Involvement of norepinephrine activity in the regulation of alpha 1 adrenergic receptors in the medial preoptic nucleus of estradiol-treated rats

To establish whether the diurnal decrease in the density of alpha 1 receptors observed in the medial preoptic nucleus (MPN) of estrogen (E2)-treated rats is related to the concomitant diurnal increase in norepinephrine (NE) turnover rates, we quantitated the density of [3H]-Prazosin binding to alpha 1 receptors after blockade of NE turnover with alpha-methyl-paratyrosine (alpha MPT). A series of preliminary studies was performed to rule out an interference of this drug with [3H]-Prazosin binding to alpha 1 adrenergic receptors in vitro and in vivo. Incubation of brain slices with alpha MPT produced a dose-dependent inhibition of [3H]-Prazosin binding to alpha 1 adrenergic receptors with an IC50 of approximately 6 mM. Scatchard analysis demonstrated that alpha MPT exhibited a simple competitive interaction with [3H]-Prazosin binding sites as shown by an increase in the apparent dissociation constant (Kd) of the ligand and no change in the number of alpha 1 receptors (Bmax). In contrast, preincubation of brain slices with alpha MPT and prior in vivo administration of alpha MPT did not affect [3H]-Prazosin binding to alpha 1 adrenergic receptors. Once we established that alpha MPT could be used to suppress NE turnover without interfering with the measurement of alpha 1 receptor densities, we repeatedly injected this drug to ovariectomized (OVX) and E2-implanted rats. The density of alpha 1 adrenergic receptors in MPN was quantitated autoradiographically. Blockade of NE turnover with alpha MPT only partially prevented the reduction in alpha 1 receptor density observed in the E2-treated rats, suggesting that the decrease in the level of [3H]-Prazosin binding sites cannot be completely ascribed to increased NE turnover rates.     

Anti-idiotypic and anti-anti-idiotypic responses to a monoclonal antibody directed to the acetylcholine receptor binding site of curaremimetic toxins.

Serotherapy, an approach currently used to protect humans against animal bites or stings, is often too specific. To broaden antiserum paraspecificity, use of antibodies directed against areas shared by all members of a toxin family was previously proposed. MST2 is a mAb that recognizes all long-chain curaremimetic toxins (Charpentier et al. (1990) J. Mol. Recog. 3, 74-81). It binds to toxin residues that make contact with the toxin's target, e.g., the nicotinic acetylcholine receptor (AcChoR). We now show that MST2 also recognizes (-) nicotine, an agonist of AcChoR. Binding properties of MST2 therefore mimick, at least partially, binding properties of AcChoR. Injection in rabbits of MST2 mixed with adjuvant, elicited anti-idiotypic (anti-Id) antibodies that inhibited binding of the toxin to AcChoR. A proportion of these anti-Id antibodies specifically bound AcChoR and thereby mimicked the toxin. Furthermore, rabbits immunized with MST2 elicited auto-anti-anti-Id antibodies capable of binding the toxin. Our data provide a molecular explanation for the previously reported signs of myasthenia gravis as triggered by antibodies raised against cholinergic antagonists. Implications in the design of antisera to toxic proteins are discussed.     

Specific Binding of [3H]Pyrilamine to Histamine H1 Receptors in Guinea Pig Brain In Vivo: Determination of Binding Parameters by a Kinetic Four-Compartment Model

The binding of [3H]pyrilamine, a selective ligand of histamine H1 receptors, to guinea pig brain in vivo was compared with its binding to a brain homogenate. The pharmacological properties (regional distribution, saturability, and stereoselectivity) of the [3H]pyrilamine binding in vivo were similar to those of the in vitro binding to brain homogenate. A dynamic four-compartment model was proposed for the analysis of the kinetics of [3H]pyrilamine binding in vivo. The receptor constants in vivo were determined by a computer-fitting method after correcting the radioactivity of arterial plasma and brain for the presence of radioactive metabolites. The in vivo association and dissociation were 213 and 42 times, respectively, slower than those of in vitro binding at 37 degrees C. A possible mechanism for slow association and dissociation in vivo is discussed.     

Interaction of hexachlorobenzene with the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in vitro and in vivo. Evidence that hexachlorobenzene is a weak Ah receptor agonist

Hexachlorobenzene (HCB) produces hepatic porphyria and induces the hepatic cytochrome P450 isozymes P450c (P450IA1) and P450d (P450IA2) in rodents. These and other effects of HCB resemble those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which acts via its binding to the aromatic hydrocarbon (Ah) receptor. We therefore examined the ability of HCB to interact with this receptor in vitro and in vivo. HCB, at concentrations of 1 microM or higher, inhibited the specific binding of [3H]TCDD (0.3 nM) to the Ah receptor in vitro, whereas the solubility of [3H]TCDD was affected only at 100 microM HCB. The inhibition was competitive, with a KI of approximately 2.1 microM. In rats fed a diet containing 3000 ppm HCB for varying times (4 h to 7 days), the specific binding of [3H]TCDD in hepatic cytosol was reduced by up to 40%, as observed previously for known Ah receptor agonists. The decrease in [3H]TCDD specific binding in cytosol of HCB-treated rats was due principally to a decrease in the number of binding sites for [3H]TCDD rather than competition from residual HCB. As shown by immunoblotting and radioimmunoassay, HCB induced the cytochrome P450 isozymes P450c and P450d, which are regulated by the Ah receptor, as well as the phenobarbital-inducible isozymes P450b and P450e. Together these results indicate that HCB is a weak agonist for the Ah receptor, and suggest that some of its effects may be mediated by its interaction with this gene-regulatory protein.     

Ontogenesis of α2-Adrenoceptor Coupling with GTP-Binding Proteins in the Rat Telencephalon

The ontogenesis of alpha 2-adrenoceptors and GTP-binding proteins and their coupling activity were investigated in telencephalon membranes of developing rats. The manganese-induced elevation of [3H]clonidine binding was increased in an age-dependent manner but the guanosine 5'-O-(3-thio)triphosphate-induced decrease in binding did not change. The extent of the binding of [3H]clonidine at 15 nM (saturable concentration) increased in an age-dependent manner and reached the adult level at 4 days after birth. Cholera toxin and pertussis toxin catalyzed ADP-ribosylation of proteins of 46 and 41/39 kilodaltons (kDa) in solubilized cholate extracts of the membranes. The 41/39-kDa proteins ADP-ribosylated by pertussis toxin (Gi alpha + Go alpha) were increased with age and reached the adult level at day 12, whereas the 46-kDa protein (Gs alpha) reached its peak on day 12 and then decreased to the fetal level at the adult stage. The immunoblot experiments of the homogenates with antiserum (specific antibody against alpha- and beta-subunit of GTP-binding proteins) demonstrated that the 39-kDa alpha-subunit of (Go alpha) and the 36-kDa beta-subunit of GTP-binding protein (beta 36) increased with postnatal age. In contrast, 35-kDa beta-subunit (beta 35) did not change. From these results, it is suggested that the coupling activity of alpha 2-adrenoceptor with GTP-binding protein gradually develops in a manner parallel with the increase of alpha 2-adrenoceptor and pertussis toxin sensitive GTP-binding proteins, Gi, and that alpha 39 beta 36 gamma may be related to the differentiation and/or growth of nerve cells in rat telencephalon.     

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase (III. Is There a Direct Interaction between the Fusicoccin Receptor and the Plasma Membrane H+-ATPase?)

A radioimmunoassay using antibodies raised against bovine serum albumin-conjugated fusicoccin (FC) was applied to measure FC bound to the plasma membrane (PM) isolated from seedlings of radish (Raphanus sativus L.) and of Arabidopsis thaliana treated in vivo plus or minus the toxin. FC bound to the PM from seedlings treated with 5 [mu]M FC was 2-fold (radish) to 7-fold (A. thaliana) higher than the binding capacity of control PM. FC binding depended on the duration of the in vivo treatment but was unaffected by cycloheximide. When FC binding and the PM H+-ATPase activity were compared under different conditions (in vivo or in vitro treatment of different lengths or with different concentrations of FC), a strict linear relation between FC binding and the activation of the PM H+-ATPase was observed in both plant materials under all the conditions tested. Comparison between the maximum binding capacity and the amount of H+-ATPase observed in PM from the two plant materials suggest a one-to-one stoichiometry between the FC receptor and the PM H+-ATPase.     

Modulation of dopaminergic neurotransmission in rat striatum upon in vitro and in vivo diclofenac treatment

Diclofenac (DCF) is a widely used non-steroidal anti-inflammatory drug, which also act as a mitochondrial toxin. As it is known that selective mitochondrial complex I inhibition combined with mild oxidative stress causes striatal dopaminergic dysfunction, we tested whether DCF also compromise dopaminergic function in the striatum. [3H]Dopamine ([3H]DA) release was measured from rat striatal slices after in vitro (2 h, 10-25 micromol/L) or in vivo (3 mg/kg i.v. for 28 days) DCF treatment. In vitro treatment significantly decreased [3H]DA uptake and dopamine (DA) content of the slices. H2O2 (0.1 mmol/L)-evoked DA release was enhanced. Intracellular reactive oxygen species production was not significantly changed in the presence of DCF. After in vivo DCF treatment no apparent decrease in striatal DA content was observed and the uptake of [3H]DA into slices was increased. The intensity of tyrosine hydroxylase immunoreactivity in the striatum was highly variable, and both decrease and increase were observed in individual rats. The H2O2-evoked [3H]DA release was significantly decreased and the effluent contained a significant amount of [3H]octopamine, [3H]tyramine, and [3H]beta-phenylethylamine. The ATP content and adenylate energy charge were decreased. In conclusion, whereas in vitro DCF pre-treatment resembles the effect of the mitochondrial toxin rotenone, in vivo it rather counteracts than aggravates dopaminergic dysfunction.     

The pharmacological action of MT-7

The mamba toxin MT-7 is the most selective ligand currently available for the muscarinic M1 receptor subtype. The toxin binds stably to the receptor and blocks the agonist-induced activation non-competitively. Although its mode of action on M1 receptors is not yet fully understood, some of the toxin properties support an allosteric mechanism. Thus, the toxin fails to elicit a complete inhibition of the binding of either the muscarinic antagonist [3H]N-methyl-scopolamine ([3H]NMS) or the agonist [3H]acetylcholine ([3H]ACh). When added to ligand-occupied M1 receptors, the toxin slows the dissociation rate of [3H]NMS and increases that of [3H]ACh. Site-directed mutagenesis studies have provided important information about the toxin amino acid residues which are critical for the stable binding to the receptor and for the allosteric modulation of antagonist dissociation. In vivo studies have shown that the intracerebral injection of MT-7 causes a long-lasting blockade of M1 receptor, thus providing a tool for the characterization of the functional role of this receptor subtype in discrete brain areas.     

Identification and characterization of the ligand-binding domain of insulin receptor by use of an anti-peptide antiserum against amino acid sequence 241-251 of the alpha subunit

We previously reported that a 23-kDa receptor proteolytic fragment containing an insulin-binding site was localized within residues 205-316 in the cysteine-rich region of the insulin receptor alpha subunit and postulated that sequence 241-251 plays a major role in insulin binding [Yip, C. C., Hsu, H., Patel, R. G., Hawley, D. M., Maddux, B. A., & Goldfine, I.D. (1988) Biochem. Biophys. Res. Commun. 157, 321-329]. In the present study, we have used an antiserum raised against a synthetic peptide containing sequence 241-251 to test this postulate and to study the role of sequence 241-251 in insulin binding. The antiserum immunoprecipitated the 23-kDa fragment, confirming our sequence assignment of this fragment. It also immunoprecipitated the intact alpha subunit of the insulin receptor that had been denatured by reduction and alkylation. However, sequence 241-251 in the native receptor was inaccessible to the antiserum since the antiserum did not block [125I]iodoinsulin binding and did not precipitate either photoaffinity-labeled insulin receptors or insulin receptors labeled with 125I. However, using a radioactive photoaffinity probe [( 125I]-AZAP-insulin) that allows cleavage and removal of insulin after photolabeling, we found that sequence 241-251 became accessible to the antiserum after removal of insulin. We conclude therefore that sequence 241-251 forms part of the insulin-binding domain of the insulin receptor and that the binding of insulin to the receptor induces a conformational change that allows exposure of this domain after removal of insulin. Such a conformational change may play a role in activation of the receptor and transmembrane signaling.     

In vitro and in vivo binding of toremifene and its metabolites in rat uterus

The in vitro binding affinities of toremifene (TOR), 4-hydroxy toremifene (4-OH-TOR) and several other metabolites for the rat uterine cytosolic estrogen receptor were compared with those of tamoxifen (TAM) and 4-hydroxy tamoxifen (4-OH-TAM). Only small differences were observed and the binding affinities of both 4-hydroxy metabolites were similar to that of estradiol (E2). Uterine uptake and subcellular distribution of [3H]TOR and [3H]TAM were then compared at 1, 8 and 72 h after administration to castrated rats. The uptake and retention of both antiestrogens were similar at all times. In each case the amount of nuclear bound radioactivity declined to low levels at 8 and 72 h but the ratios of 4-OH-TAM/TAM and 4-OH-TOR/TOR determined by HPLC analysis increased dramatically at 72 h. The level of radioactivity in both plasma and uterine cytosol at 72 h was significantly higher following [3H]TAM administration. However, most of the radioactivity appeared to be in a conjugated form since it was not extractable with solvent. Finally, the ability of prior administration of each antiestrogen (100 mg/kg) to block uterine [3H]estradiol uptake was examined at 3 and 7 days. It was found that uterine wet weights were higher than control one week after administration of both compounds. Prior administration of TOR increased nuclear uptake of [3H]E2 whereas TAM had no effect. The results of these experiments suggest that toremifene and tamoxifen have very similar in vitro and in vivo binding properties but differences in metabolism exist that may be important.     

Aflatoxin inhibition of glucocorticoid binding capacity of rat liver nuclei

The effect of aflatoxin B₁ on the binding capacity of rat liver cytoplasmic glucocorticoid receptors and the nuclear binding of the activated receptor complex was investigated. No alterations in the kinetics of [³H]dexamethasonccytosol receptor complex formation were noted 2 h after treatment with 1 mg/kg aflatoxin B₁. However, a 33% decrease in the concentration of nuclear acceptor sites and a 24% decrease in the glucocorticoid receptor-nuclear binding equilibrium constant of dissociation was observed. This response was near maximal at 2 h and persisted for at least 36 h. Inhibition of nuclear binding capacity was directly related to aflatoxin B₁ dose, with a correlation coefficient of 0.99. Actinomycin D treatment (0.1 mg/kg) resulted in a slight reduction (16%) in the concentration of nuclear acceptor sites but had no effect on the nuclear binding dissociation constant.Administration of [³H]dexamethasone to aflatoxin B₁-treated rats produced a similar pattern of glucocortocoid binding distribution in vivo to that observed in vitro. No differences in [³H]dexamethasone-cytoplasmic receptor binding between control and aflatoxin B₁-treated rats were found, whereas nuclear [³H]dexamethasone binding was reduced 34% by aflatoxin B₁ treatment.     

In vitro and in vivo pharmacological profile of 5-[2-[4-(6-fluoro-1H-indole-3-yl)piperidin-1-yl]ethyl]-4-(4-fluorophenyl)thiazole-2-carboxylic acid amide (NRA0562), a novel and putative atypical antipsychotic

In vitro and in vivo pharmacological properties of 5-[2-[4-(6-fluoro-1H-indole-3-yl)piperidin-1-yl]ethyl]-4-(4-fluorophenyl)thiazole-2-carboxylic acid amide (NRA0562), a novel atypical antipsychotic, were investigated. NRA0562 showed high affinities for human cloned dopamine D(1), D(2), D(3) and D(4) receptors with Ki values of 7.09, 2.49, 3.48 and 1.79 nM. In addition, NRA0562 had high affinities for the 5-HT(2A) receptor and the alpha(1) adrenoceptor with Ki values of 1.5 and 0.56 nM, and moderate affinity for the histamine H(1) receptor. Using in vivo and ex vivo receptor binding studies in rats, we showed NRA0562 occupied frontal cortical 5-HT(2A) receptors and alpha(1) adrenoceptor potently, while occupancy of striatal dopamine D(2) receptor was moderate as were other atypical antipsychotics. NRA0562 dose-dependently inhibited methamphetamine (MAP)-induced locomotor hyperactivity in rats. At higher dosage, NRA0562 dose-dependently antagonized MAP-induced stereotyped behavior and induced catalepsy dose-dependently and significantly in rats. But, the ED(50) value in inhibiting MAP-induced locomotion hyperactivity was 10 times lower than that in inhibiting MAP-induced stereotyped behavior, and 30 times lower than that in inducing catalepsy. In addition, the potency of NRA0562 in antagonizing MAP-induced hyperactivity in rats was higher than that of other antipsychotics, clozapine, risperidone and olanzapine. NRA0562 had favorable properties in view of prediction of extrapyramidal side effects. As this antipsychotic has a unique profile with affinity and occupancy for receptors, we propose that NRA0652 may have unique atypical antipsychotic activities, and a moderate liability of extrapyramidal motor side effects seen in the treatment with classical antipsychotics.     

Pituitary and brain D2 receptor density measured in vitro and in vivo in EEDQ treated male rats.

The effect of the alkylating compound N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) (20 mg/kg, 24 h) on dopamine D2 receptor density in rat pituitary and brain was measured using in vitro and in vivo radioligand binding techniques. In the in vitro radioligand binding experiments EEDQ was found to reduce the density (Bmax) of [3H]-spiperone binding sites in the striatum by 86% while in the pituitary the corresponding decrease was only 37%. The affinity (KD) of the remaining striatal and pituitary D2 receptors was not different in EEDQ treated animals as compared to controls. When D2 receptor density was measured in vivo the effect of EEDQ was less pronounced. Thus, in rats given EEDQ the specific binding of either of the two D2 ligands [3H]-raclopride or [3H]-spiperone (administered in a single dose) in striatum and in the limbic forebrain was reduced by 45-62%; moreover, no significant decrease in pituitary D2 receptor density was observed. The data are discussed in relation to the finding (presented in a separate paper) that the same dose of EEDQ that failed to influence pituitary D2 receptor density as measured in vivo effectively antagonizes the prolactin decreasing effect of the partial D2 agonist (-)-3-(3-hydroxyphenyl)-N-n-propyl-piperidine [(-)-3-PPP].     

On the molecular mechanisms of neutralization of a cobra neurotoxin by specific antibodies

Examination of 76 homologous neurotoxin sequences suggested that the "toxic" domain of these compounds consists of twelve highly conserved residues. Five of these, namely Lys-27, Trp-29, Asp-31, Arg-33 and Glu-38, together with a variant residue at position 36 are organized into a pattern which resembles that of d-tubocurarine. Two lines of experimental evidence are in agreement with the proposed topology of the "toxic" site in Naja nigricollis toxin alpha--Three highly conserved residues (Lys-27, Trp-29 and Lys-47) have been modified individually in toxin alpha. These modifications induce a decrease in binding affinity of toxin alpha for its target, the nicotinic acetylcholine receptor. In contrast, modifications of three residues (Leu-1, Lys-15 and Lys-51) excluded from the "toxic" domain, do not alter the binding properties of toxin alpha.--Five toxin derivatives carrying a nitroxide group at residues 1, 15, 27, 47 or 51 have been prepared. ESR spectra have been recorded for each derivative in both the free state and bound to the receptor. Mobility of the probes of the residues excluded from the "toxic" site is not altered upon receptor binding. In contrast mobility of the nitroxide of the presumed "toxic" Lys-47 becomes markedly reduced after toxin receptor complex formation. Lys-27 nitroxide is immobilized in both the free and bound state. The antigenic structure of N. nigricollis toxin alpha has been partially clarified using two different approaches. --Fifteen antigenically important residues of toxin alpha have been identified by analyzing cross-reactions between toxin alpha and eleven homologous neurotoxins, using polyclonal antibodies.--- One monoclonal antibody (M alpha 1) specific for toxin alpha has been prepared. Competition experiments, made with (3H) toxin alpha, six mono modified toxin derivatives or alpha three homologous neurotoxins, showed that the binding site of (M alpha 1) comprises the N-terminal group, Lys-15, Pro-18 and probably Thr-16. This site is topographically different from the "toxic" domain. (M alpha 1) inhibits the toxicity of toxin alpha under both in vivo and in vitro conditions. In addition, (M alpha 1) is capable of "removing" toxin molecules bound to the receptor, allowing a rapid recovery of the functional properties of the receptor.     

Aging: Effect on ex-vivo benzodiazepine binding after a diazepam injection

Ex vivo [³H]flunitrazepam receptor occupation was determined in the brain of young, mature and old male Fischer 344 rats after a single intravenous injection of a low dose of diazepam. The two benzodiazepine receptor subtypes or conformations (BZ₁ and BZ₂) were differentiated by the displacement of [³H]flunitrazepam specific binding with the triazolopyridazine, CL 218,872. The acute diazepam injection decreased ex vivo [³H]flunitrazepam binding in only the senescent rats. The [³H]flunitrazepam binding at both the BZ₁ and BZ₂ receptor or receptor conformation was significantly reduced in the old rats.     

Increase in receptor binding affinity for nimodipine in the rat brain with permanent occlusion of bilateral carotid arteries

The permanent occlusion of bilateral common carotid arteries (2VO) in rats has been shown to cause progressive and long-lasting cognitive deficits which may be due to impairment of memory retention and/or memory recall process. To clarify the function of voltage dependent calcium channels and the receptor binding of nimodipine by chronic cerebral ischemia, we examined specific (+)-[3H]PN 200-110 binding and the effect of oral administration of nimodipine in brain regions and hearts of rats, at 2 weeks to 4 months after permanent 2VO. There was no significant difference in either dissociation constant (Kd) or maximal number of binding sites (Bmax) for (+)-[3H]PN 200-110 in the cerebral cortex, hippocampus, corpus striatum and thalamus between 2VO and sham rats. In addition, in vitro inhibitory effect of nimodipine on cerebral cortical (+)-[3H]PN 200-110 binding in 2VO rats was similar to that in sham rats. Compared to control rats, oral administration of nimodipine to both 2VO and sham rats at 2 months after permanent 2VO brought about a significant increase in Kd values of specific (+)-[3H]PN 200-110 binding in the cerebral cortex, hippocampus, thalamus and myocardium, and the increase in Kd values was much larger in brain regions of 2VO rats than sham rats. However, the increase in Kd values in the myocardium did not differ between 2VO and sham rats. This observation suggests an increased in vivo binding affinity for nimodipine in chronic ischemic brain. In conclusion, the present study has shown that oral administration of nimodipine may cause a greater occupation in vivo of 1,4-dihydropyridine (DHP) calcium channel antagonist receptors in brains of permanent 2VO rats than in sham rats. Thus, nimodipine may be pharmacologically effective in preventing brain dysfunction due to cerebral ischemia in vivo.     

Aflatoxin inhibition of glucocorticoid finding capacity of rat liver nuclei.

The effect of aflatoxin B1 on the binding capacity of rat liver cytoplasmic glucocorticoid receptors and the nuclear binding of the activated receptor complex was investigated. No alterations in the kinetics of [3H]desamethasone-cytosol receptor complex formation were noted 2 h after treatment with 1 mg/kg aflatoxin B1. However, a 33% decrease in the concentration of nuclear acceptor sites and a 24% decrease in the glucocorticoid receptor-nuclear binding equilibrium constant of dissociation was observed. This response was near maximal at 2 h and persisted for at least 26 h. Inhibition of nuclear binding capacity was directly related to aflatoxin B1 dose, with a correlation coefficient of 0.99. Actinomycin D treatment (0.1 mg/kg) resulted in a slight reduction (16%) in the concentration of nuclear acceptor sites but had no effect on the nuclear binding dissociation constant. Administration of [3H]dexamethasone to alfatoxin B1 -treated rats produced a similar pattern of glucocorticoid binding distribution in vivo to that observed in vitro. No differences in [3H]dexamethasone-cytoplasmic receptor binding between control and alfatoxin B1 -treated rats were found, whereas nuclear [3H]dexanthasone binding was reduced 34% by alfatoxin B1 -treatment.     

Arrangement of the subunits of the nicotinic acetylcholine receptor of Torpedo californica as determined by alpha-neurotoxin cross-linking

[3H]Methyl-alpha-neurotoxin prereacted with dithiobis(succinimidyl propionate) (DTSP) can be covalently linked to each of the subunits of the nicotinic acetylcholine receptor in membranes from the electric tissue of Torpedo californica. Pronounced changes in the cross-linking pattern are observed upon prior incubation with receptor specific ligands and upon reduction and/or alkylation of the receptor. d-Tubocurarine has been shown to bind to two different sites in receptor-rich membranes. These sites are present in equal numbers but have different affinities [Neubig, R. R., & Cohen, J. B. (1979) Biochemistry 18, 5464-5475; Sine, S., & Taylor, P. (1981) J. Biol. Chem. 256, 6692-6699]. Using d-tubocurarine inhibition of [3H]-methyl-alpha-neurotoxin binding, we demonstrate two inhibitory constants for d-tubocurarine of 67 +/- 21 nM and 4.9 +/- 1.7 microM in unreduced membranes. We utilize the large difference in Ki's to preferentially block toxin cross-linking at the high affinity site for d-tubocurarine. Low concentrations of this competitive antagonist selectively block the cross-linking of toxin to the beta and gamma subunits of the receptor, suggesting that these subunits are located close to the toxin binding site which is also the high-affinity binding site for d-tubocurarine. Reduction of disulfide bonds alters the affinity of the receptor for alpha-neurotoxin. Alterations are also seen in the cross-linking pattern of DTSP-activated [3H]methyl-alpha-neurotoxin to reduced and alkylated membranes in the presence of tubocurarine. The constants for d-tubocurarine inhibition of [3H]methyl-alpha-neurotoxin binding to reduced and alkylated membranes are 172 +/- 52 nM and 2.4 +/- 0.4 microM. The effects of bromoacetylcholine, carbamoylcholine, gallamine, and procaine on the cross-linking pattern are also examined. Our observations are consistent with an arrangement of the subunits in the membrane of alpha beta alpha gamma delta.     

In Vivo Labeling of Serotonin Uptake Sites with [3H]Paroxetine

Previous work has shown that [3H]paroxetine is a potent and selective in vitro label for serotonin uptake sites in the mammalian brain. In the present study, [3H]paroxetine was tested in mice as an in vivo label for serotonin uptake sites. Maximum tritium concentration in the whole brain (1.4% of the intravenous dose) was reached 1 h after injection into a tail vein. Distribution of the tracer at 3 h after injection followed the distribution of serotonin uptake sites known from previous in vitro binding studies (r = 0.85). The areas of highest [3H]paroxetine concentration, in decreasing order, were: hypothalamus greater than frontal cortex greater than olfactory tubercles greater than thalamus greater than upper colliculi greater than brainstem greater than hippocampus greater than striatum greater than cerebellum. Preinjection of carrier paroxetine (1 mg/kg) significantly decreased [3H]paroxetine concentration in all areas except in the cerebellum, which is known to contain a relatively low number of specific binding sites. Kinetic studies showed highest specific [3H]paroxetine binding (tissue minus cerebellum) at 2 h after injection and slow clearance of activity thereafter (half-time of dissociation from the hypothalamus, 215 min). The specificity of in vivo [3H]paroxetine binding was studied by preinjecting monoamine uptake blockers or receptor antagonists 5 min before administration of [3H]paroxetine. Serotonergic or muscarinic cholinergic receptor antagonists and dopamine or norepinephrine uptake blockers did not reduce the in vivo binding of [3H]paroxetine. In contrast, there was an excellent correlation (r = 0.99) between the in vivo inhibitory potencies of serotonin uptake blockers in this study and previously published in vitro data on inhibition of [3H] serotonin uptake in brain synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of estrogen receptor complexes with the promoter region of genes that are negatively regulated by estrogens: the alpha 2u-globulins.

Since estrogens strongly suppress the expression of alpha 2u-globulin genes in the rat liver, we studied the binding of estrogen-receptor complexes to fragments derived from alpha 2u-globulin gene RAO 01 using a DNA-cellulose competition assay. Rat uterus cytosol labelled with [3H]estradiol was used as a source of the estrogen receptor. As a positive control in these experiments we used an oligonucleotide containing the estrogen response element (ERE) cloned into pUC18. Our experiments indicate that estrogen-receptor complexes bind specifically to the ERE and to a fragment of RAO 01 located in the 5'-upstream region (bp -606 up to -575). This fragment is conserved among other members of this gene family. This is the first time that in vitro estrogen receptor binding is observed to gene fragments derived from a gene that is repressed by this steroid in vivo.