Characterization of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to GABAA receptors in postmortem human brain

BACKGROUND AND PURPOSE: The aim of the present study was to determine whether binding of [(35)S]t-butylbicyclophosphorothionate ([(35)S]TBPS) to the convulsant binding site of GABA(A) receptors in human postmortem brain samples can be used as an in vitro index of the functional activation of these receptors. EXPERIMENTAL APPROACH: Postmortem stability of [(35)S]TBPS binding was assessed in rat brain samples harvested at various times after death and the binding properties of [(35)S]TBPS binding (K(D) and B(max)) were determined in human postmortem brain using radioligand binding studies. In addition, the ability of human brain [(35)S]TBPS binding to be allosterically modulated by compounds that bind at recognition sites distinct from the convulsant binding site was measured. KEY RESULTS: Whereas binding of [(3)H]Ro 15-1788 to the benzodiazepine binding site and [(3)H]muscimol to the agonist (GABA) binding site were retained over a 20 h postmortem interval, there was a significant decrease in the affinity and number of [(35)S]TBPS binding sites. Nevertheless, [(35)S]TBPS binding in human brain could be inhibited by TBPS, picrotoxin, loreclezole and pentobarbital and modulated by GABA with potencies comparable to those observed in rats. In addition, the GABA-induced reduction in human brain [(35)S]TBPS binding could be modulated by benzodiazepine site ligands in a manner that reflected their intrinsic efficacies. CONCLUSIONS AND IMPLICATIONS: These results suggest that allosteric coupling between the [(35)S]TBPS, GABA and benzodiazepine binding sites is preserved in postmortem human brain and that [(35)S]TBPS binding in this tissue may be used to study functional characteristics of native human GABA(A) receptors.     

Pharmacokinetics of muscarinic cholinergic drugs as determined by ex vivo (3H)-oxotremorine-M binding

The pharmacokinetic parameters of muscarinic cholinergic drugs after intravenous (IV) and oral administration to mice was determined with ex vivo (3H)-oxotremorine-M (3H-Oxt) binding to the brain. Oxotremorine had a long duration of action, and arecoline had a short one. There was a significant correlation between the ex vivo ED50 and the in vitro inhibition constants (Ki). Tremorine, a prodrug, inhibited ex vivo binding, but was relatively inactive in in vitro binding. The quaternary amines, methylscopolamine and oxotremorine-M, and the hydrophilic compound, pirenzepine, were relatively weak in inhibiting ex vivo binding because of their poor penetration of the blood-brain barrier. Oxotremorine and BM-5 were similarly bioavailable to the brain by the IV and the oral route. These results indicate that the pharmacokinetic profile of muscarinic cholinergic drugs can be determined with ex vivo (3H)-Oxt binding.     

In vitro screening of psychoactive drugs by [(35)S]GTPgammaS binding in rat brain membranes

We constructed a reproducible, simple, and small-scale determination method of the psychoactive drugs that acted directly on the monoamine receptor by measuring the activation of [(35)S]guanosine-5'-O-(3-thio)-triphosphate binding to guanine nucleotide-binding proteins (G proteins). This method can simultaneously measure the effects of three monoamines, namely dopamine (DA), serotonin (5-HT), and norepinephrine (NE), in rat brain membranes using a 96-well microplate. Activation of D(1) and D(2) receptors in striatal membranes by DA as well as 5-HT and NEalpha(2) receptors in cortical membranes could be measured. Of 12 tested phenethylamines, 2,5-dimethoxy-4-chlorophenethylamine (2C-C), 2,5-dimethoxy-4-ethylphenethylamine (2C-E), and 2,5-dimethoxy-4-iodophenethylamine (2C-I) stimulated G protein binding. The other phenethylamines did not affect G protein binding. All 7 tryptamines tested stimulated G protein binding with the following rank order of potency; 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT)>5-methoxy-N,N-diallyltryptamine (5-MeO-DALT)>5-methoxy-alpha-methyltryptamine (5-MeO-AMT)>or=5-methoxy-N,N-methylisopropyltryptamine (5-MeO-MIPT)>5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT)>N,N-dipropyltryptamine (DPT)>or=alpha-methyltryptamine (AMT). This assay system was able to designate psychoactive drugs as prohibited substances in accordance with criteria set forth by the Tokyo Metropolitan government.     

Regulation of [35S]t-butylbicyclophosphorothionate binding sites in rat brain by GABA, pyrethroid and barbiturate

GABA regulates the binding of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) within the GABA receptor-ionophore complex by decreasing the rate of radioligand association and increasing the rate of dissociation but in different ways for the EDTA/water-dialyzed rat brain membranes and a solubilized preparation obtained on treatment with the zwitterionic detergent CHAPS. In the membranes, GABA at 0.3-1 microM is a non-competitive inhibitor of [35S]TBPS binding, affecting the density of binding sites but not the affinity of the receptor, while at 5 microM both the apparent density and affinity are significantly decreased. On treatment with CHAPS the solubilized preparation and the corresponding pellet fraction become less sensitive to GABA which even at 5 microM acts only as a non-competitive inhibitor. CHAPS solubilization decreases the sensitivity of the receptor-[35S]TBPS complex to GABA-induced dissociation. GABA at micromolar levels also greatly influences the action of compounds within the TBPS domain, facilitating and modulating displacement of [35S]TBPS from EDTA/water-dialyzed membranes by the alpha-cyanopyrethroid cypermethrin and the barbiturate 5-(1',3'-dimethylbutyl)-5-ethylbarbiturate. Large differences in the Hill numbers indicate that different mechanisms may be involved in GABA modulation of the pyrethroid and barbiturate sites. In contrast, GABA does not effect [35S]TBPS displacement by photoheptachlor epoxide which acts directly at the TBPS binding site.     

Action of polychlorocycloalkane insecticides on binding of [35S]t-butylbicyclophosphorothionate to Torpedo electric organ membranes and stereospecificity of the binding site

Binding of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) to Torpedo electric organ membranes was characterized. A dose- and pH-dependent binding (100.8 pmol/mg protein) was detected with a single affinity (Kd of 0.9 microM) in the presence of 150 mM KCl at pH 6.8. Other anions such as Br- and I- also increased binding affinity, but to a lower degree than Cl-, which increased the affinity by two- to threefold. In presence of 150 mM KCl, [35S]TBPS binding was inhibited noncompetitively by Zn2+ and by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) (IC50 of 9 microM). The gamma-isomer of hexachlorocyclohexane (BHC) was much more potent in inhibiting this [35S]TBPS binding and the inhibition was competitive (Ki = 40 nM). Like binding of [35S]TBPS to the gamma-aminobutyric acid (GABA) receptor, its binding to Torpedo membranes was inhibited by pentobarbital, mephobarbital, and hexobarbital (IC50 of 85, 225, and 300 microM), respectively), but not by phenobarbital. Binding was not inhibited by diazepam, GABA, bicuculline, or avermectin B1a, ligands that bind to the GABAA receptor. [35S]TBPS binding was inhibited by BHC isomers with the following decreasing order of potency alpha = gamma greater than sigma greater than beta, and by cyclodiene insecticides. Endrin was more potent than dieldrin, but endosulfan I and II had similar effects. The data suggest that the binding site for polychlorocycloalkane insecticides on this protein is much less stereoselective than that of the Cl- channel of the GABAA receptor. Also, even though this Torpedo protein has higher affinity for insecticides, such as gamma-BHC, than does the GABAA receptor, it is the latter whose specificity correlates best with polychlorocycloalkane toxicity. Nevertheless, because of its high affinity for gamma-BHC such a protein in muscles or brain may be an important target for the action of this insecticide.     

Anxiolytic cyclopyrrolone drugs allosterically modulate the binding of [35S]t-butylbicyclophosphorothionate to the benzodiazepine/gamma-aminobutyric acid-A receptor/chloride anionophore complex

The influence of a number of anxiolytic cyclopyrrolone drugs, which include zopiclone and suriclone, on the binding of [35S]t-butylbicyclophosphorothionate (TBPS), to benzodiazepine/gamma-aminobutyric acid-A receptor/chloride anionophore complexes has been characterized in rat brain. Suriclone and its metabolites RP35,489 and RP46,166 are the most potent (IC50 approximately 3nM) inhibitors of [35S]TBPS binding thus far described, about an order of magnitude more potent than TBPS itself. The pattern of inhibition of [35S] TBPS binding by suriclone is distinctive; at approximately 10 nM there is approximately 50% inhibition of [35S]TBPS binding and inhibition "plateaus" at this level until suriclone concentrations exceed 1 microM. RP35,489 and RP46,166 display patterns of inhibition similar to suriclone. In saturation studies of [35S]TBPS binding, suriclone reduces the Bmax of [35S]TBPS-binding sites, with little or no effect on KD. Muscimol also displays a noncompetitive pattern of inhibition of [35S]TBPS binding, whereas inhibition by picrotoxinin appears competitive. [35S]TBPS dissociation is multiphasic and similar whether initiated by 10 microM TBPS or 10 microM picrotoxinin. By contrast, dissociation of [35S]TBPS is much faster (and nearly monophasic) when initiated by 10 microM TBPS/100 nM suriclone, 10 microM TBPS/1 microM muscimol, or 10 microM TBPS/1 mM pentobarbital. These results suggest that suriclone influences [35S]TBPS binding allosterically, at sites distinct from the TBPS/picrotoxinin recognition site. Inhibition of [35S]TBPS binding by suriclone varies regionally with a "plateau" at approximately 20% inhibition in the cerebellum, approximately 50% in the cerebral cortex, hippocampus and brain stem, and approximately 65% in the striatum and midbrain; by contrast, inhibition of [35S] TBPS by picrotoxinin, muscimol, and pentobarbital shows little regional variation. The inhibition of [35S]TBPS binding by suriclone is reversed by bicuculline [ED50 approximately 1 microM] in several brain regions examined. Bicuculline alone has little or no influence on [35S]TBPS binding in the cerebral cortex, hippocampus, and cerebellum, but produces a dose-dependent enhancement of [35S]TBPS binding in the striatum, midbrain, and hypothalamus. Regional differences in the effects of suriclone and bicuculline on [35S]TBPS recognition sites suggest possible heterogeneity in the coupling of cyclopyrrolone and bicuculline recognition sites to [35S]TBPS recognition sites in rat brain.     

Effect of buflomedil on epinephrine-enhanced platelet aggregation in vitro and ex vivo

The effect of buflomedil (4-(1-pyrrolidinyl)-1-(2,4,6-trimethoxyphenyl)-1-butanone) in vitro and ex vivo after intravenous and oral administration was tested upon epinephrine-enhanced platelet aggregation (PA) in platelet-rich plasma (PRP) prepared from heparinized blood of healthy volunteers. In vitro incubation of PRP with buflomedil in concentrations above 10 mumol/l resulted in a significant depression of PA to approximately one-third of the control. 30 min after a single intravenous dose of 2.5 mg/kg buflomedil a depression of epinephrine-enhanced PA to about 60% of the value before injection of the drug was observed. This effect wore off during a few hours and was no longer present 24 h thereafter. Oral ingestion of 600 mg/d buflomedil depressed PA to approximately two-thirds within 2 days, with a further decrease to some 50% after 6 days of intake. 2 days after termination of treatment epinephrine-enhanced PA had returned to premedication values. Unlike nonsteroidal antiinflammatory drugs buflomedil does not act through an inhibition of prostaglandin synthesis.     

Effects of progesterone on [35S] t-butylbicyclophosphorothionate binding in some forebrain areas of the female rat and its correlation to aggressive behavior

The antiaggressive effects of progesterone (P) were evaluated in association with alterations in [35S] t-butylbicyclophosphorothionate (TBPS; chloride ion channel antagonist) binding in some forebrain sites of the female rat using in vitro quantitative autoradiography. The administration of 4 mg P was followed by a reduction in the frequency of different aggressive behaviors such as circling, nose-to-nose and fighting (mostly of the defensive nature) in ovariectomized (OVX) sexually mature rats, housed in pairs, during male-female encounters. Quantitative autoradiography data revealed that the same P dose, at the forebrain level, was responsible for low [35S] t-butylbicyclophosphorothionate binding levels in the medial preoptic area, lateral and basolateral amygdala nucleus and oriens-pyramidalis hippocampus CA1 layer, with even lower values being obtained following the in vitro addition of the potent P metabolite 5 alpha-pregnan-3 alpha-ol-20-one. These results suggests that the probable antiaggressive role of P during heterosexual encounters may be regulated by a local potent metabolite acting at the membrane site of the GABA complex.     

20(S)-原人参二醇对SMMC-7721细胞体内外作用的研究

目的观察不同剂量的20(S)-原人参二醇(Protopanaxadiol,PPD)在体内外对人肝癌细胞株SMMC-7721抗肿瘤作用。方法建立人肝癌裸鼠皮下移植瘤模型,观察20(S)-原人参二醇的肿瘤抑制作用。MTT比色法检测20(S)-原人参二醇对SMMC-7721细胞的增殖抑制作用,Ho-echst33342核染色观察细胞凋亡形态学改变,采用FITC-An-nexinⅤ/PI双染流式细胞术分析凋亡情况,同时检测Caspase-3活性。结果在体内,PPD可抑制SMMC-7721细胞裸鼠异种移植瘤生长;在体外,PPD对SMMC-7721细胞的增殖具有明显的抑制及诱导其凋亡作用,呈时间和剂量依赖性,Hoechst33342核染色可见凋亡小体,同时伴有Caspase-3活性的增加。结论20(S)-原人参二醇在体内外均可抑制SMMC-7721细胞增殖,并诱导其凋亡,其机制可能通过活化Caspase-3诱导细胞凋亡而发挥抗肿瘤作用。     

Effect of coadministered drugs and ethanol on the binding of therapeutic drugs to human serum in vitro

The effects of coadministered drugs and ethanol on the binding of therapeutic drugs to human serum in vitro was investigated. Acetaminophen, lidocaine, phenobarbital, quinidine, theophylline, and valproic acid were added to pooled human serum at therapeutic concentrations. To each preparation was added one additional drug at three concentrations ranging from therapeutic to toxic. The following eight target drug/added drug combinations were studied: acetaminophen/phenobarbital. acetaminophen/theophylline, lidocaine/quinidine, phenobarbital/acetaminophen, phenobarbital/valproic acid, quinidine/lidocaine, theophylline/acetaminophen, and valproic acid/phenobarbital. Each serum without the other added drug as well as the serum supplemented with the other drug at the three concentrations was dialyzed against phosphate buffer. Similarly dialyzed were phenobarbital, quinidine, and theophylline, both alone at therapeutic concentrations in serum and with ethanol at three different concentrations in serum. The percentage of drug binding in each preparation was calculated. Acetaminophen diminished the binding of theophylline to human serum by a net change of 5.7% (percentage increase in free drug fraction [FDF], 11.0%) at 662 micromol/L and by a net change of 7.1% (percentage increase in FDF, 13.7%) at 1324 micromol/L. Theophylline decreased the binding of acetaminophen by a net change of 6.8% (percentage increase in FDF, 8.8%) at 277.5 micromol/L; phenobarbital reduced it by a net change of 6.6% (percentage increase in FDF, 8.5%) at 431 micromol/L. Valproic acid diminished binding of phenobarbital by a net change of 9.9% (percentage increase in FDF, 21.2%) at 1732 micromol/L. No significant effects were noted with other drug combinations or with the addition of ethanol. Coingestion of acetaminophen with theophylline, phenobarbital with acetaminophen, and valproic acid with phenobarbital at high to toxic concentrations decreases the binding of the target drug. The resulting increase in free drug concentration may lead to enhanced drug effect in vivo.     

Effect of different polymers on in vitro and ex vivo permeability of Ofloxacin from its mucoadhesive suspensions

Considering the importance of drug permeation from formulations, in vitro and ex vivo drug permeation characteristics of three oral mucoadhesive suspensions of Ofloxacin were designed and compared. Three suspensions of Ofloxacin were prepared by taking two grades of Carbopol polymer such as Carbopol 934 (C934) and Carbopol 940 (C940); and Hydroxypropyl methylcellulose. The permeability study was performed by using a Franz diffusion cell through both synthetic cellulose acetate membrane and excised goat gastrointestinal membranes in acidic as well as alkaline pH. To know the permeability of the drug from control/formulations through different membranes in acidic/alkaline pH, cumulative percentage drug permeation, apparent permeability (Papp) and flux (J) were calculated. In addition, enhancement ratio (ER) of each formulation was also determined. From our results, it is evident that formulation containing C940 was the best suspension considering Papp and J values of all formulations. Moreover, it was the most beneficial formulation for improving permeation and diffusivity of Ofloxacin even after 16 h. Hence, this suspension was probably the most suitable formulation to obtain prolonged release action of the drug. The ER values of all formulations through the excised goat intestinal mucus membrane in alkaline pH were higher than those formulations through the goat stomach mucosal membrane in acidic pH. ER values of those formulations indicate that the permeability of the drug was more enhanced by the polymers in the intestinal part, leading to more bioavailability and prolonged action in that portion of the gastrointestinal tract. It may also be concluded from our results that in addition to formulation containing C940, other formulations may also show effective controlled release action.     

Effect of chronic ethanol treatment on metabolism of drugs in vitro and in vivo.

A daily intake of ethanol ranging from 10 to 12 g/kg for 1 month in either high-fat adequate protein (HFAP) or low-fat high protein (LFHP) liquid diets resulted in significant increases in liver weight, microsomal protein and microsomal metabolism of aminopyrine, zoxazolamine, aniline and meprobamatc, when related to 100 g body weight. Morphine metabolism was increased only after the HFAP diet; pentobarbital metabolism was studied only after HFAP diet, and was increased. The increase in vitro was highest with aminopyrine and lowest with pentobarbital. The analysis of variance showed highly significant differences among the various drugs. There were significant interaction effects of drugs × ethanol and drugs × fat content, but no significant interaction between ethanol × fat or drugs × ethanol × fat except in the case of morphine. The marked between-drug variability in induction of metabolism in vitro by ethanol is also reflected in vivo among the various drugs examined. The increase in vivo was greatest with meprobamate, intermediate with aniline and zoxazolamine, low with aminopyrine and absent with pentobarbital. Moreover, there was variability in inductive effect of ethanol in vivo as compared to in vitro. It is, therefore, concluded that chronic ethanol administration does increase the metabolism of drugs in vitro and in vivo, but the diversity of effects on different drugs cannot be explained by a single mechanism based on an increase in the amount of cytochrome P-450 or other component of the mixed function oxidase system.     

Radiation inactivation of brain [35S]t-butylbicyclophosphorothionate binding sites reveals complicated molecular arrangements of the GABA/benzodiazepine receptor chloride channel complex

[35S]t-Butylbicyclophosphorothionate ([35S]TBPS), a bicyclic cage convulsant, binds to the anion gating mechanism of the GABA/benzodiazepine receptor chloride channel complex. Using a carefully calibrated radiation inactivation technique, the molecular weight of [35S]TBPS binding complexes from frozen rat cerebral cortex was estimated to be 137,000 daltons. The GABA agonist muscimol reduced [35S]TBPS binding to 0-10% of the control value, in a way which is independent of the radiation dose. This shows that the GABA receptor (Mw = 55,000 daltons) is included in the 137,000-dalton [35S]-TBPS binding complex; the [35S]TBPS binding protein alone accounts for 137,000-55,000 = 82,000 daltons. The pyrazolopyridazine etazolate (SQ 20.009) and etomidate in appropriate concentrations both reduced specific binding of [35S]TBPS. The ability of SQ 20.009 and etomidate to reduce [35S]TBPS binding was greatly reduced by exposure to low radiation doses, suggesting that SQ 20.009 and etomidate reduce [35S]TBPS binding by an allosteric mechanism requiring a molecular structure of 450,000-500,000 daltons. Benzodiazepine agonists (ethyl 4-methoxymethyl-6-benzyloxy-beta-carboline-3-carboxylate, ZK 93423) and inverse agonists (methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, DMCM) enhance and reduce [35S]TBPS binding, respectively, in repeatedly frozen and washed membrane preparations. The effects of ZK 93423 and DMCM on [35S]TBPS binding disappeared upon exposure of membranes to low radiation doses. This suggests that the benzodiazepine receptor site interacts allosterically with the [35S]TBPS binding site, requiring a molecular complex of at least c. 400,000 daltons. The [35S]TBPS site alone in these latter conditions of membrane preparation (repeatedly frozen/washed) revealed a molecular weight of 221,000 daltons (TBPS-site + GABA receptor + unknown structures). The number of binding sites for [35S]TBPS (145 pmol/g tissue) was only slightly higher than for [3H]flunitrazepam (130 pmol/g tissue) in cerebral cortex. These results are all consonant with the conclusion that the GABA/BZ receptor chloride channel complex is composed of highly integrated multimeric subunits, tentatively accounted for by a tetramic complex of molecular weight 548,000 daltons.     

In vitro and ex vivo effects of a selective nociceptin/orphanin FQ (N/OFQ) peptide receptor antagonist,CompB, on specific binding of [3H]N/OFQ and [35S]GTPgammaS in rat brain and spinal cord

1. A novel selective nociceptin/orphanin FQ (N/OFQ) peptide receptor antagonist, 1-[(3R,4R)-1-cyclooctylmethyl]-3-hydroxymethyl-4-piperidyl)-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (CompB), inhibited specific binding of [(3)H]N/OFQ to crude membranes from the rat brain and spinal cord in a concentration-dependent manner and their K(i) values were 7.11 and 4.02 nM, respectively. Rosenthal analysis indicated that there was a significant increase in the K(d) value for [(3)H]N/OFQ binding in the brain and spinal cord in the presence of CompB (10 nM). 2. There was a dose-dependent increase in K(d) values for [(3)H]N/OFQ binding in the brain and spinal cord following i.v. injection of CompB at relatively low doses (0.69-6.88 micro mol kg(-1)), compared with the control values. In the spinal cord, enhancement with each dose was constantly greater and the duration of enhancement (6.88 micro mol kg(-1)) was significantly longer. 3. The degree of increase in K(d) values for [(3)H]N/OFQ binding after i.v. injection of CompB (6.88 micro mol kg(-1)) was significantly larger in the lumbar region of the spinal cord compared to other regions. 4. CompB (0.1, 0.3 micro M) shifted the concentration-effect curves of N/OFQ-stimulated [(35)S]GTPgammaS binding in the brain and spinal cord to the right. 5. The i.v. injection of CompB (6.88 micro mol kg(-1)) significantly suppressed the N/OFQ-stimulated [(35)S]GTPgammaS binding in the rat spinal cord and shifted the concentration-effect curve to the right, while it produced little inhibitory effect in the brain. The present study has shown that CompB may exhibit pharmacological effects through a predominant blockade of N/OFQ peptide receptors in the spinal cord under in vivo conditions.