Androgen receptor and 5 alpha-reductase activity in the ductuli efferentes and epididymis of adult rhesus macaques

We measured androgen receptors (AR) and 5 alpha-reductase activity (5 alpha RA) in the ductuli efferentes and epididymides from adult rhesus macaques. Tissue samples were either assayed biochemically for AR or stained immunocytochemically (ICC) with a monoclonal antibody against AR. To estimate 5 alpha RA, tissue microsomes were incubated with [1 alpha,2 alpha-3H]testosterone, and the [3H]dihydrotestosterone formed was quantified. We found significant regional differences in the levels of both 5 alpha RA and AR in the excurrent ducts. In general, both enzyme activity and AR levels were higher in the caput and corpus epididymis than in ductuli efferentes and cauda epididymis. With ICC, positive nuclear AR staining was detected in all epithelial cell types, whereas variable numbers of stromal cells were positively stained. Our data demonstrate that there are segmental differences in the concentrations of 5 alpha RA and AR in epididymis and suggest that there may be regional differences in the regulation of epididymal functions by androgen.     

国人睾丸、附睾和输精管的氧化还原酶的组织化学观察

本文收集了１９—３８岁国人正常男性新鲜睾丸、附睾和输精管１３例,进行了氧化还原酶组织化学染色、光镜定位及定性观察。结果表明：睾丸曲细精管和输出小管上皮的ＧＤＨ,ＮＡＤＨＤ,ＮＡＤＰＨＤ,ＳＤＨ,ＧＰＤＨ,ＩＣＤＨ,ＭＤＨ,ＬＤＨ和Ｇ－６－ＰＤＨ９种酶;睾丸间质细胞和附睾管上皮的ＮＡＤＨＤ,ＮＡＤＰＨＤ,ＳＤＨ,ＩＣＤＨ,ＭＤＨ,ＧＤＨ,ＬＤＨ和Ｇ－６－ＰＤＨ８种酶;输精管的ＮＡＤＨＤ,ＮＡＤＰＨＤ,ＩＣＤＨ和ＧＤＨ４种酶的酶活性呈强阳性或极强阳性。提示输出小管和头部附睾管含有的多种氧化还原酶对精子功能成熟有极重要作用。     

Subcellular Localization and Characterization of Vasopressin Binding Sites in the Ventral Septal Area, Lateral Septum, and Hippocampus of the Rat Brain

[Arg8]-Vasopressin (AVP) has been shown to exert characteristic central physiological actions in the ventral septal area of the rat brain. This study reports the characterization of receptors for AVP in synaptic plasma membranes prepared from the ventral septal area, the lateral septum, and the hippocampus. Binding of [3H]AVP was temperature and time dependent, linearly related to protein concentration, saturable, and specific. Scatchard plot analysis suggested the presence of a population of binding sites in the three brain areas with dissociation constants and maximal binding capacities, respectively, of 1.06 +/- 0.39 nM and 24.0 +/- 7.01 fmol/mg of protein (mean +/- SEM; n = 3 for the ventral septal area, 0.92 +/- 0.13 nM and 47.0 +/- 4.96 fmol/mg of protein (n = 3) for the lateral septum, and 0.91 +/- 0.14 nM and 25 +/- 5.02 fmol/mg of protein (n = 3) for the hippocampus. In all three brain regions, the rank order of potencies of several vasopressin analogs, unrelated peptides, and other compounds for competitive displacement of ligand indicated a receptor with properties resembling those of the V1-like receptor for AVP. These data document the presence of a high-affinity, V1-like vasopressin receptor in the rat ventral septal area for which the pharmacological properties are similar to those previously reported in physiological studies.     

Estrogen and androgen receptors in the liver of cynomolgus monkeys (Macaca fascicularis)

Estrogen receptors (ER) and androgen receptors (AR) were evaluated in the hepatic cytosol from cynomolgus macaques to determine if there were differences associated with gender and endogenous hormone secretion. Saturable, high affinity binding (Kd = 0.2-0.8 nM) was demonstrated for both ER and AR from either male or female monkeys. Displacement of tritiated estradiol from the ER was estrogen specific (including ethinyl estradiol). Both androgens and the synthetic progestins (levonorgestrel and norethindrone) displaced tritiated mibolerone from the AR. Both 8S and 4S molecular forms of ER and AR were demonstrated on 5-20% sucrose density gradients. The ER levels were higher in females in the follicular phase of the menstrual cycle (40.5 +/- 1.9 fmol/mg protein) than levels in males (26.4 +/- 4.8 fmol/mg protein; P less than 0.01) or levels in luteal phase females (31.8 +/- 2.4 fmol/mg protein; P less than 0.05). AR levels were not different between females during different phases of the menstrual cycle (65.8 +/- 4.6 and 69.5 +/- 4.3 fmol/mg protein, follicular and luteal, respectively), but there was a tendency (P less than 0.10) for the levels in males (54.4 +/- 6.6 fmol/mg protein) to be lower than female levels. The demonstration of saturable, high affinity binding of androgens and estrogens in liver tissue of these primates, along with differences associated with gender and the stage of the menstrual cycle, suggests that hepatic receptors are functional and may play an important role in hepatic protein secretion.     

Estradiol binding components in intestinal mucosa of female rats.

The presence of estrogen binding components (EBC) in intestinal mucosa of female rats was investigated by competitive-binding assay using radiolabelled and nonlabelled estradiol 17 beta (E2). EBC were found exclusively in the nuclear fraction and were absent from the cytosolic and from the microsomal fractions. Two types of nuclear EBC with different binding characteristics and capacities were found: Kd1 = 4.8 +/- 0.8 nM, n1 = 18.4 +/- 4.2 fmol/mg protein (n1 = 83.4 +/- 12.5 fmol/mg DNA) and Kd2 = 31.1 +/- 6.8 nM, n2 = 91.1 +/- 18.5 fmol/mg protein (412.7 +/- 80.0 fmol/mg DNA). Type 1 component showed slightly greater affinity for estrogens as compared to progesterone and dexamethasone whereas type 2 component bound other competitors with even greater affinity than E2.     

Appendix epididymidis and aberrant ductules of the bull: light-microscopic and ultrastructural study

The appendix epididymidis and aberrant ductules possessed similar morphological characteristics. The epithelium was 31 +/- 3 microns in height and consisted primarily of ciliated and nonciliated cells, although a few lymphocytes were also present. The ultrastructure of major cell types showed most cell organelles in their cytoplasm. However, these organelles were poorly developed, suggesting that neither cell type performed either a secretory or an absorptive function. Although the vestigial organs and ductuli efferentes were similar in epithelial height and epithelial cell types, there were important morphological differences that were reliably used to differentiate between the two. First, the luminal diameter was significantly smaller in the vestigial organs (60 +/- 12 vs. 146 +/- 44 microns in the ductuli efferentes). Second, the nonciliated cells of the vestigial organs, unlike those of the ductuli efferentes, lacked both dense granules and vacuoles in the cytoplasm. Finally, the tubular cross-sections of the vestigial organs were closely packed and were located at the tip of the caput epididymidis in the case of the appendix epididymidis, and between the lobules of the ductuli efferentes in the case of the aberrant ductules.     

Identification of angiotensin II receptor and determination of its subtypes in rabbit and guinea pig fetal tissue]

Membrane angiotensin II receptors were measured in trophoblastic tissues using a 2-step procedure. The first step consisted of the relative measurement performed at a fixed 125I[Sar1 Ile8]AII concentration of 0.15 nM in order to determine which tissues had a sufficient number of binding sites for studying the competition curves. The second consisted of determining the maximal binding (Bmax) and the dissociation constant (Kd) for [Sar1 Ile8] AII and the receptor subtypes in these tissues. The relative binding measurement revealed a significant number of occupied sites in rabbit fetal placenta and chorion (159 +/- 17 and 51 +/- 10 fmol/mg proteins) and in guinea pig chorion (132 +/- 12). The mean values of the other trophoblastic tissues were 3-10-fold lower in the 2 species. The competition curves obtained from tissues with high angiotensin II binding receptors showed the predominance of the AT2 subtype in rabbit fetal placenta (AT1/AT2 = 25/75) and of the AT1 receptor in guinea pig chorion (97/3) and in rabbit chorion (90/10). The [SAR1 Ile8] AII affinity (Kd) obtained from Scatchard plot analysis was 1.2 +/- 0.2 nM (n = 5) in fetal placenta and 1.2 (n = 1) in rabbit chorion and 0.5 +/- 0.1 (n = 3) in guinea pig chorion. In these tissues, the respective Bmax values were 1,281 +/- 115 (n = 5), 263 (n = 1) and 1,188 +/- 134 fmol/mg proteins (n = 3). These findings indicate that rabbit fetal placenta and chorion and guinea pig chorion are the most important sites of action for the renin-angiotensin system present in trophoblastic tissues.     

Opposite changes in imidazoline I2 receptors and alpha2-adrenoceptors density in rat frontal cortex after induced gliosis

Opposite age-dependent changes in alpha2-adrenoceptor and imidazoline I2 receptor (I2-IRs) density have been related to brain gliosis development with aging. To check this hypothesis we applied in rats a model of reactive gliosis induced by heat. The specific binding of [3H]idazoxan (0.5-20 nM) in the presence of (-)adrenaline (5 x 10(-6) M) to membranes from rat brain cortex showed that the density of I(2)-IRs was significantly higher in membranes of injured cortex (Bmax=60+/-6 fmol/mg protein; n=9) than in control (Bmax=38+/-3 fmol/mg protein; n=9; p=0.0053). Conversely, the density of alpha2-adrenoceptors, measured by [3H]clonidine (0.25-16 nM), in the injured cortex (Bmax=75+/-4 fmol/mg protein; n=9) was significantly lower than in sham membranes (Bmax=103+/-7 fmol/mg protein; n=9; p=0.0035). No significant differences in receptor's affinity were observed between both groups. These results support the hypothesis that gliosis induces opposite changes in alpha2-adrenoceptor and I2-IR density.     

Androgen receptors in brain and pituitary of female rats: cyclic changes and comparisons with the male

The in vitro binding of a synthetic androgen, methyltrienolone ([3H]-R1881), to brain and pituitary (PIT) cytosol and nuclear extracts was determined in male and female rats. Purified cytosol was prepared from PIT or hypothalamic-preoptic area-amygdala (HPA) and incubated in the presence of 0.1 to 10 nM [3H]-R1881. Scatchard analysis revealed the presence of a single, saturable, high-affinity binding site in PIT cytosol with a dissociation constant (Kd) of 0.42 X 10(-10) M in females and 0.95 X 10(-10) M in intact males. The Kd of HPA cytosol was much less in castrated males [0.47 +/- 0.05 (SEM) X 10(-10)M, n = 7] and females (0.63 +/- 0.1 X 10(-10) M, n = 4) than in intact males (5.8 +/- 1.1 X 10(-10) M, n = 8). Treatment of castrated males with dihydrotestosterone (DHT) for 24 h (250 micrograms/100 g of body weight) increased the Kd of HPA cytosol only slightly (1.6 X 10(-10) M, mean of two replicates). Scatchard analysis of salt-extracted nuclear androgen receptor (ARn) showed a single, high-affinity binding site with similar Kd values in PIT and HPA of intact and castrated, DHT-treated male rats (PIT Kd = 7.3 X 10(-10) M, 9.3 X 10(-10) M; HPA Kd = 1.5 X 10(-9) M, 1.3 X 10(-9) M, respectively). Competition studies involving a range of several radioinert steroids revealed that the binding of [3H]-R1881 to cytosol (ARc) and nuclear extract was specific for androgen receptor when triamcinolone acetonide (10 microM) was added. The ARc and ARn levels were quantified in PIT, preoptic area (POA), hypothalamus (HT), amygdala, hippocampus, and cortex by single point estimation. Significantly (p less than 0.01) greater amounts of ARc were detected in PIT of ovariectomized females (32.7 +/- 2.9 fmol/mg of protein) than in that of orchidectomized males (22.33 +/- 1.6 fmol/mg of protein). The highest levels in the brain were seen in HT and POA. Pituitary ARc in females varied throughout the estrous cycle. Significantly (p less than 0.01) greater amounts were detected on estrus (45.8 +/- 2.2 fmol/mg of protein) and proestrus (39.0 +/- 1.9 fmol/mg of protein) than on diestrus (29.2 +/- 1.5 fmol/mg of protein). These data confirm the existence of specific receptors for androgen in male and female brain and PIT, and suggest an important role for androgen in the control of PIT hormone secretion in the female.     

Comparison of [125I]Iodolysergic Acid Diethylamide Binding in Human Frontal Cortex and Platelet Tissue

The human platelet contains a functional 5-hydroxytryptamine (5-HT) receptor that appears to resemble the 5-HT2 subtype. In this study, we have used the iodinated derivative [125I]iodolysergic acid diethylamide ([125I]iodoLSD) in an attempt to label 5-HT receptors in human platelet and frontal cortex membranes under identical assay conditions to compare the sites labelled in these two tissues. In human frontal cortex, [125I]iodoLSD labelled a single high-affinity site (KD = 0.35 +/- 0.02 nM). Displacement of specific [125I]iodoLSD binding indicated a typical 5-HT2 receptor inhibition profile, which demonstrated a significant linear correlation (r = 0.97, p less than 0.001, n = 17) with that observed using [3H]ketanserin. However, [125I]iodoLSD (Bmax = 136 +/- 7 fmol/mg of protein) labelled significantly fewer sites than [3H]ketanserin (Bmax = 258 +/- 19 fmol/mg of protein) (p less than 0.001, n = 6). In human platelet membranes, [125I]iodoLSD labelled a single site with affinity (KD = 0.37 +/- 0.03 nM) similar to that in frontal cortex. The inhibition profile in the platelet showed significant correlation with that in frontal cortex (r = 0.96, p less than 0.001, n = 16). We conclude that the site labelled by [125I]iodoLSD in human platelet membranes is biochemically similar to that in frontal cortex and most closely resembles the 5-HT2 receptor subtype, although the discrepancy in binding capacities of [125I]iodoLSD and [3H]ketanserin raises a question about the absolute nature of this receptor.     

Gallium nitrate suppresses the production of nitric oxide and liver damage in a murine model of LPS-induced septic shock.

The efficacy of gallium (Ga) nitrate was examined in a murine model of sepsis. Male Balb/c mice (6-8 weeks) were randomized into 3 groups: 1) vehicle-treated controls 2) mice with sepsis induced by treatment with 0.3 mg i.v. of Propionibacterium acnes followed one week later by 0.01 microg lipopolysaccharide (LPS) and 10 mg of D-galactosamine (GalN) 3) mice with sepsis injected with 45 mg/kg s.c. of gallium nitrate (calculated as elemental Ga) 24 hours prior to LPS/GalN. Two hours after LPS/GalN or vehicle, plasma concentrations of tumor necrosis factor (TNF-alpha) in groups 1, 2 and 3 were 54+/-31 (n=6), 21,390+/-5139 (n=4), and 21,909+/-943 (n=5) pg/ml, respectively. After 6 hours, plasma concentrations of gamma interferon (IFN-gamma) were <10 (n=8), 4771+/-1078 (n=6), and 1622+/-531 (n=15) pg/ml, respectively, and of nitrate/nitrite (products of nitric oxide) were 64+/-8 (n=7), 146+/-18 (n=8), and 57+/-8 (n=15) microM. At 18 hours, serum chemistries were; SGOT 171+/-46 (n=13), 10,986+/-3062 (n=7), and 1078+/-549 (n=8) IU/L; SGPT 165+/-59, 17,214+/-4340, and 2088+/-1097 IU/L; and total bilirubin 0.2+/-0.0, 0.9+/-0.4, and 0.2+/-0.0 mg/dl for groups 1, 2, and 3 respectively. Blinded histologic evaluation of livers at 18 hours revealed inflammatory infiltrate scores (x [range], 0=none, 1=minimal, 2=mild, 3=moderate, and 4=severe) of 0.1 [0-1] (n=8), 3.0 [2-4] (n=15), and 2.0 [0-3] (n=10), and necrosis scores of 0.0, 2.8 [0-4], and 0.9 [0-4]. Although Ga did not affect production of TNF-alpha, it ameliorated hepatocellular injury and protected against necrosis. Based on this model of sepsis, Ga may have a role in treating the human disease.     

Interactions of radiolabelled ligands with specific receptors: an analysis

The binding and displacement of beta-adrenoceptor blockers, [3H]propranolol ([3H]PRP) and [3H]dihydroalprenolol ([3H]DHA), were studied on isolated rat erythrocytes, their membranes and ghosts; the binding of [3H]DHA and a M-cholinoceptor blocker, [3H]quinuclidinylbenzylate ([3H]QNB), on cerebral cortex membranes. In all experiments, ligand-receptor interactions conformed to a model of two pools of receptors in the same effector system and the binding of two ligand molecules to the receptor. The results were similar for the displacement of [3H]PRP, [3H]DHA and [3H]QNB with propranolol, dihydroalprenolol and quinuclidinyl-benzylate, respectively. The parameters of [3H]PRP to beta-adrenoceptor binding for intact erythrocytes were: Kd1 = 0.74+/-0.07 nM, Kd2 = 14.40+/-0.41 nM, B1 = 24+/-2 unit/cell, B2 = 263+/-5 unit/cell; for ghosts, Kd1 = 0.70+/-0.17 nM, Kd2 = 19.59+/-2.59 nM, B1 = 9+/-1 fmol/mg protein, B2 = 39+/-4 fmol/mg protein. Receptor affinities were similar in erythrocytes and ghosts; on the ghost membrane, the number of receptors was considerably lower (B1 = 2 unit/cell, B2 = 6 unit/cell). The parameters of [3H]QNB to M-cholinoceptor binding of the cerebral cortex membrane were the following: Kd1 = 0.43 nM, Kd2 = 2.83 nM, B1 = 712 fmol/mg, B2 = 677 fmol/mg.     

(3)H]Adenosine uptake in brainstem membranes of CD-1 mice lacking the adenosine A(2a) receptor

Previous studies in our laboratory have demonstrated a decrease in [(3)H]nitrobenzylthioinosine binding sites in the brainstem of adenosine A(2a) receptor knockout mice, particularly in the brain nuclei involved in central control of cardiovascular function [Brain Research 877 (2000) 160]. The present study aimed to correlate this decrease, shown using autoradiography, with a functional change using a previously described method of [(3)H]adenosine uptake in a membrane preparation from the brainstem of wildtype CD - 1 and homozygous mutant mice lacking the adenosine A(2a) receptor. A statistically significant decrease was shown in the mean V(MAX) value obtained from homozygous mutant preparations (4.7 +/- 1.3 fmol/mg protein/20 s, P < 0.05, n = 4) compared to that obtained from wildtype controls (51.6 +/- 4.2 fmol/mg protein/20 s, n = 4). Competition studies using nucleoside uptake inhibitors showed a statistically significant increase in the log IC(50) values for dipyridamole (Wildtype: -4.3 +/- 0.2, Homozygous mutant: -8.3 +/- 0.4, n=5, P < 0.05) and dilazep (Wildtype: -3.9 +/- 0.8, Homozygous mutant: -8.3 +/- 0.8, n=5, P < 0.05) in the preparations using homozygous mutant tissue. The present study, in conjunction with the results of previous studies [Brain Research 877 (2000) 160], indicates that components of purinergic neurotransmission system have apparently adjusted in compensation for the lack of the A(2a) receptor.     

Evaluation of prostaglandin-receptors in human and rat liver: interspecies differences at the prostaglandin receptor-level

The properties of PGE1-, PGE2- and iloprost (stable PGI2-analogue)-binding sites on normal human and rat liver surface cell membranes were investigated. The specific binding of [3H]PGE1 to human (rat) liver surface cell membranes could be displaced most effectively by unlabeled PGE1 (IC-50:2.5 +/- 1.7, (6.1 +/- 2.1) microM) and the specific binding of [3H]PGE2 by unlabeled PGE2 (IC-50: 1.9 +/- 0.9 (2.0 +/- 0.8) microM. The Scatchard analysis on [3H]PGE1- as well as on [3H]iloprost-binding was curvilinear whereas it was clearly linear on [3H]PGE2-binding in both the species. The high-affinity [3H]PGE1-sites showed a Bmax of 36.3 +/- 5.2 (21.3 +/- 4.3) fmol/mg protein and a Kd of 2.1 +/- 1.8 (1.9 +/- 0.7) nM, the low-affinity [3H]PGE1-sites a Bmax of 93.4 +/- 18.2 (86.1 +/- 13.2) fmol/mg protein and a Kd of 10.5 +/- 2.9 (15.1 +/- 3.2) nM. The high-affinity [3H]iloprost-sites exhibited a Bmax of 71.4 +/- 13.9 (35.9 +/- 8.2) fmol/mg protein and a Kd of 4.1 +/- 1.2 (1.7 +/- 1.8) nM, the low-affinity [3H]iloprost-sites a Bmax of 217.3 +/- 42.1 (142.9 +/- 17.8) fmol/mg protein and a Kd of 16.3 +/- 4.9 (9.2 +/- 7.2) nM. The [3H]PGE2-sites showed a Bmax of 135.4 +/- 51.9 (38.8 +/- 7.4) fmol/mg protein and a Kd of 16.2 +/- 3.2 (2.5 +/- 1.2) nM. It is assumed that prostaglandins of the E-series are promising substances in the regulation of human and rat liver function since liver cells are able to bind reasonable amounts of these substances in a high affinity manner. However, interspecies differences in the affinity of the prostaglandins to their receptor-sites make it strange to assume that the same biological findings claimed several times for the rat liver are relevant for human too.     

Molecular biology of androgen action: testosterone/dihydrotestosterone receptor and androgen 5 alpha-reductase in the human foreskin

Receptors for testosterone (T) and dihydrotestosterone (DHT) as well as the tissue specific androgen-5 alpha-reductase (A5R) were studied in the foreskin of 52 healthy boys (ages 1-14 years), in order to gain molecular endocrinological data and information about the ontogeny and cytogeny, respectively, of androgen specific target organs. Enzyme determinations were carried out in tissue homogenates by an enzyme kinetic method for the evaluation of Km- and Vmax-values. Reactions velocities were calculated from the turn over rates of T to DHT, 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3,beta,17 beta-diol. The precursor (T) was used in increasing concentrations, ranging from 8 to 208 nM. Separation of reaction products was done by thin-layer chromatography and verification of specific radioactivity of metabolites by means of radio gas chromatography on capillary columns. Results of the enzyme analyses: Km = 94.9 +/- 3.5 [nM], and Vmax = 15.8 +/- 1.9 [pmol/mg.h]. Receptors were examined in the cytosolic and nuclear fractions of the tissue specimens. Saturation analyses and calculation of binding data led to specific receptors for T and DHT in the cytosolic (T: Kd = 1.56 +/- 0.12 [nM], Nmax = 122.4 +/- 11.6 [fmol/mg]; DHT: Kd = 1.9 +/- 0.1 [nM], Nmax = 493.3 +/- 77.8 [fmol/mg]) and the nuclear fractions (T: Kd = 1.43 +/- 0.13 [nM], Nmax = 28.7 +/- 3.5 [fmol/mg]; DHT: Kd = 1.37 [nM], Nmax = 196.9 +/- 22.5 [fmol/mg]), Kd-values proved to be quite homogenous (coeff. var. = 0.15-0.21), whereas maximum specific receptor binding activities (Nmax) showed age dependent fluctuations (coeff. var. = 0.35-0.45). Binding capacities of both T- and DHT-receptor, respectively, in cytosolic and nuclear fractions showed peak values in the age group 10-11 years and additional "spikes" of binding rates at age 4-5 years. It is noteworthy that Vmax-values also reached maximum levels in the latter age group. Concerning the ontogeny of the androgen receptor a change of binding properties from the cytosol to the nuclear fraction was observed with the onset of puberty. A comparison of enzyme- and receptor data lead to the theory, that subcellular hormone actions depend on interrelational regulatory mechanisms between androgen receptors (T as well as DHT) and specific enzyme systems (A5R).     

Uterine oxytocin receptors in an Australian marsupial, the brushtail possum, Trichosurus vulpecula

1. Oxytocin receptors in the uterus of the brushtail possum (T. vulpecula) were characterized by radioreceptor assay and compared with those of the sheep and rat uterus. 2. A single oxytocin binding site was found with an affinity (Kd) and receptor concentration (Ro) of 3.0 +/- 0.8 nmol/l and 200 +/- 60 fmol/mg protein, respectively (SEM; n = 5). The receptor was stable at -20 degrees C; divalent ions were required for optimum binding. 3. Competitive displacement curves with related peptides showed the following order of specificity: vasotocin greater than oxytocin greater than mesotocin = arginine-vasopressin = [Thr4, Gly7]-oxytocin greater than lysine-vasopressin = isotocin much greater than [d(CH2)5, D-Phe2, Ile4, Ala9-NH2]-AVP. 4. It was concluded that oxytocin receptors in the possum have similar characteristics to those of placental mammals.     

19-hydroxyandrostenedione does not modulate [3H]aldosterone binding to human mononuclear leucocytes and rat renal cytosol

To verify the aldosterone amplifying action of 19-hydroxyandrostenedione (19-OH-AD), we investigated [3H]aldosterone and [3H]19-OH-AD binding to type I (mineralocorticoid) receptor in the renal cytosol of adrenalectomized and ovariectomized rat, and human mononuclear leucocytes (MNL). In the [3H]aldosterone binding study, the cytosol was incubated with [3H]aldosterone and 200-fold RU28362 (11 beta,17 beta-dihydroxy-6-methyl,17 alpha-(1-propynyl)-androsta-1,4,6- trien-3-one), a pure glucocorticoid, with or without 19-OH-AD. Scatchard plots of [3H]aldosterone binding to cytosol with 0.2 or 20 nM 19-OH-AD or without 19-OH-AD were linear. Dissociation constants (Kd) and maximum bindings (Bmax) without 19-OH-AD, and with 0.2 and 20 nM 19-OH-AD were: 0.71 +/- 0.03 nM and 23.0 +/- 3.4 fmol/mg protein (mean +/- SD, n = 3), 0.72 +/- 0.05 nM and 23.1 +/- 2.3 fmol/mg protein (n = 3), and 0.77 +/- 0.04 nM and 22.9 +/- 4.8 fmol/mg protein (n = 3), respectively. 19-OH-AD did not significantly change the Kd and Bmax of [3H]aldosterone binding. A high concentration of 19-OH-AD slightly displaced 0.2 or 5 nM [3H]aldosterone bound to cytosol. In human MNL, Scatchard plots of [3H]aldosterone binding with both 0.2 and 20 nM 19-OH-AD and without 19-OH-AD were linear. Kd and Bmax were, respectively, 1.00 nM and 780 sites/cell in the absence of 19-OH-AD, and 1.07 nM and 774 sites/cell in the presence of 0.2 nM 19-OH-AD. Without 19-OH-AD they were, respectively, 0.95 nM and 551 sites/cell, and 1.10 nM and 560 sites/cell with 20 nM 19-OH-AD. A high concentration of 19-OH-AD slightly displaced 0.2 or 5 nM of [3H]aldosterone bound to MNL. In both tissues, there was no obvious specific binding of [3H]19-OH-AD within the range of 1-60 nM. The above results suggest that the amplifying effect of 19-OH-AD on aldosterone mineralocorticoid action may not occur at the binding site of aldosterone to type I receptor, and that 19-OH-AD itself may not have any direct or indirect mineralocorticoid actions on the steroid receptor-mediated process in the rat kidney and human MNL.     

Subunit composition of the molybdate-stabilized "8-9 S" nontransformed estradiol receptor purified from calf uterus

The structure of the calf uterus nontransformed molybdate-stabilized estradiol receptor (ER) has been investigated using affinity labeling with tamoxifen aziridine and several monoclonal antibodies directed either against the steroid binding protein (Mr approximately 65,000) or against the heat shock protein of Mr approximately 90,000 (hsp 90). The purification was performed using affinity chromatography and a DEAE-Sephacel column. The [3H] estradiol-ER complex was obtained as a well-defined radioactive peak, the specific activity varying between 1,600 and 3,400 pmol/mg of protein. The purified ER sediments in glycerol gradients at 9.4 S +/- 0.2 (n = 5) and at 8.1 S +/- 0.2 (n = 15) in a 0.15 M KCl containing gradient ("8-9 S" ER). From a measured Stokes radius of 7.4 +/- 0.2 nm (n = 12), an Mr of approximately 300,000 has been calculated. Studies of the purified 8-9 S ER by glycerol gradient centrifugation and by "twin antibody" assay with the JS34/32 anti-ER monoclonal antibody suggest the presence of two binding subunits in the nontransformed molecular complex. Results of immunological analysis with polyclonal and several monoclonal antibodies against hsp 90 suggest the association of two molecules of this protein to the two steroid binding subunits. In high salt medium (0.4 M KCl), the purified ER sediments at 5.2 +/- 0.3 (n = 8), has a Stokes radius of 5.7 nm +/- 0.1 (n = 2) and the Mr is approximately 129,000, values expected for a homodimer consisting of two hormone-binding subunits (Mr approximately 65,000), a result confirmed by glycerol gradient centrifugation experiments, using the monoclonal antibody JS34/32. The relationship between the nontransformed 8-9 S ER and the transformed 5 S-ER forms are discussed, the simplest possibility being the release of the already formed homodimeric ER from 8-9 S ER during transformation.     

Histochemical evaluation of glycoconjugates in the male reproductive tract with lectin-horseradish peroxidase conjugates: II. Staining of ciliated cells, basal cells, flask cells, and clear cells in the mouse

Efferent reproductive ducts of male mice, including ductuli efferentes, epididymis, and vas deferens, were fixed and embedded in paraffin, and sections were stained with a battery of lectin-horseradish peroxidase conjugates to localize specific sugars or sugar sequences in glycoconjugates. Cilia and the apical surfaces of ciliated cells in the ductuli efferentes stained intensely with lectin specific for sialic acid and terminal alpha-N-acetyl-D-galactosamine. Flask cells and clear cells in the epididymis reacted positively and similarly with most lectins used, providing evidence that these cell types are related. In contrast, disparities in lectin staining suggest that flask cells and clear cells are a cell type distinct from principal cells. Basal cells were not present in the ductuli efferentes but formed a continuous layer in the epididymis and vas deferens. Basal cells contained oligosaccharides terminated by sialic acid and alpha-D-galactose and varying amounts of terminal beta-D-galactose and alpha-N-acetyl-D-galactosamine. Basal cells also stained variably with lectins specific for the core region of complex type N-glycosidic side chains. The basal cells varied structurally, having long spinous apical processes approaching or reaching the lumen in region I of the epididymis and being low cuboidal or squamoid and lacking apical processes in epididymal regions II-V and in the vas deferens. The contiguous nature of the basal cells and the presence of glycoconjugates bearing terminal alpha-galactosyl residues in all basal cells suggest a possible role for these cells in a regulatory influence on transepithelial movement of fluid and/or ions in the epididymis and vas deferens.