic P-450 reductase with a molecular weight corresponding to that of purified liver P-450 reductase was detected at a level of 0.02 nmol/mg of microsomal protein. In the liver, the same enzyme amounts to 0.2 nmol/mg of microsomal protein. P-450 BNF-B2 was not detected in prostatic microsomes from control or PB-treated rats, whereas a protein band with a molecular weight corresponding to that of purified liver P-450 BNF-B2 was found in prostatic microsomes from BNF-treated rats at a level of 0.05 nmol P-450 per mg microsomal protein. P-450 PB-B2 was not detected in prostatic microsomes from either control, PB-treated, or BNF-treated animals.

Gel permeation chromatography, density gradient centrifugation and affinity cross-linking have been used for characterization of the structure of rat hepatic lactogen receptor.

Central 5-hydroxytryptamine (5-HT) neurons represent targets of action for antidepressant drugs, as for example is demonstrated by the presence of high densities of [3H]-imipramine binding sites in the nerve cell membranes of the central 5-HT neurons (see Langer and Briley, 1981). Quantitative receptor auto-radiography of [3H] -imipramine binding sites has indicated their presence in the nerve cell membrane of the 5-HT cell bodies, axons and nerve terminals (Fuxe et al., 1983a). The density of [3H]-imipramine binding sites seems particularly high in the 5-HT nerve cell group of the nucleus raphe dorsalis (group B7; Dahlstrom and Fuxe, 1964). In view of the fact that a high secretion of cortisol has been demonstrated in patients with a severe depression (see Carroll, 1984), it is also of particular interest that strong glucocorticoid receptor (GR) immunoreactivity (IR) has been demonstrated in the vast majority of the 5-HT nerve cells of the lower brain-stem of the male rat (Fuxe et ah, 1985a, b; Harfstrand et al., 1986a).

The present experiments on hypophysectomized and castrated male rats and on ovariectomized female rats give evidence that: 1. Estradiol-17β by direct action on the brain possibly mediated via steroid target cells within the preoptic and hypothalamic area can markedly reduce DA turnover in mesostriatal and mesolimbic DA systems. Estrogen may in this way regulate motor functions and mental activities such as mood. 2. Estradiol-17β produces its central inhibitory feedback action on LHRH secretion, at least in part, via direct action on the hypothalamus leading to an activation of the lateral tuberoinfundibular dopamine pathway which in turn via an axo-axonic influence inhibits the secretion of LHRH from the median eminence. 3. Estradiol-17β may, at least in part, influence food intake via increasing NA turnover within the paraventricular hypothalamic nucleus and the anterior periventricular hypothalamic region. 4. Estradiol-17β may, at least in part, influence emotional behaviours via reducing the NA turnover within the amygdaloid-entorhinal area. 5. Estradiol-17β (48 h, 10 μg/kg) can selectively reduce adrenaline turnover within the posterior hypothalamus indicating that also adrenergic mechanisms in brain can be sensitive, directly or indirectly, to the action of the estradiol-17β. It is suggested that following estradiol-17β treatment with or without progesterone the discrete changes in DA, NA and adrenaline turnover discovered can be produced via actions not only on estrogen target cells within the amygdaloid cortex, the preoptic area and the hypothalamus, but also via direct actions of some of the DA and NA cell bodies themselves, since they have been shown to accumulate estrogen (see Stumpf, 1979 ).

The effect of glucocorticoids on gene expression of rat class I alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) was investigated. A cDNA clone for the beta-subunit of human ADH (ADH2) was used to analyze class I ADH mRNA levels in rat hepatoma cells, which are known to contain a functional glucocorticoid receptor. RNA gel blot analysis of total cellular RNA isolated from these cells showed hybridization of the human ADH2 cDNA probe to a single approximately equal to 1500-base RNA species. Treatment of the cells with dexamethasone (0.1 nM to 1 microM) caused a dose-dependent increase in total cellular class I ADH mRNA levels by a factor of 2-4. Maximal levels were reached within 18-24 hr of treatment. This effect was reversible following withdrawal of dexamethasone. The glucocorticoid induction of class I ADH mRNA does not seem to require ongoing protein synthesis since treatment of the cells with cycloheximide did not affect the increase in class I ADH mRNA levels by dexamethasone. The human ADH2 gene contains both upstream and within the coding region sequence motifs that display homology with response elements of genes positively regulated by glucocorticoids. These data suggest a receptor-mediated transcriptional enhancement of the ADH2 gene as the mechanism of regulation. However, analysis of RNA decay in cells treated with actinomycin D indicates that the dexamethasone-induced increase in class I ADH mRNA might, at least in part, be due to enhanced ADH mRNA stability.

The affinity for the glucocorticoid receptor in rat skeletal muscle of some glucocorticoids with a new type of 16 alpha, 17 alpha-acetal substituent has been estimated and correlated to the glucocorticoid activities in three in vivo systems in rats. Budesonide (an approximately 1:1 mixture of the C(22) epimers of 11 beta, 21-dihydroxy-16 alpha, 17 alpha-[(22R,S)-propylmethylenedioxy]-pregna-1,4-diene-3,20-dione) and the isolated (22R)- and (22S)-epimers bound to the same binding site as the potent glucocorticoids dexamethasone (DEX) or triamcinolone 16 alpha, 17 alpha-acetonide (TA), but with even higher affinity than DEX or TA, despite the lack of a 9 alpha-fluoro atom in budesonide and its epimers. The (22R)-epimer was twice as active as the (22S)-epimer, 4 times more active than TA, and 14 times more active than DEX. The introduction of a 9 alpha-fluoro atom slightly decreased the binding affinity of the (22R)-epimer of budesonide, in contrast to the positive effect of 9 alpha-fluorination of, e.g., 16 alpha, 17 alpha-acetonides. The negative influence of 9 alpha-fluorination of the (22R)-epimer was partially reversed in the 6 alpha, 9 alpha-difluorinated (22R)-epimer. Nevertheless, the fluorinated compounds were more active than DEX and TA (8 and 11 times more active than DEX, and 2 and 3 times more active than TA, in case of the 9 alpha-fluoro- and 6 alpha, 9 alpha-difluoro-derivatives of the (22R)-epimer, respectively). Budesonide is metabolized mainly to 16 alpha-hydroxyprednisolone (11 beta, 16 alpha, 17 alpha, 21-tetrahydroxy-pregna-1,4-diene-3,20-dione) and 6 beta-hydroxy-budesonide. Both metabolites were very weak competitors for the ligand-binding sites on the receptor (3% and 6% of the affinity of DEX, respectively). The affinity for the receptor in vitro was closely correlated to the topical glucocorticoid activity in vivo for the 12 steroids compared (r = 0.98; R = 0.98), which supports the contention that in vitro tests for receptor affinity are useful when screening for agonists among steroids with the present type of structures. The results on receptor-ligand interaction are in accordance with X-ray crystallographic data available for some steroids.

During studies on the uptake and distribution of estramustine phosphate (Estracyt) in the rat, a major protein in the rat ventral prostate was found that binds estramustine, estromustine, and several other steroid nitrogen mustard complexes. This protein, called estramustine-binding protein, shares many physicochemical characteristics with alpha-protein, prostatic-binding protein, and prostatein, each reported to constitute a major steroid-binding protein in the rat ventral prostate. There is every indication that these four names designate one and the same protein, the binding properties of which favor the binding of lipophilic compounds, and which has shown an affinity for estramustine and closely structure-related compounds that is 100- to 1000-fold higher than for the natural steroids. Also, the human prostate has been shown to take up and bind estramustine and estromustine. However, the binding entity (or entities) is still under intense investigation.

To better understand the mode of action of Tadenan, a drug used in the treatment of benign prostatic hyperplasia, the effect of its active principle docosanol, IK.2, was investigated in rats. IK.2 had no effects on the weight and histologic appearance of the prostate in intact rats but increased the RNA/DNA quotient in the ventral prostate. The plasma concentrations of luteinizing hormone and testosterone were reduced. In orchiectomized animals IK.2 increased the weight of the prostate and the adrenals. In adrenalectomized, orchiectomized animals IK.2 did not increase prostatic weight but on the contrary caused a further weight reduction. IK.2 had a thymolytic effect in intact rats but not in adrenalectomized rats in which the thymus weight was increased. The results indicate that IK.2 increases adrenal steroid secretion. The supposedly higher concentration of adrenal androgens causes a stimulation of the prostate most easily discernible in orchiectomized animals. The further weight reduction of the ventral prostate in orchiectomized, adrenalectomized animals, and the increased thymus weight in adrenalectomized animals after IK.2 administration may suggest that IK.2 has effects other than the stimulatory effect on the adrenals.

Prostatic secretion protein (PSP) or estramustine-binding protein is a major protein in rat ventral prostate. The amount of PSP was measured per mg of cytosolic protein at different ages and after castration or administration of sex hormones. The amount of PSP is relatively low before puberty (25 microgram/mg of protein) but increases at about 28 days of age to about 670 microgram/mg of protein and then decreases to a constant level of about 300 to 400 microgram/mg of protein, which is stable until at least 9 months of age. Following castration, the amount of PSP decreased relatively slowly, but 6 days after castration less than 20% of the original amount of PSP was detected. Treatment with testosterone propionate (1 mg/day) for 2 weeks (starting 2 weeks after castration) restored precastration levels of PSP. It is concluded that PSP is an androgen-sensitive protein, and it is suggested that PSP should be considered as a probe for estimation of androgenic action on the prostate. PSP is similar to the so-called prostatic binding protein as well as to prostatein, and it is quite possible that the three proteins represent one and the same entity.

The present investigation was undertaken to evaluate whether treatment of gallstone patients with chenodeoxycholic acid is associated with changes of the hepatic metabolism of steroids. Altogether 37 patients with cholesterol gallstones undergoing cholecystectomy were included in the study. Nine of them had been treated with chenodeoxycholic acid (15 mg/kg/day) for about 8 weeks prior to operation. Two hydroxylations involved in cholic acid biosynthesis, 12 alpha-hydroxylation of 7 alpha-hydroxycholest-4-en-3-one and 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, and the metabolism of a physiological steroid hormone, androst-4-ene-3, 17-dione, were studied in the microsomal fraction of liver homogenates. The 12 alpha-hydroxylase was inhibited about 50%, which is in accordance with a regulatory function of this enzyme. The 25-hydroxylase and the metabolism of androst-4-ene,3, 17-dione were unaffected. It is concluded that chenodeoxycholic acid treatment is not associated with general influences on hepatic steroid metabolism.

Publisher Summary This chapter discusses the regulation of hepatic steroid metabolism by the hypothalamo-pituitary-liver axis. The pituitary gland is essential for the maintenance of the female type of steroid metabolism in the adult animal. The chapter compares the effects of continuous infusion of growth hormone (GH) with injections of the hormone at different intervals to investigate the effects of the secretory pattern of GH. hGH had to be used in this experiment because of low amounts of rGH available. Hypophysectomized female rats received 120 μg hGH/24h. The results seem to indicate that the secretory pattern of GH is of great importance for its effects on hepatic steroid metabolism. The present hypothesis is that the effects of androgens and estrogens with regard to hepatic steroid metabolism are mediated via their central regulation of the secretory pattern of the feminizing factor. This factor is probably identifiable with GH or a GH-related peptide.

Monoamines, GABA and acetylcholine are the best-known transmitters of the hypothalamus (Fuxe and Hokfelt, 1969; Fuxe and Jonsson, 1974: Bjorklund et al., 1973; Carlsson et al., 1962; Dahlstrom and Fuxe, 1964; Hokfelt et al., 1974b; Ungerstedt, 1971; Roberts, 1975; Roberts et al., 1975; Tappaz et al., 1976; Vogt, 1954; Snyder and Taylor, 1972; Brownstein et al., 1975b; Fuxe et al., 1976b; Hokfelt et al., 1978). Recently evidence has accumulated that hypothalamic hormones such as luteinising hormone releasing hormone (LH-RH), somatostatin, thyrotrophin releasing hormone (TRH) and other types of peptides such as substance P and enkephalin can also subserve a transmitter or a modulator role (changing the action of a transmitter) at synaptic junctions within the hypothalamus (Barry and Dubois, 1974; Brownstein et al., 1975a; Brownstein et al., 1974; Elde et al., 1976a; von Euler and Gaddum, 1931; Fuxe et al., 1976a; Fuxe et al., 1976b; Ganten et al., 1975; Giachetti et al., 1976; Guillemin, 1978; Guillemin et al., 1976; Elde et al., 1976b; Hokfelt et al., 1974a; Hokfelt et al., 1975b; Hokfelt et al., 1975c; Hokfelt et al., 1975e; Reichlin et al., 1976; Renaud et al., 1975; Said and Rosenberg, 1976 Vanderhaeghen et al., 1975; Zimmerman, 1976; Larsson et al., 1976; Fuxe et al., 1977d; Hokfelt et al., 1978). In the present article the distribution of the various monoamine-, GABA-, acetylcholine- and peptide-containing neurones within the hypothalamus will be described. The article will then describe how monoamine systems can control activity in hypothalamic hormone-containing pathways and in other types of peptide-containing pathways. Finally, how the various types of transmitters and/or modulators can influence activity in the catecholamine (CA) systems of the hypothalamus, will be discussed with particular reference to the dopamine (DA) systems in the median eminence.