Use of a mammalian gonadotropin‐releasing hormone (GnRH) agonist to characterize pituitary GnRH receptors in white sturgeon (Acipenser transmontanus Richardson)

Aim of the present study was to characterize the pituitary GnRH receptor in white sturgeon, Acipenser transmontanus, using a superagonist analog of mammalian GnRH [D‐Ala⁶, des‐Gly¹⁰–Pro⁹]‐ethylamide and to investigate the possible effect of estradiol‐17β treatment on the concentration and affinity of the GnRH receptor in immature white sturgeon. The binding of ¹²⁵I‐GnRH‐A to sturgeon pituitary receptors was rapid and saturable at 4°C and 20°C. However, maximal binding at 20°C was almost two‐fold greater than the highest binding noted at 4°C. Specific binding of radioligand was directly related to the amount of tissue included in the assay system over the range of 5–20 mg fresh tissue equivalents per ml. The binding capacity of ¹²⁵I‐GnRH‐A with sturgeon pituitary tissue was much greater than radiolabeled GnRH. Administration of E₂ to immature sturgeon caused an almost two‐fold increase in GnRH‐A binding capacity (E₂ treated: Bmax = 2.87 fmoles 3 mg^?1 FTE; control: Bmax = 1.70 fmoles 3 mg^?1), and did not affect GnRH‐A binding affinity (E₂ treated: Ka = 0.13 × 10¹¹ m ^?1; control: Ka = 0.15 × 10¹¹ m ^?1). Overall, the study provides evidence that the GnRH analog is effective for characterizing the GnRH receptor in white sturgeon; however, more experimentation is necessary to determine whether E₂ administration to immature white sturgeon can increase the GnRH receptor capacity.     

Absence of correlation between antiestrogenic activity and binding affinity for the estrogen receptor.

We have compared the binding to the estrogen receptor (R) of different androgens and antiestrogens with their antiestrogenic activities on uterine growth. We found that estradiol (E₂)¹ and hydroxytamoxifen, a potent antiestrogen, displayed the same affinity for R. Conversely, androgens which have a much lower affinity for R and a much higher dissociation rate than E₂, behave at high doses as full estrogens, with no significant antiestrogenic activity. We conclude that there is no correlation between the dissociation rate from R and the antiestrogenic activity of R ligands and that one cannot discriminate between estrogen and antiestrogen ligands by simply evaluating their $\text{in vitro}$ binding to the cytosol R.     

Characterization of estrogen and progesterone receptors in monkey endometrium: methodology and effects of estradiol and/or progesterone on endometrium of castrate monkeys.

Appropriate methods for repeated surgical collection of endometrial tissue from rhesus monkeys, and characterization of cytosol and nuclear estrogen (E₂) and progesterone (P) receptors (R) are described. Tissue collection was made in the mid-luteal phase at abdominal fundal hysterotomy. Functional status of the ovaries was determined by visual inspection and RIA of E₂ and P in serum. Receptor assay procedures were devised permitting the measurement of total cytosol and nuclear receptor concentration. Sucrose density gradients of labelled cytosol were made and a 4S saturable binding component for ³H P and for ³H E₂ were found. Equilibrium dissociation constants of ³H E₂ and ³H R5020 were 2.1×10^?10M and 3.6 × 10^?9M, respectively. These binding characteristics are similar to those found in human endometrium and suggest that these surrogate primates have extensive utility in investigation of factors influencing E₂R and PR concentrations in endometrial tissue during the menstrual cycle and implantation. Simulated menstrual cycle were produced in 20 castrate monkeys by sequential treatment with estradiol and progesterone in silastic capsules. RIA of E₂ and P, and gonadotropins in peripheral serum provided assuredness of the hormonal status of each monkey under treatment. Cytosol and nuclear receptors for E₂ and P were measured in the endometrium after different intervals of the treatment. E₂ receptor (E₂R) levels were not changed during the estrogen sequence, but were lowered by progesterone therapy in both cytosol and nuclear components. Progesterone receptor (PR) synthesis in cytosol was induced by exogenous estrogen. The total concentration of PR decreased with the uptake of P by the cell; meanwhile, the ratio of cytosol to nuclear P receptors declined. These data suggest that this sequential estrogen-progesterone regimen induces the changes in E₂R and PR patterns in the endometrium of ovariectomized monkeys which occurs due to ovarian cyclicity in the normal menstrual cycle.     

Biochemical properties of the 1 alpha, 25-dihydroxyvitamin D3 cytoplasmic receptors from human and chick parathyroid glands

Cytoplasmic receptors for 1α, 25-dihydroxyvitamin D₃ from human parathyroid adenoma tissue and rachitic chick parathyroid glands have been characterized with regard to a number of physical, chemical, and ligand binding properties. Both receptors are 3.6–3.7 S proteins with molecular weights of approximately 75,000 and Stoke's molecular radii of 36 Å. It was found that the receptors possess a cysteine residue in or near the 1α, 25-dihydroxyvitamin D₃ binding site which is critical for ligand binding activity. The receptors both have equilibrium dissociation constants for 1α, 25-dihydroxyvitamin D₃ in the range of 2 to 5 × 10^?10m at 4 °C and second-order association rate constants for their seco-steroid ligand of 1 × 10⁷, m^?1 min^?1 (0 °C). The dissociation rate constants were found to be 5.3 × 10^?4 min^?1 (4 °C) for the human receptor and 1.3 × 10^?5 min^?1 (4 °C) for the chick receptor. The great deal of similarity which exists between the cytoplasmic 1α, 25-dihydroxyvitamin D₃ receptors from avian and mammalian parathyroid glands suggests a homologous function for these molecules in the two tissues.     

Similarities and differences of the binding of estradiol and 4-hydroxytamoxifen (an antiestrogen) in the chick oviduct cytosol

The binding characteristics of [³H]estradiol and 4-[³H]hydroxytamoxifen (a powerful estradiol antagonist) in the chick oviduct cytosol was analyzed by sucrose gradient centrifugation and dissociation kinetics experiments at 28°C. Heating the cytoplasmic estradiol-estrogen receptor complexes led to the ‘transformation’ of the receptor; as with the estrogen receptor in other target tissues and species, the transformed receptor sedimented in the 5 S region of sucrose gradients containing 0.4 M KCI and had a slower rate of dissociation of bound estradiol. Upon heating, the cytoplasmic 4-hydroxytamoxifen complexes also appeared to undergo similar changes in their physical states as analyzed by sedimentation rates and dissociation kinetics, and we conclude that antiestrogen can transform the receptor. Sodium molybdate inhibited the temperature mediated changes with both estrogen and antiestrogen complexes. Slight but consistent differences in the sedimentation coefficient and rate of ligand dissociation were observed between the complexes formed by estradiol and 4-hydroxytamoxifen but the relevance to opposite biological activities remains unknown.     

The role of arginyl residues in estrogen receptor activation and transformation

Receptor-estradiol complexes (RE2) formed at 0 degree C in hypotonic buffers bind poorly to nuclei (nonactivated state); their sedimentation coefficient in low or high salt sucrose density gradients (SDG) is 8 S or 4 S, respectively (untransformed state); estradiol dissociates from untransformed RE2 at a high rate (k-1 = 0.44 min-1). Brief heating (28 degrees C, 30 min) induces activation (increased binding of RE2 to nuclei and polyanions), transformation (formation of receptor dimers which sediment at 6 S in 0.4 M KCl/borate SDG) and RE2 transition into a state from which E2 dissociates at a lower rate (k-2 = 8 X 10(-3) min-1). We have examined the role of arginyl residues in the above changes in receptor properties. It is well established (Patthy, L., and Smith, E. L. (1975) J. Biol. Chem. 250, 557-564; 565-569) that 1,2-cyclohexanedione (1,2-CHD) is a highly specific arginine-modifying agent; in borate buffer at 28 degrees C, but not at 0 degrees C, peptide arginyls are covalently modified. RE2 complexes heated in the presence of 1,2-CHD (50 mM) bind poorly to nuclei; 1,4-cyclohexamedione and 1,2-cyclohexanediol had no effect. This reagent also prevents the temperature-induced transition of RE2 into a state with slow E2 dissociation rates although it does not interfere with heat transformation (formation of 6 S dimer). Modification of heat-activated and transformed RE2 by 1,2-CHD causes a loss in receptor binding to nuclei and alters RE2 from a state with slow into a state with fast E2 dissociation rates, although the receptor remains unaltered in the transformed 6 S state. At 0 degree C, i.e. in the absence of covalent arginyl modification, 1,2-CHD promotes dissociation of the 8 S aggregate into 4.6 S subunits which bind to nuclei to the same extent as heat-transformed control RE2. Heating of the molybdate-stabilized 8 S receptor in the presence of 1,2-CHD yields a nonactivated 8 S receptor (4.6 S on high salt SDG); removal of molybdate and unreacted 1,2-CHD by gel filtration at 0 degree C followed by exposure to high ionic strength causes 8 S to 4 S dissociation; these 4 S subunits, however, do not bind to nuclei, suggesting that their nucleotropic domain was accessible to 1,2-CHD modification while the receptor was in the aggregated 8 S state. It is proposed that the nuclear binding site of the estrogen receptor contains arginyl residues. Furthermore, a distinct set of arginyl residues appears to be related to the estrogen-binding domain; its integrity is required for the heat-induced formation and maintenance of the RE2 state with slow E2 dissociation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Simultaneous Measurements of Estrogen Nuclear (E2Rn) and Progestin Cytosol (PRc) Receptors in the Hypothalamus and Pituitary Gland of Rats

Abstract

The present methods used to measure estrogen nuclear (E₂Rn) and progestin cytosol (PRc) receptors in the hypothalamus and pituitary gland require that separate assays be performed to determine the concentrations of each receptor. In the present studies we describe a method which simultaneously measures both E₂Rn and PRc in hypothalamic and pituitary tissue. Tissue samples were homogenized in tris-EDTA-glycerol-dithiothreitol buffer and centrifuged at 1500 × g for 5 min. The supernatant was purified for the PRc assay while the nuclear pellet was extracted for the E₂Rn exchange assay.

For the PRc assay, the supernatant was centrifuged at 106,500 × g for 30 min and aliquots from the resultant supernatant then were incubated with ³H-R5020. For the E₂Rn exchange assay, the original pellet was purified further by successively resuspending and centrifuging it through several sucrose solutions. Estrogen-receptor complexes were extracted from the chromatin pellet with a 0.4M KC1 solution and aliquots of the final supernatant were incubated with ³H-estradiol.

In both assays, the samples were placed onto Sephadex LH20 columns and the receptor bound ³H-steroid was eluted directly into scintillation vials. Scatchard analyses revealed that these assays measure a single class of binding sites for E₂Rn and PRc with dissociation constants (K_D) and maximal number of binding sites (Bmax) similar to those previously reported using a separate assay for each receptor.     

Buprenorphine: Characteristics of binding sites in the rat central nervous system

The binding of the mixed opiate agonist-antagonist ³H-buprenorphine to rat CNS membranes was stereospecific, saturable and had high affinity. Maximal specific binding of ³H-buprenorphine at 25°C was reached by 30 minutes and dissociation from the receptor was slow. ³H-Buprenorphine labelled a single class of high affinity binding sites (K_D = 0.86nM, B_max = 30.2pmole/g tissue). The B_max for ³H-buprenorphine was about two times that for the μ-opiate receptor drugs ³H-naloxone and ³H-dihydromorphine, and three times the B_max for the σ-opiate receptor ligand ³H-D-Ala², L-Met⁵-enkephalinamide. The regional distribution of ³H-buprenorphine binding was qualitatively similar to the distribution of ³H-naloxone and ³H-dihydromorphine binding. Changing the incubation temperature from 25°C to 37°C increased ³H-buprenorphine binding in all regions of the CNS yet decreased ³H-naloxone and ³H-dihydromorphine binding in most regions. These effects of increasing temperature were a result of changes in ³H-opiate affinity for the receptor with no significant changes in receptor number. Sodium chloride (154mM) enhanced both ³H-buprenorphine and ³H-naloxone binding, and decreased ³H-dihydromorphine binding. The potency of opiate alkaloids and peptides in displacing ³H-buprenorphine was relatively weak with IC₅₀ values ranging between 40nM and 600nM. Furthermore displacement curves were shallow, yielding curvilinear Scatchard plots. Buprenorphine was very potent in displacing ³H-naloxone (IC₅₀ = 0.52nM), ³H-dihydromorphine (IC₅₀ = 1.17nM) and ³H-D-Ala², L-Met⁵-enkephalinamide (IC₅₀ = 0.47nM). These findings suggest that buprenorphine binds to both μ- and δ-opiate receptors.     

The effects of long-term estrogen exposure on the induction of sexual behavior and measurements of brain estrogen and progestin receptors in the female rat

The purpose of this study was to investigate long-term effects of estradiol-17β (E₂) on sexual receptivity and to study the molecular basis for estrogen potentiated changes in receptivity. We therefore examined the long-term effects of E₂ on E₂ and progestin receptors in the hypothalamus-preoptic area-septum (HPS) and pituitary (PIT) of the female rat. Twenty-one days following ovariectomy, females received a 5-mm Silastic capsule of E₂ or cholesterol (C) for 1 week (pretreatment). Some animals were sacrificed for chemical analyses (i). The remainder had their capsules removed and 5 days later these animals either were sacrificed for chemical analyses (ii), or received E₂ (reimplantation). Forty-six hours after reimplantation, females either were sacrificed for chemical analyses (iii), or were tested for receptivity. When tested with sexually active males or by a manual stimulation method, animals pretreated with E₂ showed significantly better lordosis scores than animals pretreated with C. The tests for lordosis were carried out after administration of E₂ or E₂ + P to all subjects tested. During E₂ pretreatment, HPS and PIT cytosol progestin receptors increased significantly, while available estrogen receptor levels decreased significantly, as compared with C controls. Five days after E₂ pretreatment, HPS progestin receptor levels had decreased to the level observed in C controls, while PIT progestin receptors were slightly elevated. In HPS and PIT, levels of available E₂ receptor in E₂ and C pretreated animals were indistinguishable from each other. Neither the saturation capacity of the estrogen receptor nor the dissociation constant for binding [³H]E₂ was altered by E₂ pretreatment, as shown by Scatchard equilibrium analysis. Forty-six hours following E₂ reimplantation, progestin HPS and PIT receptor levels in E₂ and C pretreated animals were identical. Long-term potentiation of lordosis by E₂ does not result from a change in estrogen or progestin receptor dynamics in HPS of female rats.     

1,25-Dihydroxyvitamin D3 receptors in rat kidney cytosol

Rat kidney cytosol contains a 3.3 S high affinity binding component for 1,25-dihydroxyvitamin D₃ as detected by DNA-cellulose chromatography and subsequent sucrose gradient analysis. The semipurified aporeceptor demonstrates specificity for 1,25-dihydroxyvitamin D₃ and an apparent dissociation constant for this sterol-hormone of 3.4 × 10^?10M at 25°C. The physicochemical properties of this binding component are in agreement with those observed for the chick intestinal 1,25-dihydroxyvitamin D₃ receptor, suggesting that this component may function as a specific receptor for the hormone in the kidney.     

Exchange assays using sodium thiocyanate to measure total nuclear content of estrogen receptors in the hypothalamic pituitary axis of mice

We studied the effect of sodium thiocyanate (NaSCN) in the determination of specific nuclear estrogen receptors from the hypothalamic-pituitary axis (HPA) of mice. We compared the results with those of the exchange assay using either suspensions of whole nuclei or KC1-nuclear extracts. Our findings demonstrated that 0.5 M NaSCN was more efficient than 0.5 M KC1 in extracting [³H] E₂ nuclear content from HPA (91% vs 79.3%). Nuclear fractions extracted with 0.5 M NaSCN revealed the presence of a single class of low-capacity-high affinity binding sites that sedimented in the 4.0 S area. Nuclear binding was T° dependent and reached maximum levels of 450 ± 156 (S.E.) fmol/mg DNA after an overnight incubation at 4°C. Such levels were comparable to those observed in whole nuclei suspensions after 1 hour incubation at 37°C (618 ± 71 fmol/mg DNA, p > 0.05) but two-fold higher (p <0.01) than the concentration of binding sites measured in KC1-extracted nuclear fractions under similar experimental conditions. We conclude that NaSCN extracted the total content of nuclear estrogen receptors in HPA of mature mice.     

Presence of D2-dofamine Receptors in Human Term Placenta

Abstract

We studied the binding of [³H]-spiperone on human term placental membranes. This binding reached plateau level after 30 min incubation at 37°C and was reversed (t_1/2 ～ 5 min) by addition of an excess of unlabeled spiperone. Scatchard analysis of saturation experiments with increasing doses of [³H]-spiperone (0–25 nM) showed one class of high affinity binding sites with a dissociation constant (Kd) of 14 ± 2 nM and a maximal binding capacity (Bmax) of 222 ± 9 fmoles/mg protein. The affinity of 5 competitors was determined in competitive binding assays. The D₂-dopamine antagonists were the most potent inhibitors: Ki for spiperone and haloperidol were 8 ± 2 and 56 ± 22 nM respectively. Dopamine inhibited [³H]-spiperone binding with a Ki of 570 ± 50 μM whereas Schering 23390 (D₁ antagonist) and propranolol (β-adrenergic antagonist) were without effect. The binding was also inhibited by 100 μM GTPγS (38 ± 8% inhibition), indicating that the dopamine receptor is coupled with a GTP binding protein. These results demonstrate for the first time the presence of D₂-dopamine receptors in human placenta.     

Biology and receptor interactions of estriol and estriol derivatives in vitro and in vivo

The biological effects of estriol (E3) have been studied in three estrogen targets, namely, the rat uterus in vivo and in vitro, in primary human endometrial cell cultures and in MCF-7 human breast cancer cells in culture. Studies on the temporal relationships between estrogen receptor binding and biological responses in the uterus using estriol and several more long-acting estriol derivatives, namely, 17α-ethynyl estriol, estriol-3-cyclopentyl ether, and 17α-ethynyl estriol-3-cyclopentyl ether, indicate that estriol is a short-acting compound with a brief duration of action. Estriol is a poor stimulator of uterine growth and plasminogen activator activity in vivo. Chemical modifications of the estriol molecule produce long-acting derivatives that result in a prolonged input of hormone receptor complexes into the nucleus and a prolonged and marked stimulation of uterine growth. In human endometrial cells in primary tissue culture, E₃ has 12% the affinity of estradiol (E₂) for cytosol estrogen receptor and it is quite effective yet slightly less potent than estradiol in stimulation of progesterone receptor synthesis. Low concentrations of E₃(10⁻¹⁰ M) stimulate growth of MCF-7 cells in vitro and dose-response curves show E₃ to be only slightly less effective than E₂. In these endometrial and breast cancer cell systems in vitro, there is no metabolism of E₃ while E₂ is metabolized to estrone.Hence, estriol is an effective estrogen in vitro. In vivo, it is short-acting, but it can be made a full estrogen agonist when given at a sufficiently high concentration or in a chemically modified form which prolongs its activity by enabling effective concentrations of the compound to be maintained in the blood and in target tissues.     

Metabolic and proliferative responses to estrogen by hepatocytes selected for plasma membrane binding-sites specific for estradiol-17beta.

Hepatocytes were isolated by established procedures from freshly-excised livers of ovariectomized rats. Integrity of the cells was verified by DNA, protein, and calcium contents, and by dye exclusion. The cells also showed progressive increments in oxidation to ¹⁴CO₂ of [26-¹⁴C]cholesterol during one to five hours' incubation. Analysis was undertaken of cellular reactivities toward estrogen and the hepatocarcinogen dibutylnitrosamine (DBN). Binding and retention of [³H]estradiol-17β (E₂β) by isolated liver cells was specific for E₂β, saturable, temperature-dependent, and maximal after 30-minute incubation. The apparent dissociation constant for the binding process at 22°C is 2 × 10^?9 M, and the total number of binding-sites at saturation corresponds to approximately 3,400 E₂β molecules per liver cell. To probe for steroid binding-sites at their external surfaces, cells were incubated 30 minutes with mounted 17β-estradiol-17-hemisuccinyl:albumin:nylon fibers. The covalentlyimmobilized estrogen (1 ng/mg albumin) was accessible for interaction with antiserum directed against 17β-estradiol-17-hemisuccinyl:albumin. Significant numbers of isolated liver cells were retained by estrogen-derivatized fibers at 22°C after extensive washes. Binding was markedly reduced by incubation at 4°C and by prior exposure to free E₂β (× 10^?8 M), but not to the relatively inert estradiol-17α (E₂α). Fiber-bound cells could be dislodged by brief incubation in 150 mOsM saline with 2 × 10^?7 M E₂β or diethylstilbestrol, but not E₂α, cortisol, progesterone, or testosterone, and recovered intact. Cells that had been retained by the fibers and those that were not adherent were collected and washed under identical conditions, then plated in serum-free, chemically-defined medium at 37°C. After 72 hours, specific binding of E₂β by the fiber-binding cells during 30 minutes' incubation was 2.5-fold that of cells which had not bound the immobilized steroid. Similarly, stimulation of the oxidation to ¹⁴CO₂ of [26-¹⁴C]cholesterol by E₂β was greater in fiber-binding than in non-binding liver cells after three hours' incubation. In the absence of added mitogen, thymidine incorporation into macromolecular form (20 hours), and cell proliferation (48 hours) were significantly greater in fiber-binding cells as compared to non-binding hepatocytes. Moreover, in parallel experiments, when cells were exposed to 1 × 10^?9 M estrogens or to 1 × 10^?4 M nitrosamines to assess the capacities of these substances to increase basal thymidine incorporation, total DNA, and cell numbers, only those cells with estrogen-binding sites at their surfaces showed significant E₂β- and DBN-induced increments in these parameters as compared with paired controls that had been treated with E₂α or the noncarcinogen diphenylnitrosamine. These data indicate that the accessibility of hormone-binding components at the plasma membrane may contribute to the capacity of a given liver cell to respond to E₂β, as well as to other known hepatocarcinogens.     

Characterization of the Binding of the GABA Agonist [3H]Piperidine-4-Sulphonic Acid to Bovine Brain Synaptic Membranes

Abstract: The binding of radioactive piperidine-4-sulphonic acid ([³H]P4S) to thoroughly washed, frozen, and thawed membranes isolated from cow and rat brains has been studied. Quantitative computer analysis of the binding curves for four regions of bovine brain revealed the general presence of two binding sites. In these brain regions less satisfactory computer fits were obtained for receptor models showing one or three binding sites or negative cooperativity. With the use of Tris-citrate buffer at 0°C the two affinity classes for P4S in bovine cortex membranes revealed the following binding parameters: K_D= 17 ± 7 nM (B_max= 0.15 ± 0.07 pmol/mg protein) and K_D= 237 ± 100 nM (B_max= 0.80 ± 0.20 pmol/mg protein). Heterogeneity was also observed for association and dissociation rates of [³H]P4S. The slow binding component (k_on= 5.6 × 10⁷ or 8.8 × 10⁷ M^-1 min^-1, k_Off= 0.83 min^-1, and K_D= 14.7 or 9.4 nM, determined by two different methods in phosphate buffer containing potassium chloride) corresponds to the high-affinity component of the equilibrium binding curve (K_D= 11 nM, B_max= 0.12 pmol/mg protein in the same buffer system). The association and dissociation rates for the subpopulation of rapidly dissociating sites, apparently corresponding to the low-affinity sites, were too rapid to be measured accurately. The binding of [³H]P4S appears to involve the same two populations of sites with B_max values similar to those for [³H]GABA binding to the same tissue, although the kinetic parameters for the two ligands are somewhat different. Furthermore, comparative studies on the inhibition of [³H]P4S and [³H]GABA binding by various GABA analogues, strongly suggest that P4S binds to the GABA receptors. The different effects of P4S and GABA on benzodiazepine binding are discussed.     

Characterization of receptors for α2-macroglobulin-trypsin complex in rat hepatocytes

High-affinity receptors for α₂-macroglobulin-trypsin complex were demonstrated in rat hepatocytes at 4°C. The dissociation rate constant for the labelled complex was very small at low receptor occupancies, approx. 4·10⁻⁴ min⁻¹. Dissociation was biphasic at high receptor occupancies with a rate constant for the rapid phase of about 2·10⁻² min⁻¹. At near-equilibrium, half of the receptors were saturated at a complex concentration of 150 pM, and the Scatchard plot was concave upwards. Thus, the binding shows complex kinetics with the probable involvement of negative cooperativity. Binding of the labelled complex was not influenced by galactose, mannose, mannose phosphate or fucoidin, whereas it was abolished in the absence of extracellular Ca²⁺ and inhibited by bacitracin. Approx. 70% of the labelled complex bound at 4°C was rapidly internalized (k_int about 3·10⁻¹ min⁻¹) after being warmed to 37°C. Radioactivity released from the cells at 37°C comprised intact labelled complex and iodide. The complex was initially released at a rapid rate (k₋₁ about 1·10⁻¹ min⁻¹) from about 25% of the cell-bound pool. This probably represents dissociation from the receptors. A slow phase of release followed, so that half of the bound pool was finally released as intact complex. Iodide release followed a sigmoidal curve after a 20 min lag period. Thus, specific high-affinity receptors mediate the internalization and eventual degradation of α₂-macroglobulin-proteinase complex into hepatocytes.     

Synthesis,structural characterization,and properties of the organothorium alkylthiolate complex [(CH3)5C5]2Th(SCH2CH2CH3)2

This contribution reports the synthesis and characterization of the organothorium alkylthiolate complex [(CH₃)₅C₅]₂Th(SCH₂CH₂CH₃)₂. This compound crystallizes in the monoclinic space group C2/c (#15) with four molecules in a cell of dimensions a=19.066(2), b=11.603(1), c=16.379(2) Å, and β=130.08(1)°. Least-squares refinement led to a value for the conventional R index (on F_o) of 0.040 for 132 variables and 2030 observations having F_o²⩾3σ(F_o²). The molecular structure consists of an unexceptional ‘bent sandwich’ [(CH₃)₅C₅]₂Th fragment coordinated to two n-propylthiolate ligands. The ThS bond distance is 2.718(3) Å; the SC(α) distance, 1.78(2) Å; the ThSC(α) angle, 108.3(5)°; and the SThS′ angle, 102.5(2)°. Contrasts are drawn with the structures of analogous actinide alkoxides     

[125I]LSD binding to serotonin and dopamine receptors in bovine caudate membranes

[¹²⁵I]LSD (labeled at the 2 position) has been introduced as the first ¹²⁵I-labeled ligand for serotonin 5-HT₂ (S2) receptors. In the present study we examined the binding of [¹²⁵I]LSD and its non-radioactive homologue, 2I-LSD, to bovine caudate homogenates. The binding of [¹²⁵I]LSD is saturable, reversible, stereospecific and is destroyed by boiling the membranes. The specific to total binding ratio in this tissue is 75–80% and Scatchard plots of the binding data reveal K_d = 1.1 nM, B_max = 9.6 fmol/mg wet weight tissue. The association and dissociation rate constants are highly temperature dependent. At 0°C the net dissociation is less than 5% after 1 h and the association rate is proportionately slow. IC₅₀ values for a variety of compounds show a clear 5-HT₂ (S2) serotonergic pattern at this [¹²⁵I]LSD site. Blockage of this primary 5-HT₂ (S2) caudate binding site by 0.3 μM mianserin reveals the presence of a weaker [¹²⁵I]LSD binding site with a K_d = 9.1 nM, B_max = 7.6 fmol/mg tissue. This secondary site is a D3 dopaminergic receptor site, as shown by the relative abilities of various displacers to inhibit this binding. Binding studies with nonradioactive 2I-LSD reveal a clear preference for D2 over D3 dopamine receptor sites. [¹²⁵I]LSD is a sensitive and selective label for 5-HT₂ (S2) serotonin receptor sites in both rat frontal cortex and bovine caudate membranes. Blockage of the primary bovine caudate [¹²⁵I]LSD binding site with mianserin allows the high sensitivity of [¹²⁵I]LSD to be applied to D2 dopamine receptor studies as well.     

Hydrocortisone binding receptor in the rat pituitary GH3 cell line.

A receptor with specificity and high affinity for hydrocortisone (HC) has been found in the cytosol of GH₃ cells, a growth hormone (GH) producing culture. Scatchard analysis indicated that the interaction of [³H]HC with the receptor has an apparent dissociation constant (K_d) of about 6.0 × 10^?9M and a concentration of binding sites of approx. 1 × 10^?13 mol/mg cytosol protein. The second order association rate constant was determined to be 1.5 × 10⁶ M^?1 min^?1. The receptor activity is stable at 2°C for several hours, but is destroyed completely by heating at 37°C for 1 hour, or by treatment with pronase, only partially by RNase, but not by DNase. The binding of [³H]HC to the cytosol receptor is inhibited by unlabeled progesterone (PR) or dexamethasone to the same extent as the inhibition by unlabeled HC. However, it is only partially inhibited by testosterone, 17-methyl-testosterone, 17α and 17β-estradiol, and 4-pregnen-20β-ol-3-one, and is unaffected by 5α-pregnan-3β,20β-diol. The biological role for these receptors in the regulation of GH synthesis is supported by the observations that the HC-stimulated production of GH is antagonized by PR, which competes with the binding of HC to the receptor.