Multiple [35S]t-Butylbicyclophosphorothionate Binding Sites in Rat and Chicken Cerebral Hemispheres

Specific binding of [35S]t-butylbicyclophosphorothionate (TBPS) to membranes from cerebral hemispheres of adult rat and chicken was determined over a range of radioligand concentrations from 0.25 to 500 nM. Scatchard plots of these data were curvilinear and nonlinear regression analysis indicated binding to two sites that differ in affinity. For rat cerebrum, KD(1) = 1.15 nM, Bmax(1) = 0.085 pmol/mg; KD(2) = 232 nM, Bmax(2) = 16.9 pmol/mg. For chicken cerebrum, KD(1) = 1.39 nM, Bmax(1) = 0.111 pmol/mg; KD(2) = 166 nM, Bmax(2) = 17.6 pmol/mg. This multiplicity of [35S]TBPS binding was further confirmed when unlabeled TBPS or picrotoxinin displaced radioligand. The displacement curves were biphasic and yielded Hill coefficients from 0.65 to 0.70. These displacement curves were also resolved into two components with distinct IC50 values for unlabeled TBPS (rat, 1.55 and 271 nM; chicken, 2.40 and 224 nM). The IC50 values were similar to the dissociation constants obtained from equilibrium binding measurements.     

Further characterisation of the [35S]-TBPS binding site of the GABA receptor complex in locust (Schistocerca gregaria) ganglia membranes

1. Using homogenates of supraoesophageal ganglia from locust we observed specific binding of [35S]-TBPS which was linear with protein concentration up to 7 mg/ml, showed a pH optimum at pH 9.0 and was linear with NaCl concentration up to 350 mM. 2. Kinetic analysis of the binding showed positive cooperativity as a result of changes in on and off-rates with occupation of the binding site by the ligand. The apparent KD = 417 nM and Bmax = 1083 fmol/mg of membrane protein were calculated using a computer program for dose-response curve fitting. 3. The binding was enhanced by GABA, pentobarbital and benzodiazepines. Picrotoxinin had no effect on the binding at 0.1 mM. Only the cage convulsants TBPS and IBP were able to displace the binding. 4. Whilst the characteristics of the binding are similar to those reported for house fly thorax and abdomen preparations they are significantly different from those reported for house fly head, cockroach nerve cord and rat brain.     

Solubilization of convulsant/barbiturate binding activity on the gamma-aminobutyric acid/benzodiazepine receptor complex

Binding activity of the radioactive cage convulsant [35S]t-butylbicyclophosphorothionate was solubilized from rat brain membranes using the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio] propanesulfonate. Binding (KD = 26 nM, Bmax = 0.4 pmol/mg protein) was inhibited by picrotoxin and related convulsants and by barbiturates and related depressants that interact with gamma-aminobutyric acid and benzodiazepine receptors via the picrotoxinin binding site. The convulsant/barbiturate binding activity chromatographed on gel filtration as a single peak coinciding with the benzodiazepine/gamma-aminobutyric acid receptor protein complex.     

Actions of Insecticides on the Insect Gaba Receptor Complex

Abstract

The actions of insecticides on the insect γ-aminobutyric acid (GABA) receptor were investigated using [³⁵S]t-butylbicyclophosphorothionate ([³⁵S]TBPS) binding and voltage-clamp techniques. Specific binding of [³⁵S]TBPS to a membrane homogenate derived from the brain of Locusta migratoria locusts is characterised by a Kd value of 79.3 ± 2.9 nM and a B_max value of 1770 ± 40 fmol/mg protein. [³⁵S]TBPS binding is inhibited by mM concentrations of barbiturates and benzodiazepines. In contrast dieldrin, ivermectin, lindane, picrotoxin and TBPS are inhibitors of [³⁵S]TBPS binding at the nanomolar range. Bicuculline, baclofen and pyrethroid insecticides have no effect on [³⁵S]TBPS binding. These results are similar to those obtained in electrophysiological studies of the current elicited by GABA in both Locusta and Periplaneta americana central neurones. Noise analysis of the effects of lindane, TBPS, dieldrin and picrotoxin on the cockroach GABA responses reveals that these compounds decrease the variance of the GABA-induced current but have no effect on its mean open time. All these compounds, with the exception of dieldrin, significantly decrease the conductance of GABA-evoked single current.     

35S]t-butylbicyclophosphorothionate binding sites in susceptible and cyclodiene-resistant houseflies.

4-aminobutyric acid (GABA)-gated chloride ion channels are important molecular targets for a number of polychlorocycloalkane compounds including cyclodiene insecticides. Previous radioligand binding studies have indicated that cyclodiene insecticides are potent inhibitors of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to housefly thorax and abdomen membranes. In the present study, a laboratory-reared, cyclodiene-resistant (CYW) housefly strain (Musca domestica) showed resistance to a number of cyclodiene insecticides. Specific, saturable [35S]TBPS binding was detected in thorax and abdomen membranes prepared from housefly strains susceptible (CSMA) and resistant (CYW) to cyclodienes. Scatchard analysis of [35S]TBPS binding data from CSMA and CYW membranes revealed no significant differences between the two strains in either the affinity (Kd) or the density (Bmax) of specific, saturable binding sites. There were no differences in the comparative effectiveness of a range of polychlorocycloalkanes, including cyclodiene insecticides, as inhibitors of specific [35S]TBPS binding to CSMA and CYW thorax and abdomen membranes. Therefore, if an alteration in target site is a mechanism for resistance to cyclodienes in the CYW strain, it is not readily measurable using [35S]TBPS.     

Similar properties of [35S]t-butylbicyclophosphorothionate receptor and coupled components of the GABA receptor-ionophore complex in brains of human, cow, rat, chicken and fish

No significant differences are evident in the specific binding characteristics of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) to EDTA/water-dialyzed P2 membranes of human, cow, rat, chicken and fish brain. This species similarity includes dissociation constants of 61-77 nM at 37 degrees C, maximum receptor densities of 3-7 pmol/mg protein, and sensitivity to inhibition or displacement by gamma-aminobutyric acid (GABA), two cage convulsants (picrotoxinin and t-butylbicycloorthobenzoate) and the insecticide [1R,cis, alpha S]-cypermethrin, indicating a constancy during vertebrate evolution of the [35S]TBPS binding site and its coupling with other components of the GABA receptor-ionophore complex. As a possible exception, chicken and fish brain membranes appear to be less sensitive than the others to the insecticide alpha-endosulfan. Human and rat preparations are also essentially identical relative to the inhibition of radioligand binding by two GABA mimetics (muscimol and 3-amino-propanesulfonic acid), six other cage convulsants (including examples of three classes of polychlorocycloalkane insecticides), a potent anthelmintic agent (Ivermectin), dimethylbutylbarbiturate, the convulsant benzodiazepine Ro 5-3663, and ethanol. The findings to date with [35S]TBPS and the GABA receptor-ionophore complex in rat brain membranes are therefore generally applicable to human preparations. Cow brain is an appropriate source for large scale preparations in receptor purification studies since it is essentially identical to human and rat preparations in all parameters examined. Species differences in sensitivity to the toxic effects of the convulsants and polychlorocycloalkane insecticides considered are apparently not attributable to receptor site specificity.     

Polychlorinated Convulsant Insecticides Potentiate the Protective Effect of NaCl Against Heat Inactivation of [3H]Flunitrazepam Binding Sites

Six polychlorinated convulsant insecticides that potently inhibit t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to rat brain membranes also potentiate the protective effect of NaCl (200 mM) against heat inactivation of [3H]flunitrazepam binding sites on the same membranes. Similar effects were obtained with all "cage" convulsants tested. The rank order of potencies as heat protection potentiators was similar to the rank order of potencies as inhibitors of [35S]TBPS binding (alpha-endosulfan greater than endrin greater than dieldrin greater than toxaphene greater than lindane). alpha-Endosulfan and endrin are more potent in both respects than any previously reported picrotoxin-like (cage) convulsant, but are much less toxic to mammals. The greatly reduced toxicities of alpha-endosulfan and endrin in mammals may reflect partial gamma-aminobutyric acid (GABA)-neutral properties of these compounds. Time courses of heat inactivation of [3H]flunitrazepam binding sites in the presence of 200 mM NaCl plus saturating concentrations of endrin or picrotoxin revealed monophasic components constituting about 88% of the binding sites, suggesting that virtually all [3H]flunitrazepam binding sites are coupled to picrotoxin binding sites in the GABA/benzodiazepine/picrotoxin receptor complex. Protection against heat inactivation constitutes a useful tool for characterizing the various allosterically linked binding sites in neurotransmitter receptor complexes.     

t-[3H]Butylbicycloorthobenzoate: New Radioligand Probe for the γ-Aminobutyric Acid–Regulated Chloride Ionophore

t-[3H]Butylbicycloorthobenzoate [( 3H]TBOB; 22 Ci/mmol) was prepared by reductive dechlorination of its 4-chlorophenyl analog with tritium gas. This new radioligand binds reversibly to fresh washed rat brain P2 membranes in 500 mM NaCl plus 50 mM sodium-potassium phosphate buffer (pH 7.4) at 25 degrees C, with 80-90% specific relative to total binding, a KD of 61 +/- 15 nM, and a Bmax of 1.6 +/- 0.5 pmol/mg of protein. [3H]TBOB association with its binding site(s) is monophasic, but its dissociation is biphasic. The binding characteristics of [3H]TBOB are essentially identical to those of t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) with respect to pH dependence, stimulation by anions, regional distribution in the brain, and pharmacological profile. Saturation analyses and dissociation studies further indicate that TBOB and TBPS have a common binding site. However, binding of the two radioligands differs in respect to temperature effects. In contrast to [35S]TBPS, which exhibits negligible binding at 0 degrees C, [3H]TBOB binds to rat brain membranes at 0, 25, and 37 degrees C with similar KD values. [3H]TBOB with its long radioactive half-life and temperature-independent KD is a valuable supplement to [35S]TBPS in further biochemical and pharmacological characterization of the gamma-aminobutyric acid receptor-ionophore complex.     

Effect of Halide Ions on t-[35S]Butylbicyclophosphorothionate Binding

The binding of t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) to a site on the GABAA receptor complex is ion dependent. This study was conducted to determine the effects of ion species and concentration on the time course, affinity, and number of sites of [35S]TBPS binding. At a concentration of 200 mM ion, the time to equilibrium for [35S]TBPS binding was shortest for I-, followed by Br- less than Cl- less than F-. A similar rank order was observed for the concentration of ion required to produce half-maximal [35S]TBPS binding. Saturation binding experiments were conducted to evaluate the effect of increasing ion concentration on the KD and Bmax of [35S]TBPS binding. The Bmax was independent of both ion species and concentration. The receptor affinity, however, increased with increasing concentration for each ion. Calculated maximal affinity values were not different between ions; however, the EC50 to produce those values was different among ions and ranked in the same order as that for time course and maximal binding data. Association and dissociation rates for [35S]TBPS binding were greater in I- than in Cl-. These data emphasize the importance of ion selection and incubation times on [35S]TBPS binding.     

Physicochemical Properties of Serotonin 5-HT3 Binding Sites Solubilized from Membranes of NG 108-15 Neuroblastoma-Glioma Cells

Specific binding sites with pharmacological properties typical of serotonin 5-HT3 receptors were identified in membranes of the murine hybridoma cell line NG 108-15, using [3H]zacopride as a ligand. Optimal solubilization of these sites (yield, 50%) could be achieved using the detergent 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) at 24 mM plus 0.5 M NaCl in 25 mM Tris-HCl, pH 7.4. Specific [3H]zacopride binding to soluble sites in the 100,000-g CHAPS extract was saturable and showed characteristics (Bmax = 425 +/- 81 fmol/mg of protein; KD = 0.19 +/- 0.02 nM) closely related to those of membrane-bound sites (Bmax = 932 +/- 183 fmol/mg of protein; KD = 0.60 +/- 0.03 nM). Determination of association (k+1 = 0.17 nM min-1) and dissociation (k-1 = 0.02 min-1) rate constants for the soluble sites gave a KD value of 0.12 nM, a result consistent with that calculated from saturation studies. As assessed from the displacement potencies (IC50) of 10 different drugs, the pharmacological profile of [3H]zacopride specific binding sites was essentially the same (r = 0.99) in the CHAPS-soluble extract and in cell membranes, although some increase in the affinity for 5-HT3 antagonists (zacopride, ICS 205-930, and MDL 72222) and decrease in the affinity for 5-HT3 agonists (2-methyl-5-hydroxytryptamine and phenylbiguanide) were noted for the soluble sites. Sucrose density gradient sedimentation of the CHAPS-soluble extract gave a Svedberg coefficient of 12S for the material with [3H]zacopride specific binding capacity. Chromatographic analyses using Sephacryl S-400 and wheat germ agglutinin-agarose columns indicated marked enrichment (by 2.5- and 10-fold, respectively) in [3H]zacopride specific binding activity in the corresponding eluates compared with the starting soluble extract, a finding suggesting that both steps are of potential interest for the partial purification of solubilized 5-HT3 receptors. Two soluble materials with apparent molecular masses of approximately 600 and approximately 36 kDa were found to bind [3H]zacopride specifically in the Sephacryl S-400 eluate. Interestingly, molecular mass determination by radiation inactivation of [3H]zacopride binding sites in frozen NG 108-15 cells gave a value of approximately 35 kDa.     

Presynaptic Cholinergic Mechanisms in the Rat Cerebellum: Evidence for Nicotinic, but Not Muscarinic Autoreceptors

The present study shows that N-[3H]methylcarbamylcholine ([3H]MCC) binds to a single population of high-affinity/low-density (KD = 5.0 nM; Bmax = 8.2 fmol/mg of protein) nicotinic binding sites in the rat cerebellum. Also, there exists a single class of high-affinity binding sites (KD = 4.8 nM; Bmax = 24.2 fmol/mg of protein) in the cerebellum for the M1 specific muscarinic ligand [3H]pirenzepine. In contrast, the M2 ligand, [3H]AF-DX 116, appears to bind to two classes of binding sites, i.e., a high-affinity (KD = 3 nM)/low-capacity (Bmax = 11.7 fmol/mg of protein) class, and a second class of lower affinity (KD = 28.4 nM) and higher capacity (Bmax = 36.3 fmol/mg of protein) sites. The putative M3 selective ligand [3H]4-diphenylacetoxy-N-methylpiperidine also binds to two distinct classes of binding sites in cerebellar homogenates, one of high affinity (KD = 0.5 nM)/low capacity (Bmax = 19.5 fmol/mg of protein) and one of low affinity (KD = 57.5 nM)/high capacity (Bmax = 140.6 fmol/mg of protein). In experiments which tested the effects of cholinergic drugs on acetylcholine release from cerebellar brain slices, the nicotinic agonist MCC enhanced spontaneous acetylcholine release in a concentration-dependent manner, and the maximal increase in acetylcholine release (59.0-68.0%) occurred at 10(-7) M. The effect of MCC to increase acetylcholine release was Ca2+-dependent and tetrodotoxin-insensitive, suggesting an action on cholinergic terminals. Also, the MCC-induced increase in acetylcholine release was effectively antagonized by dihydro-beta-erythroidine, d-tubocurarine, and kappa-bungarotoxin, but was insensitive to either atropine or alpha-bungarotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

(+)-PN200-l 10 and Ouabain Binding Sites in Purified Bovine Adrenomedullary Plasma Membranes and Chromaffin Cells

Bovine adrenal medulla plasma membranes were purified by a differential centrifugation procedure using sucrose and Urografin discontinuous density gradients; the membranes were enriched 10-12-fold in acetylcholinesterase activity and [3H]ouabain binding sites. Specific (+)-[3H]PN200-110 binding to these membranes amounted to 90% of total binding and was saturable and of high affinity (KD = 41 pM; Bmax = 119 fmol/mg of protein) with a Hill coefficient close to 1, a result suggesting the presence of a single, homogeneous population of dihydropyridine receptors. The association and dissociation rate constants were, respectively, 7.5 X 108 M-1 min-1 and 0.023 min-1. Unlabeled (+)-PN200-110 displaced (+)-[3H]PN200-110 binding with a potency 100-fold higher than (-)-PN200-110 (IC50,0.5 and 45nM, respectively). Although the two enantiomers of BAY K 8644 completely displaced (+)-[3H]PN200-110 binding, they exhibited no stereoselectivity (IC50, 69 and 83 nM,respectively). Whereas ( +/- )-nitrendipine very potently displaced (+)-[3H]PN200-110 binding (IC50 = 1.3 nM) verapamil and cinnarizine displaced the binding by only 30 and 40% at 1 microM, and diltiazem increased it by 20% at 10 microM. [3H]Ouabain bound to plasma membranes with a KD of 34 nM and a Bmax of 9.75 pmol/mg of protein, a figure 80-fold higher than the Bmax for (+)-PN200-110. [3H]Ouabain also bound to intact chromaffin cells with a Bmax of 244 fmol/10(6) cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific inhibition of [3H] saxitoxin binding to skeletal muscle sodium channels by geographutoxin II, a polypeptide channel blocker

Geographutoxin II (GTX II), a peptide toxin isolated from Conus geographus, inhibited [3H]saxitoxin binding to receptor sites associated with voltage-sensitive Na channels in rat skeletal muscle homogenates and rabbit T-tubular membranes with K0.5 values of 60 nM for homogenates and 35 nM for T-tubular membranes in close agreement with concentrations that block muscle contraction. Scatchard analysis of [3H]saxitoxin binding to T-tubular membranes gave values of KD = 9.3 nM and Bmax = 300 fmol/mg of protein and revealed a primarily competitive mode of inhibition of saxitoxin binding by GTX II. The calculated KD values for GTX II were 24 nM for T-tubules and 35 nM for homogenates, respectively. In rat brain synaptosomes, GTX II caused a similar inhibitory effect on [3H]saxitoxin binding at substantially higher concentrations (K0.5 = 2 microM). In contrast, binding of [3H]batrachotoxin A 20-alpha-benzoate and 125I-labeled scorpion toxin to receptor sites associated with Na channels in synaptosomes was not affected by GTX II at concentrations up to 10 microM. Furthermore, [3H]saxitoxin binding to membranes of rat superior cervical ganglion was only blocked 10% by GTX II at 10 microM. These results indicate that GTX II interacts competitively with saxitoxin in binding at neurotoxin receptor site 1 on the sodium channel in a highly tissue-specific manner. GTX II is the first polypeptide ligand for this receptor site and the first to discriminate between this site on nerve and adult muscle sodium channels.     

Binding Characteristics of t-[35S]Butylbicyclophosphorothionate in Discrete Brain Regions of Rats Made Tolerant to and Dependent on Pentobarbital

The effects of acute and continuous pentobarbital administration by pellet implantation on binding characteristics of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) in discrete regions of rat brains were examined. Acute administration of pentobarbital (60 mg/kg, s.c.) affected neither the KD nor the Bmax values of [35S]TBPS binding in any of the regions studied. The cerebella of pentobarbital-tolerant rats had an increased density of [35S]TBPS binding sites with no change in their apparent affinity. There were no significant changes in the binding characteristics in the frontal cortex (FC), the striatum (ST), and the substantia nigra (SN) of these animals. Twenty-four hours after removal of the pentobarbital pellets, a significant decrease in the latency of onset of first twitch response induced by pentylenetetrazol (PTZ) (50 mg/kg, i.p.) was observed. In addition, the density of [35S]TBPS binding sites was significantly increased in the FC, the SN, and the cerebellum but not in the ST. In all brain regions studied, placebo pellet implantation and pentobarbital tolerance and dependence caused no changes in the apparent affinity of [35S]TBPS binding or the IC50 of pentobarbital for the inhibition of [35S]TBPS binding. These results suggest that [35S]TBPS binding was significantly increased following the withdrawal of the pentobarbital pellets without altering intrinsic coupling activity of barbiturate recognition sites and convulsant binding sites and that these increases in [35S]TBPS binding are related to the increased susceptibility to seizures induced by PTZ in rats made dependent on pentobarbital.     

Solubilization and Detergent Effects on Interactions of Some Drugs and Insecticides with the t-Butylbicyclophosphorothionate Binding Site Within the γ-Aminobutyric Acid Receptor-Ionophore Complex

Specific binding of [35S]t-butylbicyclophosphorothionate (TBPS) to rat brain membranes (RBM) is enhanced nine-fold by EDTA/water dialysis and 1.3- to 4.2-fold by 50 nM ketosteroid R 5135, or 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or related piperazine-N-alkanesulfonate buffers, or extensive washing with NaCl/Na phosphate or Na phosphate/citrate solution. About one-fifth of the [35S]TBPS binding capacity appears in the soluble fraction whereas the rest remains in particulate form on treatment of the EDTA/water-dialyzed RBM with 20 mM CHAPS. Similar KD values (64-86 nM) are obtained for the original EDTA/water-dialyzed membranes and the CHAPS-treated and/or -solubilized preparations. The Bmax of the EDTA-treated RBM is reduced five-fold on solubilization with CHAPS. The potency for displacement of [35S]TBPS changes in the presence of CHAPS or on CHAPS solubilization: gamma-aminobutyric acid (GABA) and muscimol inhibit specific [35S]TBPS binding more strongly in the absence than in the presence of CHAPS: TBPS, picrotoxinin, and photoheptachlor epoxide are almost equally active with RBM, RBM + CHAPS, and RBM solubilized with CHAPS. Levels of (1R, alpha S)-cis-cypermethrin and dimethylbutylbarbiturate which are inhibitory with RBM are moderately stimulatory after TBPS receptor solubilization. Thus CHAPS defines three regions of the GABA receptor-ionophore complex, i.e., the GABA and benzodiazepine receptors, the TBPS/picrotoxinin/polychlorocycloalkane receptor(s), and the sites at which the alpha-cyano pyrethroid and the barbiturate interact with TBPS binding.     

Stress-Induced Changes in t-[35S]Butylbicyclophosphorothionate Binding to γ-Aminobutyric Acid-Gated Chloride Channels Are Mimicked by In Vitro Occupation of Benzodiazepine Receptors

The allosteric modulation of t-[35S]butylbicyclophosphorothionate binding by flunitrazepam was studied in well-washed brain membranes prepared from control and swim-stressed rats. Swim stress has been reported to decrease the KD and increase the Bmax of this radioligand. Flunitrazepam increased radioligand binding with equal potency (EC50 approximately 11 nM) in both groups, but the maximal enhancement (efficacy) produced by this drug was significantly greater in control than in swim-stressed rats. Ro 15-1788 (a benzodiazepine receptor antagonist) blocked the effect of flunitrazepam on t-[35S]butylbicyclophosphorothionate binding in both groups. This increase in t-[35S]butylbicyclophosphorothionate binding resulted from a significant reduction in KD with no alteration in Bmax. The KD values obtained in cortical membranes of control rats after addition of flunitrazepam were not significantly different from those in the swim-stressed group. Preincubation of cortical homogenates from control animals with flunitrazepam prior to extensive tissue washing resulted in Bmax and KD values of t-[35S]butylbicyclophosphorothionate similar to those obtained in stressed animals. These findings suggest that stress and flunitrazepam may share a common mechanism in regulating t-[35S]butylbicyclophosphorothionate binding and support the concept that stress-induced modification of gamma-aminobutyric acid (GABA)-gated chloride channels in the CNS results from the release of an endogenous modulator (with benzodiazepine-like properties) of the benzodiazepine-GABA receptor chloride ionophore receptor complex.     

α-[3H]Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid Binding to Human Cerebral Cortical Membranes: Minimal Changes in Postmortem Brains of Chronic Schizophrenics

The binding of alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA), a selective ligand for the ion channel-linked quisqualate receptor, was evaluated in Triton X-100-treated membranes of human cerebral cortex. The presence of chaotropic ions produced divergent effects on specific [3H]AMPA binding: A twofold increase in the binding was observed with thiocyanide at 100 mM, although iodide (100 mM) and perchlorate (100 mM) reduced the binding. Chemical modifications of the sulfhydryl group with p-chloromercuriphenylsulfonic acid (PCMBS) produced threefold increases in specific [3H]-AMPA binding in the absence of KSCN as well as in the presence of KSCN. Treatment with dithiothreitol restored the enhanced specific [3H]AMPA binding by PCMBS to the basal level. Although specific [3H]AMPA binding in the absence of KSCN showed a single site (KD = 220 nM, Bmax = 235 fmol/mg of protein), curvilinear Scatchard plots of specific [3H]AMPA binding in the presence of 100 mM KSCN can be resolved into two binding sites with the following parameters: KD1 = 5.82 nM, Bmax1 = 247 fmol/mg of protein; KD2 = 214 nM, Bmax2 = 424 fmol/mg of protein. Quisqualate and AMPA were the most potent inhibitors of the [3H]AMPA binding in the presence of KSCN. Potent inhibitors of the binding included beta-N-oxalylamino-L-alanine (L-BOAA), cysteine-S-sulfate, L-glutamate, 6-cyano-7-nitroquinoxaline-2,3-dione, and 6,7-dinitroquinoxaline-2,3-dione. Kainate, L-homocysteine sulfinic acid, and L-homocysteic acid were active with an IC50 value of a micromolar concentration, whereas L-cysteic acid and L-cysteine sulfinic acid were weakly active.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chlorocyclohexane insecticides and male medfly attractants: similar stereospecificity for neuroactivity and interactions with a housefly [35S]t-butylbicyclophosphorothionate binding site

[35S]t-Butylbicyclophosphorothionate [( 35S]TBPS) undergoes saturable specific binding to a membrane preparation from housefly thoraxes and abdomens with apparent Kd and Bmax values at equilibrium of 0.17 microM and 2.2 pmol/mg protein at 20 degrees C. Lindane is more potent than three other isomers of hexachlorocyclohexane as a toxicant for houseflies and in displacing [35S]TBPS from this housefly membrane preparation. This correlation of similar stereospecificity for neuroactivity and interaction with the TBPS receptor extends to the Mediterranean fruit fly male attractant trimedlure and its components and analogs. The relative attractancy of t-butyl cis-4-chloro-trans-2-methylcyclohexanecarboxylate and of three less active isomers of this trans-chlorocyclohexane-carboxylate parallel their potency in the [35S]TBPS binding assay. With both trimedlure and the related cyclohexene attractant siglure the trans-isomers are more potent than the cis-isomers as attractants and in displacing [35S]TBPS. Scatchard analyses indicate that lindane binds at the same site as [35S]TBPS. The hexachlorocyclohexane isomers and trimedlure components are much more potent inhibitors with membrane preparations from houseflies than from rat brain. The housefly TBPS receptor possibly serves as a model for the primary target sites, thereby suggesting that both the insecticide and the attractant may interact with some component of the putative GABAergic nervous or neuromuscular system.     

Convulsant potencies of tetrazoles are highly correlated with actions on GABA/benzodiazepine/picrotoxin receptor complexes in brain

A series of tetrazole convulsants were examined for their potencies in displacing [35S]-t-butylbicyclophosphorothionate (TBPS) from the picrotoxin site on the benzodiazepine-GABA-chloride ionophore receptor complex. All of the tetrazole derivatives tested inhibited [35S]-TBPS binding from rat forebrain membranes, and except for one (undecamethylenetetrazole), had Hill coefficients near unity. Similar to other chemically unrelated convulsants the inhibition of [35S]-TBPS binding by the various tetrazole derivatives was unaffected by the addition of the bicucculine-like GABA antagonist, R 5135. To ascertain whether the inhibition of specific [35S]-TBPS binding by the tetrazole derivatives was related to their convulsant properties, we compared their in vitro potencies in displacing [35S]-TBPS binding with their minimum convulsant potencies in mice. A very good correlation was observed (r = 0.96, p less than 0.001) between their relative affinities for the [35S]-TBPS binding site and their convulsant potencies, indicating that pentamethylenetetrazol and related tetrazoles may produce their convulsant and anxiogenic actions via the GABA-benzodiazepine-chloride ionophore receptor complex.     

Effects of chronic nicotine treatment on nicotinic receptors in the rabbit urinary bladder

Nicotine induced a phasic contraction in the rabbit urinary bladder. The response was abolished by hexamethonium and partially reduced by atropine and capsaicin. Simultaneous atropine and capsaicin treatment did not abolish the contraction. These findings suggest that the response to nicotine is due to acetylcholine, tachykinins, and unknown mediator release. In contrast, nicotine-induced contraction diminished following the chronic nicotine treatment without a change of its pharmacological properties. These results suggest the possibility that chronic nicotine treatment causes a decrease in nicotinic receptor numbers. Therefore, the binding properties of (-)-[3H]nicotine on rabbit urinary detrusor muscle membrane fractions were studied to evaluate the effects of chronic nicotine treatment on nicotinic receptors. Specific (-)-[3H]nicotine binding reached saturation and Scatchard plots were curvilinear, suggesting the existence of two different affinity sites for (-)-[3H]nicotine. Dissociation constants (KD) and maximum binding sites (Bmax) were KD1 = 4.91 +/- 1.88 nM, Bmax1 = 2.42 +/- 0.22 fmol/mg protein and KD2 = 263 +/- 56 nM, Bmax2 = 25.0 +/- 4.3 fmol/mg protein. In urinary bladder membrane fractions from chronic nicotine-treated rabbits, KD and Bmax values were KD1 = 3.96 +/- 0.38 nM, Bmax1 = 1.07 +/- 0.25 fmol/mg protein and KD2 = 249 +/- 12 nM, Bmax2 = 10.8 +/- 1.5 fmol/mg protein. Dissociation constants for both sites following chronic nicotine treatment did not change but maximum binding site numbers for both sites significantly decreased (p less than 0.05). These results suggest that the decrease in contractile response evoked by nicotine after chronic nicotine treatment in rabbit urinary bladder is due to a decrease in numbers of nicotinic receptors.