Binding of [3H]methyltrienolone to androgen receptor in rat liver

The synthetic androgen methyltrienolone is superior to testosterone and androstenedione for the measurement of androgen receptor in tissues where the native ligands are metabolized into inactive derivatives. [³H]Methyltrienolone binds with a high affinity to androgen receptor in cytosol prepared from male rat livers, as the Scatchard analysis revealed that the K_d value was 3.3 · 10^?8 M and the number of binding sites was 35.5 fmol/mg protein. Since methyltrienolone also binds glucocorticoid receptor which exists in rat liver, the apparent binding of androgen receptor is faulty when measured in the presence of glucocorticoid receptor. The binding of methyltrienolone to glucocorticoid receptor can be blocked by the presence of a 100-fold molar excess of unlabeled synthetic glucocorticoid, triamcinolone acetonide, without interfering in its binding to androgen receptor, because triamcinolone does not bind to androgen receptor. Triamcinolone-blocked cytosol exhibited that the K_d value was 2.5 · 10^?8 M and the number of binding sites was 26.3 fmol/mg protein, indicating a reduction to $\text{3}\text{4}$ of that in the untreated cytosol. The profile of glycerol gradient centrifiguration indicated that [³H]methyltriemolone-bound receptor migrated in the 8–9 S region in both untreated and triamcinolone-blocked cytosols, but the 8–9 S peak in triamcinolone-blocked cytosol was reduced to about $\text{3}\text{4}$ of that of untreated cytosol.     

Macromolecular binding of (5,6-3H) prostaglandin E1 in liver cytosol in vitro

[³H] Prostaglandin E₁ (PGE₁) is bound extensively to macromolecules in liver cytosol $\text{in vitro}$. A principal binding protein accounts for 80% of the binding. This macromolecule is saturated at about 10^?10 M PGE₁. The partially purified protein has a molecular weight of 50,000 by gel filtration and a pI of about 3.5 by isoelectrofocusing. Binding is primarily noncovalent and the dissociated ligand behaves similarly to the parental [³H] PGE₁ on thin layer chromatography. Possible significance of this interaction is discussed.     

Identification of opiate receptor binding in intact animals.

After intravenous administration of ³H-naloxone to rats, particulate bound radioactivity accumulated in the brain is selectively associated with opiate receptor binding sites, providing a means of labeling the opiate receptor $\text{in vivo}$. The regional distribution of ³H-naloxone bound $\text{in vivo}$ closely parallels regional differences in opiate receptor binding $\text{in vitro}$ with highest levels in the corpus striatum, negligible receptor-associated binding in the cerebellum and intermediate levels in other regions. ³H-Naloxone binding $\text{in vivo}$ is saturable with the same total number of binding sites determined $\text{in vivo}$ as by $\text{in vitro}$ procedures. Nalorphine is markedly more potent than morphine in inhibiting ³H-naloxone binding $\text{in vivo}$ and non-opiates are ineffective. The half-life for dissociation of ³H-naloxone bound to particles $\text{in vivo}$ is the same as its dissociation rate after binding occurs $\text{in vitro}$, and sodium stabilizes ³H-naloxone bound $\text{in vivo}$ from initial rapid dissociation as predicted from the known properties of the opiate receptor $\text{in vitro}$.     

Characterization of a progestin-binding macromolecule in the amphibian oocyte cytosol.

A [³H]-progestin-binding macromolecule has been isolated from $\text{R}\text{.}\text{pipiens}$ oocyte cytosol and characterized using binding assays, gel filtration, DEAE-cellulose chromatography and ultracentrifugal techniques. Macromolecules present in the prophase oocyte cytosol have a high affinity and specificity for the synthetic progestin R5020 and the intact oocyte will concentrate both R5020 and progesterone 20–40 fold from the medium. This may be the first published case of a cytosol steroid binding macromolecule in a cell system in which the steroid appears to act at an extranuclear level.     

Kinetics of in vivo binding of antagonist to muscarinic cholinergic receptor in the human heart studied by positron emission tomography

Positron Emission Tomography (PET) was used to analyse $\text{in vivo}$ antagonist binding to human myocardial muscarinic cholinergic receptor. The methiodide salt of the muscarinic antagonist, quinuclidinyl benzilate (MQNB), was labeled with the positron emitter, Carbon-11, and injected intravenously to 8 normal subjects. ¹¹C-MONB concentration was determined $\text{in vivo}$ in the ventricular septum from 40 cross-sectional images acquired at the same transverse level over a period of 70 minutes. In 4 subjects, various amounts of unlabeled atropine were rapidly injected at 20 minutes to study whether atropine competitively inhibited MQNB.The kinetics of binding of ¹¹C-MQNB were not the same $\text{in vivo}$ and $\text{in vitro}$. The apparent dissociation rate of ¹¹C-MQNB $\text{in vivo}$ was much slower (by 1 to 2 orders of magnitude) than that observed $\text{in vitro}$ with ³H-QNB. After atropine injection, ¹¹C-MNQB dissociated from its binding sites at a rate that apparently depended on the amount of atropine present. ¹¹C-MQNB kinetics were analysed with a mathematical model which assumes the existence of a boundary layer containing free ligand in the vicinity of the binding sites. The dissociation rate of the radioligand depends on the probability of its rebinding to a free receptor site.     

Binding of opiates and endogenous opioid peptides to neuroleptic receptor sites in the corpus striatum.

Some opiates with morphinan- and benzomorphan-structures possess affinities for neuroleptic receptors as revealed by their abilities to compete with ³H-spiroperidol for common binding sites in rat striatum $\text{in vitro}$ (IC₅₀ in the range between 10^?6 and 10^?5M). The binding of these opiates to neuroleptic receptors appears to be of pharmacological significance, since $\text{in vivo}$ studies in mice revealed a small but significant displacement of spiroperidol by high doses of the opiate antagonist levallorphan from specific binding sites in the striatum. In addition, there exists some correlation between the ability of opiates to bind to neuroleptic receptor sites $\text{in vitro}$ and their potency to evoke “bizarre behavior” in rats $\text{in vivo}$. In contrast, a wide variety of other opiates having morphine-, morphinone- or oripavine-structure showed no affinity for neuroleptic binding sites $\text{in vitro}$ (IC₅₀ greater than 10^?4 M). Of the opioid peptides (methionine-enkephalin, leucine-enkephalin and β-endorphin) none has an affinity for neuroleptic binding sites. A variety of other peptides were also investigated but did not interfere with spiroperidol binding. Only ACTH showed a moderate affinity for neuroleptic binding sites.     

Specific in vivo binding of 77Br-p-bromospiroperidol in rat brain: A potential tool for gamma ray imaging

The binding of the gamma labeled neuroleptic, ⁷⁷Br-$\text{p}$-bromosprioperidol, in the rat brain was examined $\text{in vivo}$. This binding parallels the binding of ³H-spiroperidol, in that binding is especially high in dopaminergically innervated areas, is saturable, and is displaced by high doses of unlabeled spiroperidol (1–5). Thus, ⁷⁷Br-$\text{p}$-bromospiroperidol is a suitable ligand for use in gamma ray imaging techniques for $\text{in vivo}$ monitoring of receptor binding.     

Complex formation by 3 H-aldosterone in rat kidney and liver

Low molecular weight polar complexes were shown to be formed $\text{in vivo}$ from ³H-aldosterone in both kidney and liver subcellular fractions, the majority being present in the cytosol fractions. Significant differences were observed between the quantities of polar complexes present in kidney subcellular fractions from intact and adrenalectomized male rats and also between the quantities of these kidney polar complexes from spironolactone treated male rats. ³H-aldosterone macro-molecule complexes were shown to exist in appreciable quantities only in the kidney cytosol fractions of adrenalectomized male rats. These gel filtration studies also showed the ³H-aldosterone labeled macromolecule complexes to consist of two protein peaks; one of high molecular weight and the other of lower molecular weight (～50,000 mol. wt.). The amount of ³H-aldosterone labeled protein complexes in kidney cytosol was greatly reduced when adrenalectomized rats were pretreated $\text{in vivo}$ with spironolactone.     

The role of cAMP on neoplastic cell growth and regression. III. Altered cAMP-binding in DBcAMP-unresponsive Walker 256 mammary carcinoma.

An exogenous supply of N⁶,O^2′-dibutyryl cyclic adenosine 3′,5′-monophosphate (DBcAMP) $\text{in vivo}$ produces regression of one type of Walker 256 mammary carcinoma cell population (DBcAMP-responsive); a second type of cell population continues to grow despite DBcAMP treatment (DBcAMP-unresponsive). A correlation was found between altered cAMP-binding of the tumor cytosol and DBcAMP-unresponsiveness. It was found that there was: a) a higher apparent dissociation constant (Kd) for cAMP-binding in unresponsive tumor cytosol $\text{in vitro}$, and b) unsaturability of cAMP-binding by unresponsive tumor cytosol in response to elevated cAMP levels $\text{in vivo}$. Cycloheximide abolished the saturation of cAMP binding $\text{in vivo}$ as well as tumor regression produced by DBcAMP.     

Benzodiazepine binding in human brain: characterization using [3H]flunitrazepam.

[³H]Flunitrazepam was used to characterize benzodiazepine binding sites in human brain. The benzodiazepine binding sites exhibited high affinity, pharmacological specificity and saturability in their binding of [³H]flunitrazepam. The dissociation constant (K_D) of [³H]flunitrazepam binding was determined by three different methods and found to be in the range of 2–3 nM. The potency of several benzodiazepine analogs to inhibit specific [³H]-flunitrazepam binding $\text{in}$$\text{vitro}$ correlated well with their potency in several $\text{in}$$\text{vivo}$ human and animal tests. The density of [³H]-flunitrazepam binding sites was highest in the cerebrocortical and rhinencephalic areas, intermediate in the cerebellum, and lowest in the brain stem and commissural tracts.     

Differences in macromolecular binding between cyclic AMP and its dibutyryl derivative in vitro

Rat liver cytosol binds ³H-cAMP and ³H-DBcAMP $\text{in vitro}$. Fractionation of bound radioactivity by DEAE-Sephadex chromatography shows that ³H-cAMP is associated with a different cytosolic protein than is ³H-DBcAMP. The pI's of the cAMP-protein and the ³H-DBcAMP-protein complexes are 6.7 and 3.9, respectively. Competition studies between ³H-cAMP and its structural analogues have shown the following order of effectiveness in competing for binding sites in rat liver cytosol: cAMP > N⁶-MBcAMP > O²′-MBcAMP. No inhibition of ³H-cAMP binding was observed with 5′-AMP, adenosine, cGMP or DBcAMP. $\text{In vitro}$ binding experiments with rat serum has shown that only ³H-DBcAMP binds to any significant extent.     

Carcinogen-protein complexes in mammary gland after administration of 3-methylcholanthrene

Twenty-four hrs after i.p. administration of ³H-3-methylcholanthrene (³H-MCA) to 8–10 weeks old virgin or 14 days pregnant $\text{BALB}\text{c}$ mice, the macromolecules and ³H-hydrocarbon-protein complexes in their mammary cytosols were extensively resolved according to molecular size. The profiles of the macromolecules at both stages of mammary development were generally similar, and contained at least five families of molecular size of modes: > 300,000, 181,000, 88,000, 44,000 and 27,000 daltons. At least three species of ³H-hydrocarbon-protein complex were present. The principal complex had a modal molecular weight of 83,000, and the two minor species > 300,000 and 47,000. Only a small amount of complex similar to the principal species was produced $\text{in vitro}$ by incubation of ³H-MCA with cytosol at 1–4°, indicating that the carcinogen is probably metabolized prior to interaction $\text{in vivo}$ with its principal target protein.     

Preliminary characterization of a small intestinal binding component for retinol and fatty acids in the rat

A component which can bind retinol and fatty acids was detected in the rat's intestinal cell cytosol following intestinal perfusion $\text{in}$$\text{vivo}$ with ³H-all-trans retinol. Following Sephadex G-100 filtration of the cytosol, the void volume concentrate was treated with 2-mercaptoethanol and SDS. Sephadex G-100 filtration of the concentrate disclosed the presence of a cytosol binder of an approximate molecular weight of 12,000–17,000. The binder contained most of the ³H-retinol eluted off the column. $\text{In}$$\text{vitro}$ incubation experiments disclosed that ³H-retinol could be displaced from its bindinf cytosol fraction by the addition of nonradioactive retinol, retinyl acetate, and the fatty acids octanoic, linoleic, and linolenic. Butyric acid addition did not displace ³H-retinol from its binding fraction. The intestinal cytosol binding fraction may be involved in the trans-cytosol transport of lipid compounds from the lipid cell membrane to the intracellular organelles.     

In vitro and in vivo inhibition by zopiclone of benzodiazepine binding to rodent brain receptors.

Up to now the only drugs known to be able to inhibit the binding of benzodiazepines to rodent brain receptors are members of this chemical family.Zopiclone (RP 27 267), a new drug with a pharmacological profile similar to that of chlordiazepoxide and nitrazepam but entirely different chemically from benzodiazepines, has been tested for its ability to inhibit benzodiazepine binding. $\text{In vitro}$ and $\text{in vivo}$ studies have shown that zopiclone is able to inhibit the binding of [³H] diazepam and [³H] flunitrazepam to brain receptors. The potency of zopiclone is quite comparable to that of diazepam and nitrazepam $\text{in vitro}$ and to that of chlordiazepoxide $\text{in vivo}$.These results confirm the pharmacological similarities existing between zopiclone and the benzodiazepines.     

Specific binding protein for 1,25-dihydroxyvitamin D3 in bovine mammary gland

Specific binding proteins for 1,25-dihydroxyvitamin D₃ were identified in bovine mammary tissue obtained from lactating and non-lactating mammary glands by sucrose density gradient centrifugation. The macromolecules had characteristic sedimentation coefficients of 3.5-3.7 S. The interaction of l,25-dihydroxy[³H]vitamin D₃ with the macromolecule of the mammary gland cytosol occurred at low concentrations, was saturable, and was a high affinity interaction (K_d = 4.2 × 10^?10M at 25 °C). Binding was reversed by excess unlabeled 1,25-dihydroxyvitamin D₃, was destroyed by heat and/or incubation with trypsin. It is thus inferred that this macromolecule is protein as it is not destroyed by ribonuclease or deoxyribonuclease. 25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃, and vitamin D₃ did not effectively compete with 1,25-dihydroxyvitamin D₃ for binding to cytosol of mammary tissue at near physiological concentrations of these analogs, thus demonstrating the specificity of the binding protein for 1,25-dihydroxyvitamin D₃. In vitro subcellular distribution of 1,25-dihydroxy[³H]vitamin D₃ demonstrated a time- and temperature-dependent movement of the hormone from the cytoplasm to the nucleus. By 90 min at 25 °C 72% of the 1,25-dihydroxy[³H]vitamin D₃ was associated with the nucleus. In addition a 5–6 S macromolecule which binds 25-hydroxy[³H]vitamin D₃ was demonstrated in mammary tissue. Finally, it is possible that the receptor-hormone complex present in mammary tissue may function in a manner analogous to intestinal tissue, resulting in the control of calcium transport by 1,25-dihydroxyvitamin D₃ in this tissue.     

Naloxone antagonism of phenoxybenzamine antinociception in the mouse tail stimulation test.

Phenoxybenzamine was antinociceptive in the mouse tail electrical stimulation assay (ED50, 36.8 mg/kg) with a peak effect at $\text{2}\text{1}\text{2}$ hours after subcutaneous injection. Naloxone antagonized this antinociception action of phenoxybenzamine in a dose-related manner. Dose-ratio analysis of naloxone's antagonism of phenoxybenzamine antinociception gave a pA₂ value of 6.15, similar to that found for the benzomorphinan mixed agonist-antagonists. This is in agreement with the sodium response ratio found for phenoxybenzamine, 4.3, in $\text{in vitro}$ assays of phenoxybenzamine inhibition of ³H-naloxone binding to mouse brain homogenate (5). These findings suggest that phenoxybenzamine binds to the opiate receptor both $\text{in vivo}$ as well as $\text{in vitro}$ in a manner similar to the mixed agonist-antagonists.     

Demonstration of [3H] diazepam binding to benzodiazepine receptors in vivo.

Benzodiazepine receptors were labeled with [³H] diazepam following intravenous injection in rats. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ to rat forebrain membranes was displaceable by co-injection of clonazepam or the pharmacologically active enantiomers of two benzodiazepines, B9 and B10, but was not displaced by equal doses of the pharmacologically in-active enantiomers. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ was bserved in kidney, liver, and abdominal muscle, but was not stereospecifically diplaced in any peripheral tissue studied. The regional distribution of benzodiazepine receptors in brain was uneven, with specific [³H] diazepam binding being highest in the cerebral cortex and lowest in the ponsmedulla. Preliminary studies of the subcellular distribution of [³H] diazepam binding demonstrated highest specific binding to synaptosomal membranes. These data demonstrate the feasibility of labeling benzodiazepine receptors in rat brain $\text{in}$$\text{vivo}$.     

Androgen on the estrogen receptor I — Binding and in vivo nuclear translocation

In the immature rat uterus, high concentrations of androgens competed specifically with estradiol on the estrogen receptor (RE). This competition was stereospecific for C₁₉ steroids bearing a 17β and/or 3 hydroxyl group. Very low affinity ligands, such as testosterone, could not compete with estradiol at equilibrium but decreased the association rate of estradiol on its receptor. High doses (> 0.4mg) of 5 α aihydrotestosterone provoked $\text{in vivo}$ as $\text{in vitro}$ the nuclear translocation of RE. The nuclear receptor thus formed displayed the same 5.2 S sedimentation constant as that induced by estradiol. We conclude that the weak affinity binding of androgens to the estrogen receptor is sufficient to induce its nuclear translocation $\text{in vivo}$ provided androgen concentration is high enough in uterus to occupy the estradiol binding site. Conversely, progesterone which does not bind RE could not provoke its nuclear translocation.     

Interactions of chlorinated hydrocarbon pesticides with the 8S estrogen-binding protein in rat testes

The effect of certain DDT analogs on the binding of ³H-estradiol to the 8–9S estrogen binding protein of rat testicular cytosol was studied by sucrose sedimentation analysis. The binding of ³H-estradiol to testicular cytosol was inhibited by o,p'DDT, a DDT analog which is estrogenic in the intact female, but not by p,p'DDE which is a nonestrogen in the female. The pesticide methoxychlor, which is estrogenic $\text{in vivo}$ in the female, failed to inhibit ³H-estradiol binding, presumably requiring metabolic activation for binding to the testicular cytosol. In fact, its di-demethylated metabolite 2,2-bis(p-hydroxyphenyl)-1, 1,1-trichloroethane (HPTE), also estrogenic $\text{in vivo}$, caused $\text{marked}$ suppression of ³H-estradiol binding.     

Norharman inhibition of [3H]diazepam binding in mouse brain

Norharman competitively inhibits specific binding of [³H]-diazepam in mouse brain homogenates. $\text{In vivo}$ this β-carboline produces a striking rigid catatonic-like appearance which is abolished by diazepam. It also causes a rapid tremor but has little anticonvulsant effect. Measurement of $\text{in vivo}$ concentrations and receptor occupancy demonstrate that these biological effects occur at doses which occupy a large proportion of benzo-diazepine receptors. It may represent a ligand of the benzo-diazepine receptors whose effects are opposite those of diazepam.