Ontogenetic changes in porcine adrenocortical adrenocorticotropic hormone receptors.

1. Binding of [125I]ACTH(1-38) analog to adrenal receptors was measured in fetal pigs (Sus domesticus) at 15-day intervals from midpregnancy (60 days) to near term (105 days; pregnancy length 114 days). 2. Binding was greatest at day 60 (0.42 +/- 0.03 fmol/200 micrograms protein or 0.50 +/- 0.08 fmol/50 micrograms DNA), and least at day 105 (0.13 +/- 0.03 fmol/200 micrograms protein or 0.16 +/- 0.04 fmol/50 micrograms DNA). Total adrenal binding was constant (0.61 +/- 0.02 fmol/paired adrenals). 3. Scatchard analyses at day 60 and day 105 showed comparable apparent affinities of ACTH receptors (Ka day 60 = 1.51 +/- 0.72 x 10(9) M-1 vs Ka day 105 = 1.94 +/- 0.78 x 10(9) M-1). 4. DNA per paired adrenals and membrane-associated protein increased 1.6-fold, providing a constant protein: DNA ratio. Concentrations of adrenal cortisol were constant from 60 to 90 days of gestation age but increased dramatically by day 105. 5. These data suggest that during 60-105 days of gestation age the number of ACTH receptors per cell is reduced.     

Evidence for oxytocin receptors in cultured bovine luteal cells.

Specific receptors for oxytocin (OT) on intact luteal cells are demonstrated. Cultured cells from bovine corpora lutea (CL) at different stages (Days 3-5, 8-12, and 15-18 of the estrous cycle) were examined for OT receptors by a radioreceptor assay using the 125I-labeled OT antagonist [d(CH2)5,Tyr(Me)2,Thr4,Tyr-NH2(9)] -vasotocin. Binding specificity was demonstrated in displacement studies with various related peptides. Scatchard analysis revealed the presence of a binding site with an association constant of Ka = 2.6 x 10(9) M-1 and a capacity of 5.9 fmol/micrograms DNA. Additionally, in 50% of the experiments (n = 6) two different binding sites were observed. The Ka of the high-affinity site was 2.6 x 10(10) M-1; its capacity was 0.73 fmol/micrograms DNA. The low-affinity site had an apparent Ka of 4.9 x 10(8) M-1 and a capacity of 8.8 fmol/micrograms DNA. Observation of one versus two binding sites related neither to the assay conditions nor to the state of the individual CL used for the cell culture and therefore appeared to reflect individual variation within the OT receptor population. Significant binding of OT was observed at all luteal stages. OT binding was maximal at the mid-luteal stage (Days 8-12). We conclude that a direct action of OT on the bovine CL is mediated by the OT receptor, supporting the hypothesis that luteal OT plays an important physiological role in the regulation of progesterone release and/or other luteal functions in a paracrine or autocrine fashion.     

Nuclear thyroxine and triiodothyronine binding in mononuclear cells in dependence of age

Nuclear binding of thyroxine (T4) and triiodothyronine (T3) in mononuclear blood cells was investigated in 12 young (age 16-30 years) healthy subjects (group A), in 12 middle-aged (age 31-60 years) healthy subjects (group B) and in 12 elderly (61-90 years) healthy subjects. Serum free T3 was depressed in group C as compared to the younger age groups, whereas serum free T4 and TSH did not differ between the groups. Maximal specific nuclear binding capacity for both T4 and T3 decreased with increasing age, T4 group A: 1.2 fmol T4/100 micrograms DNA, group B: 1.2 fmol T4/100 micrograms DNA, group C: 0.7 fmol T4/100 micrograms DNA; T3 group A: 1.7 fmol T3/100 micrograms DNA, group B: 1.0 fmol T3/100 micrograms DNA, group C: 0.9 fmol T3/100 micrograms DNA. The equilibrium association constant (Ka) for T4 increased with age, group A: Ka = 3.3 X 10(9) l/mol, group B: Ka = 3.2 X 10(9) l/mol, group C: Ka = 6.4 X 10(9) l/mol, whereas Ka for nuclear binding of T3 decreased with age group A: Ka = 3.9 X 10(9) l/mol, group B: Ka = 5.9 X 10(9) l/mol, group C: Ka = 1.8 X 10(9) l/mol. We conclude that, whereas the opposite variations of nuclear capacity and binding affinity for T4 tend to preserve the nuclear T4 concentration, the nuclear T3 concentration definitely decreases with age. The unaltered serum levels of TSH suggest that the decrease of both serum levels of free T3 and the nuclear T3 concentration might represent physiologically changes in old age.     

Effects of adrenaline pretreatment on in vitro binding of 125I-triiodothyronine to nuclear receptor, intracellular distribution of endogenous triiodothyronine and activities of alfa-glycerophosphate dehydrogenase and malic enzyme in rat liver

Especially coated adrenaline tablets (A) or placebo tablets (P) which release linearly the hormone were implanted in male Wistar rats. Six hours later animals were sacrificed and kinetic parameters of T3-125I binding to nuclear receptor, intracellular distribution of endogenous T3 and activities of alfa-GPD and ME were investigated. The association constant values (Ka) of nuclear receptor were increased after pretreatment with 7.5, 15 and 45 mg A tablets and were 1.07, 1.35 and 1.48 X 10(9) M-1 vs 0.85 X 10(8) M-1 value seen after P. The maximal binding capacity (MBC) values decreased after pretreatment with the same doses of A and were 0.044, 0.036 and 0.025 pmol T3/100 micrograms DNA vs. 0.065 pmol T3/100 micrograms DNA in P pretreated. Adrenaline pretreatment significantly increased the amount of endogenous T3 present in liver nuclei while the amount of T3 present in cytosol decreased. Activity of mitochondrial alfa-GPD was increased after 15 and 45 mg of A. Significant rise of activity of cytosol ME was seen only after pretreatment with 45 mg of A.     

High-affinity angiotensin receptors in rat adrenal medulla

Angiotensin II receptors have been quantitated in single rat adrenal medullas by incubation of tissue sections with 125I-[Sar1]-AII, autoradiography with exposure to 3H-sensitive Ultrofilm, computerized densitometry and comparison with 125I-labelled standards. Rat adrenal medulla contains a single class of high affinity AII receptors with a Ka of 0.84 +/- 0.02 X 10(9) M-1 and a Bmax of 3259 +/- 502 fmol/mg protein, one of the highest densities in AII receptors found in rat tissues. These observations provide evidence for a local site of action of AII in the release of adrenal medullary catecholamines.     

Increase in concentration of uterine oxytocin receptors and decrease in response to 13,14-dihydro-15-keto prostaglandin F2 alpha in ewes after withdrawal of exogenous progesterone.

Ovariectomized ewes were treated with progesterone and oestradiol to induce oestrus (day of expected oestrus = day 0) and with progesterone on days 1 to 12. The concentrations of endometrial oxytocin receptors and the 13,14-dihydro-15-keto prostaglandin F2 alpha (PGFM) response induced by oxytocin were measured on days 12, 14, 16 and 18 after the cessation of progesterone treatment on day 12, by a receptor binding assay and direct radioimmunoassay, respectively. During the period of treatment, the concentrations of plasma progesterone were high and remained above 2 ng ml-1 until day 13 when they dropped rapidly to less than 0.5 ng ml-1 by day 14. The concentrations of oxytocin receptors in endometrium of control ewes were high (820.7 +/- 91.7 (SEM) fmol mg-1 protein). Treatment with progesterone significantly (P < 0.01) reduced the concentrations of the receptors on days 12 and 14 (144.1 +/- 65.0 and 200.4 +/- 45.4 fmol mg-1 protein, respectively). The receptor concentrations then increased to relatively high values on day 16 (1021.4 +/- 216.6 fmol mg-1 protein) and remained high until day 18 (677.7 +/- 103.4 fmol mg-1 protein).(ABSTRACT TRUNCATED AT 250 WORDS)     

Thionamides and arsenite inhibit specific T3 binding to the hepatic nuclear receptor

Methimazole (MMI) and propylthiouracil (PTU) are widely used for the treatment of Graves' disease. However, no studies have been reported on the action of these drugs on binding of L-triiodothyronine (T3) to the nuclear receptor. T3 receptors of rat liver nuclei, prepared by differential centrifugation, were extracted with 0.4 M KCl and 5 mM dithiothreitol (DTT). In the assessment of T3 binding to the DTT-reduced receptor, the hepatic nuclear extract was chromatographed on Superose 6 to remove DTT and isolate proteins of relative mass approximately 50,000 (chromatographed nuclear receptors (CNRs)), prior to the addition of [125I]T3 of high specific activity (3300 microCi/micrograms; 1 Ci = 37 GBq). MMI or PTU at 2 mM reduced specific T3 binding to CNR by 84% and 85%, respectively. The inhibitory effects of these reagents and 2 mM sodium arsenite (which complexes dithiols) were additive. Scatchard analyses indicated that neither MMI nor PTU (at 2 mM) significantly altered the affinity constant (Ka) (from 2.41 x 10(9) to 1.74 x 10(9) M-1 for PTU and 1.79 x 10(9) M-1 for MMI), while they both decreased (p less than 0.02) maximal binding capacity (from 0.36 +/- 0.02 to 0.19 +/- 0.02 pmol/mg protein for MMI and 0.17 +/- 0.02 pmol/mg protein for PTU). Dose-response curves showed that 50% inhibition was attained at 0.6 mM PTU or 1.0 mM MMI with approximately 25% inhibition by both at 0.1 mM. Artefactual binding effects by MMI and PTU on [125I]T3 were excluded by chromatography experiments. Similar results were obtained using nuclear receptors prepared from livers of hyperthyroid rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of vasopressin administration and deficiency upon 3H-AVP binding sites in the CNS and periphery during development.

Arginine8-vasopressin (AVP, 40 micrograms/100 g b.wt., SC) was administered to male Long-Evans (LE) pups from day 1 to 7 of life and the pups were sacrificed on day 8 or 60. 3H-AVP binding was performed on membranes prepared from the liver, kidney, and septum. No significant changes were observed in the kidney or septum of animals 8 or 60 days old. However, the chronic AVP treatment did result in a significant increase in the density of 3H-AVP binding sites in the liver when compared to control day 8 pups (control 44 +/- 2 vs. AVP 56 +/- 3 fmol/mg protein), with no change in affinity. This effect was maintained into adulthood, as the day 60 AVP-treated LE rats also showed a significant increase in liver 3H-AVP binding sites compared to control (control 186 +/- 9 vs. AVP 239 +/- 14 fmol/mg protein), with no change in affinity. A comparison of 3H-AVP binding sites in 8-day-old LE, heterozygous Brattleboro (HET-BB), and homozygous Brattleboro rats (HOM-BB) was performed to assess the effect of complete (HOM-BB) and partial (HET-BB) VP deficiency on binding sites in the CNS and periphery. The liver again was the only tissue in which a change in 3H-AVP binding characteristics was noted. The HOM-BB rat (Bmax 144 +/- 6 fmol/mg protein) displayed a significant increase in AVP binding sites from the LE rat (Bmax 100 +/- 7 fmol/mg protein), while the 3H-AVP binding sites in the HET-BB rat liver (Bmax 69.8 +/- 9 fmol/mg protein) were significantly lower than LE rats. Thus hepatic AVP receptors appear most sensitive to the presence or absence of vasopressin during the early postnatal period.     

3,5,3'-triiodo-L-thyronine binding sites in nuclei of human trophoblastic cells

Nuclear binding sites of T3 in human trophoblastic cells were biochemically characterized. Nuclei were isolated by a combination procedure with mild homogenization of the freshly obtained trophoblastic tissue aged term gestation, centrifugations and Triton X-100 treatment. The isolated nuclei were incubated with various concentrations of 125I-T3 at 20 degrees C for 3 h. The total number of T3 binding sites per nucleus was approximately 650. The apparent association constant (Ka) was 6.0 X 10(9)M-1. Nuclear proteins extracted from purified nuclei with 0.4M KCl were able to bind T3 giving rise to nuclear thyroid hormone binding protein-T3 complexes and they were precipitated with bovine IgG, as a carrier protein, by 12.5% polyethylene glycol. Binding was maximum in 3 h incubation at 20 degrees C or in 18 h at 0 degrees C, while it dropped quickly at 37 degrees C. The binding characteristics were analyzed by Scatchard plots. In nuclear proteins obtained from 8 term placentae there was a single set of high affinity-low capacity T3 binding sites with Ka of 7.0 X 10(9)M-1. The capacity is about 62.7 fmol T3/mg DNA. The binding sites were found to be specific for L-T3, while L-T4 was about 100-fold less effective, rT3 ineffective, and D-T3 and D-T4 were roughly 1/8 and 1/5 as active as L-T3 and L-T4, respectively in displacing 125I-T3 from the binding sites. These data confirmed that human placenta is a target organ of thyroid hormones; trophoblastic cells contain T3 nuclear receptors which are biochemically similar to those isolated from liver, although the capacity is low.     

Effect of male accessory glands autoaggression on androgenic cytosolic and nuclear receptors of rat prostate.

The effect of immunization against male accessory gland (MAG) homogenates over androgenic cytosolic and nuclear receptors of rat prostate was studied. In the MAG-immunized rats the Bmax of cytosolic receptors was significantly increased (120.3 +/- 44.3 vs 47.7 +/- 24.9 fmol/mg protein, p less than 0.01, mean +/- SD). In contrast, the Bmax of nuclear receptors in the MAG-immunized rats showed no significant difference as regarded controls (kidney immunized rats) when expressed as fmol/100 micrograms DNA (196.1 +/- 84.8 vs 148.3 +/- 88.9) but it show to slight differences (p less than 0.1) when data were reported as percent of weight of tissue (2,189 +/- 918.6 vs 1,303 +/- 611.2 fmol/g wet issue). Results (mean +/- SD) on binding affinity of cytosolic receptors showed no significant differences in MAG-immunized rats as compared with controls (Kd: 1.98 +/- 0.66 vs 1.92 +/- 0.20 nM). Likewise, only a slight difference between both groups was attained for Kds of nuclear receptors (2.34 +/- 0.28 vs 1.80 +/- 0.62 nM, p less than 0.2). On the other hand, 5 alpha 1-dihydrotestosterone (DHT) values obtained in prostate homogenates were significantly decreased in MAG-immunized rats as compared with controls (17.4 +/- 2.0 vs 7.1 +/- 0.9 ng/g tissue, mean +/- SD, p less than 0.01). However, testosterone (T) levels in gland tissue showed no significant differences between both groups (2.4 +/- 0.5 vs 2.6 +/- 0.3 ng/g tissue) with an increase in the T: DHT ratio from 0.14 to 0.37.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-adrenergic receptor in heart of starved rats

Female Wistar-strain rats were starved for 14-19 days by feeding approximately 1/4 of the amount consumed by ad libitum fed controls. The body weight was reduced by 41% and the heart weight by 38% in these starving periods. The 125I-iodocyanopindrol (ICYP) binding capacity of heart preparations from the starved rats was 35.3 +/- 11.1 (mean +/- SD) fmol/mg protein in comparison with 69.3 +/- 14.9 for the controls. Serum 3,5,3'-triiodothyronine (T3), thyroxine and TSH levels as well as pituitary TSH contents were markedly lower in the starved rats. One group of them further received 20 ng of T3 daily after the 8th day of the experiment. The body weight decreased by 47% of the controls but the ICYP binding capacity recovered to 56.3 +/- 10.9 fmol/mg protein. There was no difference in association constants of the receptors in these three groups. It was concluded that quasi-chronic starvation in rats caused a remarkable decrease in the number of beta-adrenergic receptors in heart and this was partly offset by the substitution of T3.     

Studies on the nuclear 3, 5, 3'-triiodo-L-thyronine binding sites in cytotrophoblast

Human placental trophoblasts contain 3, 5, 3'-triiodo-L-thyronine (T3) nuclear receptors not only at term but all throughout pregnancy. We determined which of the trophoblast, cytotrophoblast (C-cell) or syncytiotrophoblasts (S-cell) respond to thyroid hormone, and whether the T3 binding capacity changes with placental aging. Nuclear protein of mononuclear cells purified from term chorionic tissue by enzymatic digestion and Percoll gradient centrifugation had an apparent association constant (Ka) of 5.2 x 10(9)M-1 and a binding capacity of 445 fmol T3/mg DNA, 8 times greater than that of term trophoblasts. Primary harvested mononuclear cells reacted against neither anti-hCG-beta nor anti-hPL antibodies, although some of them reacted immunocytochemically against anti-hCG-alpha antibody. These cells aggregated with each other and transformed into multinuclear cells in culture after 96 hrs of incubation, showing that these primary harvested cells were C cells that had morphologically transformed into S cells. The transformed cells secreted hCG and hPL and immunocytochemically stained for these markers, suggesting that the C cells had functionally transformed into S cells. Nuclear binding of T3 in trophoblastic tissue is present not only at term but throughout pregnancy. Although each nuclei had a similar Ka value, the binding capacity decreased towards term. These findings suggest that nuclear T3 receptors of placental trophoblast change with placental aging and this change is mainly due to the change in the C/S cell ratio. We concluded that the cytotrophoblast is an active target cell of thyroid hormone.     

Ontogeny, regional distribution and properties of thyroid-hormone receptors in the developing chick brain

Studies on the thyroid-hormone receptors in the nuclei of developing chick brain revealed a single class of binding sites for tri-iodothyronine (T3) and thyroxine (T4) at all embryonic and adult ages. High-affinity [Ka = (1.85-3.3) X 10(9)M-1 and (0.3-0.6 X 10(9)M-1 for T3 and T4 respectively] receptors were detected in the brain as early as day 7 of embryonic development; their level increased progressively rapidly until day 13, and thereafter the value remained essentially constant during development. Occupancy of the receptor site with endogenous hormone was 75-90% at 7-11 days, 50-60% during the late phase of embryogenesis (13-17 days), and 80% after hatching. Comparison of the binding properties of the receptors with T3 and T4 indicates that, although the binding capacities per nucleus are almost identical, T4 has four to five times less binding affinity than T3. The half-lives of dissociation of solubilized T3- receptor complexes were 20-30h between 0 degrees and 7 degrees C, about 4h at 20 degrees C and less than 15 min at 37 degrees C. Studies of the regional distribution of receptors in the brain indicate that cerebrum has the highest concentration of T3 receptors (4000-7000 sites per nucleus); this concentration is 2-4-fold higher than that in the cerebellum, optic lobe or medulla oblongata. The overall results indicate that between 7 and 13 days of embryonic development the thyroid-hormone receptors in the embryonic chick brain, particularly in the cerebrum, assume a very high level and appear to be mostly saturated with endogenous hormone. This, and the temporal correspondence of the phenomenon with the period of neuronal growth and synaptogenesis, strongly indicate the influence of the hormone in the maturation of the developing brain.     

Androgen metabolism in hirsute patients treated with cyproterone acetate

Cyproterone acetate (CPA) in association with percutaneously administered estradiol has been used for the treatment of 150 hirsute patients for periods ranging from 6 months to 3 years. A spectacular clinical improvement ensued. Plasma testosterone (T) and androstenedione (A) fell from 69.0 +/- 24 to 33.0 +/- 8 and 210 +/- 103 to 119 +/- 25 ng/dl (mean +/- SD) respectively after 3 months of treatment and remained low thereafter. In contrast, T glucuronide (TG) and 3 alpha-androstanediol (Adiol) remained high during the whole course of treatment: 37 +/- 9 and 115 +/- 43 micrograms/24 h respectively. In vitro T 5 alpha-reductase activity (5 alpha-R) in pubic skin decreased from 147 +/- 34 to 79 +/- 17 fmol/mg skin after 1 year of treatment. To elucidate the discrepancy between plasma and urinary androgens levels, T production rate (PR) and metabolic clearance rate (MCR) were measured with the constant infusion technique in 7 patients before and after 6 months of treatment. PR decreased from 988 +/- 205 to 380 +/- 140 micrograms/24 h (mean +/- SD). In contrast MCRT increased from 1275 +/- 200 to 1632 +/- 360 1/24 h; this increase in MCRT explains the striking plasma T concentration fall and the high TG and Adiol excretion relative to the decrease in PR. Antipyrine clearance rate (n = 8) increased from 36.3 +/- 5.2 to 51.5 +/- 7.4 ml/min whereas 6 beta hydroxycortisol remained unchanged. In conclusion, CPA acts through several mechanisms: (1) it lowers the androgen input to the target cells by (a) depressing T production through its antigonadotropic effect and (b) accelerating T metabolic inactivation due to a partial enzymatic inducer effect on the liver; (2) at the target cell level it competes with any remaining T for the receptor binding sites; (3) the decrease in the androgen-dependent skin 5 alpha-R is a consequence of both actions of androgen suppression and androgen receptor blockade; it reinforces the antiandrogenic effect of CPA.     

Development of preovulatory follicles expected to form short-lived corpora lutea in beef cows

Oestrus, expected to be followed by a short luteal phase, was induced in post-partum cows by weaning their calves at 35 days after parturition. Ovaries containing the first preovulatory follicles (Type F) formed after parturition were collected 3 h after the onset of oestrus. For comparison, preovulatory follicles (Type C) were collected 3 h after the onset of oestrus in normally cycling cows. The number of granulosa cells was determined and the concentrations of receptors for follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in granulosa cells and for LH in theca cells were measured. Concentrations of oestradiol-17 beta, testosterone, androstenedione and progesterone in follicular fluid were also measured. Type F follicles contained about twice the number of granulosa cells (based on DNA) as did Type C follicles (45.8 +/- 11.3 and 24.5 +/- 3.9 micrograms DNA/follicle, respectively; P less than 0.05) but these cells had fewer receptors for LH (0.13 +/- 0.02 vs 0.29 +/- 0.03 fmol/micrograms DNA; P less than 0.01) and FSH (0.61 +/- 0.08 vs 1.3 +/- 0.29 fmol/micrograms DNA; P less than 0.08) than did those from Type C follicles. Additionally, there were fewer receptors for LH in theca tissue from Type F than from Type C follicles (28.3 +/- 5.2 vs 51.3 +/- 6.1 fmol/follicle; P less than 0.01). Concentrations of oestradiol-17 beta (475.8 +/- 85.6 vs 112.9 +/- 40.0 ng/ml; P less than 0.01) and androstenedione (214.1 +/- 48.7 vs 24.7 +/- 7.7 ng/ml; P less than 0.01) in follicular fluid were higher in Type C than in Type F follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the intensity of the suckling stimulus and ovarian steroids on pituitary gonadotropin-releasing hormone receptors during lactation

These studies examined whether the decrease in pituitary responsiveness to gonadotropin-releasing hormone (GnRH) observed during lactation in the rat results from a change in pituitary GnRH receptors. GnRH binding capacity was determined by saturation analysis using D-Ala6 as both ligand and tracer. During the estrous cycle, the number of GnRH binding sites increased from 199 +/- 38 fmol/mg protein on estrus to 527 +/- 31 fmol/mg protein on the morning of proestrus, whereas there was no change in receptor affinity (Ka, 6-10 X 10(9) M-1), During lactation, females nursing 8 pups on Days 5 or 10 postpartum had 50% fewer GnRH receptors (109-120 fmol/mg protein) than observed during estrus or diestrus 1 (199-242 fmol/mg protein) although receptor affinity was similar among all the groups. No deficits in pituitary GnRH receptors were observed in females nursing 2 pups on Day 10 postpartum. Removal of the 8-pup suckling stimulus for 24 or 48 h resulted in a dramatic increase in GnRH receptor capacity by 24 h from 120 +/- 16 to 355 +/- 39 fmol/mg protein. The rise in GnRH receptors after pup removal was accompanied by an increase in serum luteinizing hormone (LH) and estradiol concentrations. To assess the role of ovarian steroids in determining GnRH receptor capacity during lactation, females were ovariectomized (OVX) on Day 2 postpartum. Suckling of a large litter (8 pups) completely blocked the postcastration rise in serum LH and in pituitary GnRH receptors on Day 10 postpartum (OVX+ 8, 77 +/- 12 fmol/mg protein; OVX+ 0, 442 +/- 38 fmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of the hydrophilic beta-adrenergic antagonist [3H]CGP-12177 to cardiac tissue slices: characterization and ontogenetic studies in dogs

A new technique was developed to characterize the binding of a hydrophilic beta-adrenergic antagonist, [3H]CGP-12177, to 1-mm thick slices of canine cardiac tissue. This technique was used to quantify the density (Bmax) and the affinity (Kd) of these receptors in the right ventricular conus (RVC) and the left ventricle (LV) at day 1 to 6 weeks of age, and in the adult. Binding was found to be reversible, saturable, stereospecific, of high affinity, and thermolabile. There was an increase in the density of beta-adrenergic receptors between day 1 (Bmax = 2.2 +/- 0.3 fmol/mg tissue in RVC and 2.9 +/- 0.8 fmol/mg tissue in the LV) and 2 weeks of age postnatally, after which it remained constant until 6 weeks of age (Bmax = 7.5 +/- 0.4 and 6.8 +/- 0.9 fmol/mg tissue in RVC and LV, respectively); however, by 6 weeks of age it had not reached adult levels (10.3 +/- 1.0 fmol/mg tissue). The affinity of these receptors did not change between early neonatal life (Kd = 1.3 +/- 0.4 nM) and adulthood (Kd = 1.4 +/- 0.2 nM). The density of beta-adrenergic receptors in the RVC was similar to that in the LV. This new method of quantifying beta-adrenergic receptors in cardiac tissue is simple and fast, and requires minimal tissue handling. It proved to be useful in studying the development of cardiac beta-adrenergic receptors with age.     

Specific binding of atrial natriuretic factor to renal glomeruli during day or night antiorthostatic suspension in the rat

We observed a significant increase in plasma atrial natriuretic factor (ANF) in antiorthostatic hypokinetic suspension (AOH) rats after 2 h of suspension when the experiment was made during day. Plasma ANF was investigated in relation to renal glomerular ANF receptors during AOH at night. The aim of this study was 1) to compare the day and night ANF responses to AOH 2) to determine whether the renal glomerular ANF receptors are involved. The rats were divided into 2 groups: i) 24 population cage (PC), and ii) 24 were attached by the tail (Morey's model) and remained in the horizontal position (attached horizontal-AH). Six AH were suspended (30 degrees) for 2 hours (AOH) and sacrificed with the controls: PC and AH (12.00h). The same experiment was made during the night (24.00h). A significant increase in plasma ANF was found in both AOH and AH after 2 h of suspension during day and night (19 +/- 2.3 pg/ml vs 9 +/- 0.95 and 18 +/- 3 pg/ml vs 10.2 +/- 1.8 respectively). PC rats had a significantly higher ANF level (38 +/- 5 pg/ml) than AH or AOH. The glomerular ANF receptor population was slightly lower in AOH than in AH (429 +/- 12 fmol/mg protein vs 507 +/- 5) during day. During night, a significantly lower number of ANF receptors was observed in AOH animals as compared to AH (168 +/- 2 fmol/mg protein vs 455 +/- 3). A decrease in glomerular receptors was also noted in PC during night. Day-time head-down tilt, bed rest or head-out water induced a natriuretic and diuretic response, whereas the normal recumbency at night does not lead to such effects. We conclude that the natriuretic and diuretic response not observed during night was associated with elevated plasma ANF levels and decreased ANF receptor density.     

Effects of fasting on the distribution of cytoplasmic and nuclear oestrogen receptors in rat liver, uterus, pituitary and hypothalamus before and after exogenous oestrogen administration

The accumulation of oestrogen receptors in the liver cell nuclei of intact female rats 45 min after administration of 100 micrograms 17 alpha-ethynyloestradiol-17 beta i.p., decreased progressively during a 72-h fast from 2550 +/- 860 to 257 +/- 67 fmol/mg DNA, a level not significantly different from that in uninjected animals. Cytoplasmic oestrogen receptor concentrations also decreased, but only to about 60% of the original level (from 84.1 +/- 27.5 to 50.3 +/- 2.09 fmol/mg protein during the fast). Similar differences were found when these parameters were examined in normally fed and 72-h-fasted ovariectomized rats. On the other hand these parameters were unaffected in uterus, pituitary and hypothalamus. Uterine cytoplasmic receptor concentrations remained at about 500 fmol/mg protein during the fasting period, those in the pituitary and hypothalamus at about 230 and 30 fmol/mg protein, respectively. Nor was in vivo translocation in these organs affected by fasting. Regardless of nutritional status, the nuclear oestrogen receptor concentrations in uterus rose from about 500 to 2000 fmol/mg DNA after ethynyloestradiol administration, those in the pituitary and hypothalamus from approximately 250 to 2000 and from 250 to 500 fmol/mg DNA respectively.