Ovarian steroid regulation of serotonin reuptake transporter (SERT) binding, distribution, and function in female macaques.

The serotonin reuptake transporter (SERT) plays an important role in serotonin neurotransmission and in several psychopathological disorders such as depression and anxiety disorders. In this study, we investigated whether the ovarian steroids, estrogen (E) and progesterone (P) regulate SERT binding, intracellular distribution, and function using [(3)H]citalopram ligand binding with quantitative autoradiography, immunofluorescence histochemistry with confocal microscopy and [(3)H]serotonin uptake, respectively. Ovariectomized macaques received either placebo, E alone, P alone or E plus P for 28 days. In the raphe, E, P, and E+P treatments did not change SERT binding density. In several hypothalamic nuclei, [(3)H]citalopram binding was increased by E, P, and E+P. Immunofluorescent SERT in serotonin soma was intracellular and similar among treatments. In the hypothalamus, immunofluorescent SERT was located along the serotonergic axons and there was a significant proliferation of immunofluorescent fibers in hormone-treated animals. In addition, E and E+P treatment increased serotonin uptake in the basal ganglia. These findings suggest that ovarian hormones regulate SERT protein expression and distribution, perhaps via extracellular serotonin or mRNA stability, but not solely at the level of gene transcription. Further investigation on the possible action of ovarian steroids on the directionality of SERT transport is indicated.

Neurotransmitter action in osteoblasts: expression of a functional system for serotonin receptor activation and reuptake.

Neurotransmitter regulation of bone metabolism has been the subject of increasing interest and investigation. Because serotonin (5-HT) plays a role as a regulator of craniofacial morphogenesis, we investigated the expression and function of 5-HT receptors and the 5-HT transporter (5-HTT) in bone. Primary cultures of rat osteoblasts (rOB) and a variety of clonal osteoblastic cell lines, including ROS 17/2.8, UMR 106-H5, and Py1a, showed mRNA expression for 5-HTT as well as the 5-HT(1A), 5-HT(1D), 5-HT(2A), and 5-HT(2B) receptors by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Protein expression of the 5-HT(1A), 5-HT(2A), and 5-HT(2B) receptors was confirmed by immunoblot. 5-HTT binding sites were assessed in ROS 17/2.8 and UMR 106-H5 cells by binding of the stable cocaine analog [125I]RTI-55, which showed a relatively high density of nanomolar affinity binding sites. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, showed the highest potency to antagonize [125I]RTI-55 binding in ROS and UMR cells. GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5-HT uptake rate in ROS cells was 110 pmol/10 min per well, with a K(m) value of 1.13 micromol/L. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC(50) values in the nanomolar range. In normal differentiating rOB cultures, 5-HTT functional activity was observed initially at day 25, and activity increased almost eightfold by day 31. In mature rOB cultures, the estimated density of [125I]RTI-55 binding sites was 600 fmol/mg protein. Functional downregulation of transporter activity was assessed after PMA treatment, which caused a significant 40% reduction in the maximal uptake rate of [3H]5-HT, an effect that was prevented by pretreatment with staurosporine. The affinity of 5-HT for the transporter was significantly increased following PMA treatment. We assessed the functional significance of expression of the 5-HT receptors by investigating the interaction between 5-HT and parathyroid hormone (PTH) signaling. 5-HT potentiates the PTH-induced increase in AP-1 activity in UMR cells. These results demonstrate that osteoblastic cells express a functional serotonin system, with mechanisms for responding to and regulating uptake of 5-HT.

Mapping genes that regulate density of dopamine transporters and correlated behaviors in recombinant inbred mice.

Binding of 3beta-(4-iodophenyl) tropane-2beta-carboxylic acid methyl ester ([125I]RTI-55) to the dopamine transporter (DAT) in neostriatum from C57BL/6J, DBA/2J, and 21 BXD recombinant inbred (RI) mouse strains indicated highly significant strain differences in DAT density (Bmax) but no significant differences in affinity (Kd) for this radioligand. Strain mean Bmax values and the known genomic locations of 1390 marker loci were used to carry out a genome-wide search for quantitative trait loci (QTLs), which are chromosomal sites containing genes that influence DAT expression. This search revealed an unusually large effect QTL on chromosome 19 in the region of the proopiomelanocortin pseudogene Pomc-ps1 (8-11 cM), homologous to regions of human chromosomes 9q21 and 11q12-13. This QTL (logarithm of the odds 4.7, df = 1, p = 3 x 10(-6)) by conservative estimates accounts for just over half of the genetic variation in DAT binding site density. The QTL is not the DAT gene itself (Dat1, chromosome 13), but a powerful modulator of DAT expression in neostriatum. Furthermore, DAT expression levels in 20 of the BXD RI strains and the chromosome 19 QTL were correlated with cocaine and methamphetamine-induced locomotor activation and thermic responses (hypo- or hyperthermia), but were not correlated with behaviors related to sensitization, reward, voluntary consumption, stereotypy, or seizures induced by these two psychostimulant drugs. The results suggest that there is a gene(s) on proximal chromosome 19 that strongly influences DAT expression in neostriatum and may influence psychostimulant-induced activity and thermal responses.

NMDA receptor subunit mRNA and protein expression in ethanol-withdrawal seizure-prone and -resistant mice.

BACKGROUND: Ethanol withdrawal seizure-prone (WSP) and -resistant (WSR) mice have been genetically selected for differences in handling-induced convulsion severity during withdrawal from chronic ethanol administration. Importantly, drug-naïve mice from these selected lines also differ in handling-induced convulsion severity. Different N-methyl-D-aspartate (NMDA) receptor subunit and splice variant associations confer varying sensitivities to ethanol, and may play a role in the different behavioral responses of the WSP and WSR mice. METHODS: In situ hybridization of riboprobes was used to characterize NMDA receptor subunit and splice variant mRNA expression in cortex and hippocampus from WSP and WSR mice. In addition, immunoblotting and immunohistochemistry were used to examine the expression of specific NMDA receptor subunits and splice variants in hippocampus and cortex from the selected mouse lines. RESULTS: In situ hybridization of riboprobes indicated that, in brain sections from both WSP and WSR mice, there was a differential regional distribution of mRNA for the mouse NR1, NR2A, NR2B, and NR2C NMDA receptor subunits. However, there were no differences between the selected lines in the hybridization of riboprobes to hippocampal subfields or cortical layers. In addition, hybridization of the probe for a 63-base N1-terminal cassette of ethanol-sensitive NR1 splice variants labeled both cortex and hippocampus. The level of hybridization did not differ across subfields of the hippocampus. Results from Western blot and immunohistochemical experiments also indicated that there were no differences between selected lines in NMDA receptor subunit protein expression. However, there was a correlation between mRNA and protein expression in hippocampus and cortex for each NMDA receptor subunit that was examined. CONCLUSIONS: The data suggest that at the level of both mRNA and protein, NMDA receptor subunit and splice variant expression can be uncoupled from convulsion severity in mice that have been selectively bred for symptoms of ethanol withdrawal.

Drug interactions with the dopamine transporter in cryopreserved human caudate.

Although several model systems have been developed to characterize the function of the dopamine transporter (DAT), there is a relative lack of data regarding dopamine (DA) uptake by human caudate, as contrasted to binding studies. Cryopreserved human brain tissue can be used for functional as well as radioligand binding studies of neuronal proteins. The drug-induced inhibition of [125I]RTI-55 binding to, and [3H]DA uptake by, cryopreserved human caudate preparations has now been characterized. Using human caudate membranes, a single site for [125I]RTI-55 binding was observed in association and saturation experiments. The relative potencies of 22 drugs for inhibition of [125I]RTI-55 binding to membranes prepared from cryopreserved human caudate, fresh rat striatum, and HEK293 cells expressing the recombinant human DAT (HEK-hDAT) were highly correlated. The affinity of DA for the DAT, as measured by [3H]DA uptake experiments, was higher in both the cryopreserved human caudate and freshly prepared rat striatum than in HEK-hDAT cells. Although affinities were similar in rat and human brain tissue preparations, the maximal uptake rate in the cryopreserved human caudate was approximately 1 to 4% and 7% of the rate in fresh and cryopreserved rat striatal preparations, respectively. The relative potencies of 22 drugs for inhibition of [3H]DA uptake were similar for tissue prepared from cryopreserved human caudate, nonfrozen rat striatum, and intact HEK-hDAT cells. These data suggest that cryopreserved human caudate can be used to characterize drug interactions with the DAT, and that HEK-hDAT cells provide a comparable system for modeling the initial interaction of drugs with native hDAT.

Uptake and release of neurotransmitters.

The availability of clonal cell lines for norepinephrine, dopamine, and serotonin transporters allows the characterization of drug interactions with transporter recognition sites using radioligands, as well as the characterization of drug effects on selective transporter-mediated uptake and release of substrate. In addition to clonal cell lines, synaptosomes prepared from specific brain regions can be used to conduct these studies without interference by endogenous transporters or binding proteins that are present in other tissues. This unit presents protocols for uptake and release of tritiated substrates using intact cells (either detached or in suspension) or synaptosomes. An HPLC procedure for electrochemical detection of nonradiolabeled substrates is also provided. Time-dependent release can also be measured in assays involving real-time sampling.

Characteristics of drug interactions with recombinant biogenic amine transporters expressed in the same cell type.

We characterized the effects of drugs on the uptake of [3H]neurotransmitter by and the binding of [125I](3beta-(4-iodophenyl)tropane-2beta-carboxylic acid methyl ester ([125I]RTI-55) to the recombinant human dopamine (hDAT), serotonin (hSERT), or norepinephrine (hNET) transporters stably expressed in human embryonic kidney 293 cells. RTI-55 had similar affinity for the hDAT and hSERT and lower affinity for hNET (Kd = 1. 83, 0.98, and 12.1 nM, respectively). Kinetic analysis of [125I]RTI-55 binding indicated that the dissociation rate (k-1) was significantly lower for hSERT and the association rate (k+1) was significantly lower for hNET compared with the hDAT. The potency of drugs at blocking [3H]neurotransmitter uptake was highly correlated with potency at blocking radioligand binding for hDAT and hSERT. Substrates were more potent at the inhibition of [3H]neurotransmitter uptake than radioligand binding. The potency of drugs was highly correlated between displacement of [3H]nisoxetine (Kd = 6.0 nM) and [125I]RTI-55 from the hNET, suggesting that these radioligands recognize similar sites on the transporter protein. The correlation observed between inhibitory potency for uptake and binding of either ligand at the hNET was lower than correlations between uptake and binding for hDAT and hSERT. The present results indicate that the cocaine analog [125I]RTI-55 has unique binding properties at each of the transporters and that the use of recombinant transporters expressed by a single cell type can provide a powerful screening tool for drugs interacting with biogenic amine transporters, such as possible cocaine antagonists.

[3H]substrate- and cell-specific effects of uptake inhibitors on human dopamine and serotonin transporter-mediated efflux.

Drug-induced efflux of substrates was characterized in C6 rat glioma cells stably expressing a recombinant human dopamine (DA) or serotonin (5-HT) transporter (C6-hDAT and C6-hSERT, respectively). In the absence of Ca2+, these cells spontaneously and rapidly released preloaded [3H]DA or [3H]5-HT, respectively, but maintained constant levels of [3H]N-methy-4-phenylpyridinium (MPP+) for up to 90 minutes. In C6-hSERT cells, transporter substrates such as methamphetamine, amphetamine, and dopamine induced relatively rapid release of [3H]MPP+, with t1/2 values of approximately 15 minutes, while the t1/2 value for serotonin was about 30 minutes. Similar results were obtained with C6-hDAT cells. Uptake blockers that are not substrates at the transporters had considerably greater t1/2 values, as compared to substrates, suggesting different mechanisms for altering transporter function. Dose-response curves for each drug, conducted at each drug's t1/2, indicated considerable differences in potency (EC50) at stimulating [3H]MPP+ release from C6-hSERT cells [3beta-(4-iodophenyl)tropane-2beta-carboxylic acid methyl ester (RTI-55) > imipramine > 1-[2-diphenylmethoxy]ethyl-4-(3-phenylpropyl)-piperazine (GBR-12935) threo-(+/-)-methylphenidate > cocaine > mazindol > 2-beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT) > (+)methamphetamine > amphetamine > DA > fenfluramine > norepinephrine (NE) > 5-HT]. A different rank order of potency was observed for the effects of drugs on [3H]MPP+ release from C6-hDAT cells [imipramine > RTI-55 > cocaine > mazindol > CFT > GBR-12935 > threo-(+/-)-methylphenidate > amphetamine > (+)methamphetamine > fenfluramine > DA > NE > 5-HT]. Based on efficacies for stimulating [3H]MPP+ release from C6-hDAT cells, drugs could be grouped into three categories, with substrates causing release of approximately 75% of loaded [3H]MPP+, cocaine analogues causing approximately 50% release, and other drugs causing an average release of approximately 25% of loaded [3H]MPP+. The results, taken together with results from previous reports, suggest that the transfected cell type contributes to the characteristics of transporter-mediated release, that drugs interact with different sites on the transporters in the uptake and release process, and that the mechanism of transporter-mediated release may not be a simple reversal of substrate uptake.

Metabolism of catecholamines by catechol-O-methyltransferase in cells expressing recombinant catecholamine transporters.

To determine if catechol-O-methyltransferase (COMT) metabolizes catecholamines within cell lines used for heterologous expression of plasmalemmal transporters and alters the measured characteristics of 3H-substrate transport, the uptake of monoamine transporter substrates was assessed in three cell lines (C6 glioma, L-M fibroblast, and HEK293 cells) that had been transfected with the recombinant human transporters. Uptake and cellular retention of 3H-catecholamines was increased by up to fourfold by two COMT inhibitors, tropolone and Ro 41-0960, with potencies similar to those for inhibition of COMT activity, whereas the uptake of two transporter substrates that are not substrates for COMT, [3H]serotonin and [3H]MPP+, was unaffected. Direct measurement of monoamine substrates by HPLC confirmed that tropolone (1 mM) increased the retention of the catecholamines dopamine and norepinephrine, but not the retention of serotonin in HEK293 cells. Saturation analysis of the uptake of [3H]dopamine by C6 cells expressing the dopamine transporter demonstrated that tropolone (1 mM) decreased the apparent Km of transport from 0.61 microM to 0.34 microM without significantly altering the maximal velocity of transport. These data suggest that endogenous COMT activity in mammalian cells may alter neurotransmitter deposition and thus the apparent kinetic characteristics of transport.

Characterization of a recombinant human dopamine transporter in multiple cell lines.

A 3.5-kilobase cDNA encoding the dopamine transporter was isolated from a human substantia nigra cDNA library. Sequence analysis of the coding region of the transporter identified two nucleotide differences between the cDNA and published human dopamine transporter sequences. One of the substitutions changed an amino acid conserved among previously cloned dopamine (DA) and norepinephrine transporters, Arg-344, to a methionine. C6 glioma cells or COS-7 cells transfected with the cDNA (C6-hDAT and Cos7-hDAT cells) accumulated [3H]DA with high affinity (Km = 1.2 and 1.5 microM, respectively), and DA uptake inhibitors had similar potencies in both cell lines. [3H]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane ([3H]CFT) bound to membranes prepared from both cell lines with high affinity (Kd = 2-6 nM), although some experiments with C6-hDAT cell membranes indicated the presence of a second class of binding sites with lower affinity for the radioligand. Using the high-affinity Kd value for [3H]CFT binding determined in Cos7-hDAT cells to calculate Ki values, drug affinity for inhibition was highly correlated (r = .92) with affinity for inhibition of [3H]DA uptake, although transporter substrates were significantly more potent inhibitors of uptake than of [3H]CFT binding. The binding of [3H]1-[2-diphenylmethoxy]ethyl-4-(3-phenylpropyl)-piperazine ([3H]GBR-12935) to C6-hDAT cells could not be characterized due to high binding to untransfected C6 cells, but on Cos7-hDAT cells the radioligand labeled a single population of binding sites (Kd = 1 nM). Inhibition of [3H]GBR-12935 binding by drugs correlated highly with inhibition of either [3H]CFT binding (r = .98) or of [3H]DA uptake (r = .95).(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of substituted 3-carbamoylecgonine methyl ester analogues: irreversible and photoaffinity ligands for the cocaine receptor/dopamine transporter.

Photoaffinity ligands are useful tools for the isolation, purification, and characterization of proteins. As a step toward the goal of producing a photoaffinity probe for the dopamine transporter, isocyanato and azido derivatives of 3-[(phenylcarbamoyl)oxy]ecgonine methyl ester were synthesized and tested for their ability to interact with the cocaine receptor of mammalian brain via two different assays. The ability of two isothiocyanato (N=C=S) (para and meta) and two azido (N3) (para and meta) derivatives, as well as (-)-cocaine, to inhibit [3H]cocaine binding and [3H]dopamine uptake and to covalently interact with the cocaine-binding site was tested. The p-N=C=S was the most potent, with IC50 values of 0.23 and 0.49 microM for [3H]cocaine binding and [3H]dopamine uptake. The m-N3 and p-N3 inhibited [3H]cocaine binding with IC50 values of 0.63 and 1.00 microM and inhibited [3H]dopamine uptake with IC50 values of 5.08 and 1.32 microM, respectively. Preincubation of synaptosomal membranes with the m- or p-N=C=S isomer either in reduced lighting or under ultraviolet light followed by two washes resulted in inhibition of 70% and 85% of [3H]cocaine binding, respectively, indicating the highly reactive properties of these compounds. After preincubation in reduced lighting, m-N3 and p-N3 inhibited 0% and 13% of [3H]cocaine binding, while following preincubation under ultraviolet light, the inhibition increased to 61% and 68%, respectively. Thus, the isothiocyanato derivatives appear to bind irreversibly to the cocaine receptor in the presence or absence of ultraviolet light, whereas the azido derivatives are photoreactive compounds which may prove useful in the purification of the receptor.

Release of dopamine via the human transporter.

A human dopamine transporter cDNA was cloned and transfected into COS-7 cells, a cell line that lacks vesicular storage and release mechanisms. Cells expressing the dopamine transporter acquired the capacity to take up and release dopamine via the transporter. Ionic conditions that stimulate inside-out transport in vivo, such as depolarizing concentrations of K+ or low concentrations of extracellular Na+, were found to stimulate Ca(2+)-independent release of [3H]dopamine from transfected COS-7 cells. Dopamine uptake inhibitors had one of three effects on transporter-mediated efflux. Some drugs, in addition to inhibiting uptake, inhibited spontaneous release of dopamine. Drugs in this class included mazindol, GBR-12935, bupropion, nomifensine, and benztropine. All of the drugs with the potential for abuse by humans either enhanced release (methamphetamine, amphetamine, and ethanol) or had no effect on release (phencyclidine, cocaine, and WIN 35,428). The ability to define classes of uptake blockers based on their effects on human transporter-mediated dopamine efflux may lead to the identification of structural features of the transporter that differentiate abused from nonabused drugs.

Allosteric regulation by sodium of the binding of [3H]cocaine and [3H]GBR 12935 to rat and bovine striata.

Sodium regulation of ligand binding to the dopamine transporter of rat and/or bovine striata was investigated using a filtration binding assay. In low Na+ phosphate or bicarbonate-buffered sucrose (300 mOsm), the tissue exhibited high affinity for [3H]cocaine which was reduced by the addition of Na+ in a dose-dependent manner. However, [3H]GBR 12935 binding was insensitive to Na+ in these physiological buffers. Although binding of [3H]GBR 12935 was displaced by cocaine in a manner consistent with competitive displacement, a non-linear affinity shift of the displacement of [3H]GBR 12935 by cocaine suggests that the two ligands bind to distinct sites. Binding of both radioligands was suppressed when measured in sodium-free 50 nM Tris-sucrose and increased with the addition of Na+. Scatchard analysis indicated that Bmax for [3H]cocaine binding in Tris plus 120 mM NaCl reached the same level as in the physiological buffers. In Krebs-Ringer buffer with phosphate, bicarbonate or Tris, which contained 120 nM NaCl, both [3H]cocaine and [3H]WIN 35428 binding exhibited lower affinities than in Na(+)-deficient phosphate buffer. It is suggested that the cation form of Tris binds to the dopamine transporter and that the Tris-receptor complex does not bind [3H]cocaine or [3H]GBR 12935. Na+ displaces Tris, forming a Na(+)-receptor complex which binds these ligands. Thus, it is suggested that the Na(+)-dependent binding of cocaine to the dopamine transporter is observed only in Tris.

Pyrethroid insecticides indirectly inhibit GABA-dependent 36Cl- influx in synaptoneurosomes from the trout brain.

Rainbow trout (Oncorhynchus mykiss) are extremely sensitive to the neurotoxic activity of pyrethroid insecticides. One possible target for pyrethroids is the GABA(A) receptor of brain of the trout, the function of which can be tested by measurement of influx of 36Cl- into synaptoneurosomes, in response to the application of agonists. gamma-Aminobutyric acid produced a time- and concentration-dependent increase in influx of 36Cl- in synaptoneurosomes from the brain of the trout, which exhibited the pharmacology characteristic of a response mediated by activation of a GABAA receptor. Deltamethrin, (1R alpha S)-cis-cypermethrin and permethrin produced a dose-dependent increase in the basal uptake and a corresponding decrease in GABA-dependent influx, with a maximum inhibition of 70-82%. This effect of pyrethroid was stereospecific, of high potency and inhibited by tetrodotoxin (TTX) and t-butylbicyclophosphorothionate (TBPS). The sensitivity of the effect of the pyrethroid to TTX suggested an activation by pyrethroid of the voltage-dependent sodium channel. Veratridine, a sodium channel activator, elicited similar changes in the basal uptake of chloride, which were TTX-sensitive. Neither deltamethrin nor veratridine had a measurable effect on the efflux of 36Cl- from synaptoneurosomes. Thus, pyrethroid insecticides may interfere with the function of GABAA receptors indirectly through an interaction with the voltage-dependent sodium channel in the brain of the trout and consequently perturb chloride influx, possibly through a voltage-dependent chloride channel.

Synthesis of 3-carbamoylecgonine methyl ester analogues as inhibitors of cocaine binding and dopamine uptake.

Five (1R-2-exo-3-exo)-3-(N-phenylcarbamoyl)ecgonine methyl ester analogues were synthesized and characterized by 1H and 13C NMR, IR, and thermospray MS. The compounds were synthesized in two or three steps as (-)-stereoisomers from (-)-ecgonine in good yield (56% overall). These cocaine derivatives were assessed for their ability to inhibit [3H]cocaine binding to rat striatal tissue and to inhibit [3H]dopamine uptake into synaptosomes prepared from the same tissue. The most potent of the analogues was (1R-2-exo-3-exo)-2-(carbomethoxy)-8-methyl-8-azabicyclo[3.2.1]octyl 3-N-(3'-nitrophenyl)carbamate. IC50 values for inhibition of cocaine binding and dopamine uptake were 37 and 178 nM, respectively. Amino derivatives were less active than the nitro and (1R-2-exo-3-exo)-2-(carbomethoxy)-8-methyl-8-azabicyclo [3.2.1]octyl 3-N-(4'-aminophenyl)carbamate had the lowest affinity for the receptor with IC50 values of 63 and greater than 100 microM in the aforementioned assays, respectively.

Dependence on gamma-aminobutyric acid of pyrethroid and 4'-chlorodiazepam modulation of the binding of t-[35S]butylbicyclophosphorothionate in piscine brain.

Binding sites for t-[35S]butylbicyclophosphorothionate ([35S]TBPS) were detected in well-washed membranes from the brain of trout; gamma-aminobutyric acid (GABA) acted as an uncompetitive inhibitor of the binding of [35S]TBPS, decreasing both the number of binding sites and the affinity of TBPS. Inhibition of the binding of [35S]TBPS by deltamethrin, a Type II pyrethroid, was modulated by GABA; both the affinity and the efficacy of this insecticide increased with incremental concentrations of GABA. Deltamethrin also enhanced the potency of GABA as an inhibitor of the binding of [35S]TBPS. The interaction of 4'-chlorodiazepam (Ro5-4864) with [35S]TBPS was dependent on GABA: in the absence of GABA, Ro5-4864 inhibited up to 40% of the binding; in the presence of 10 microM GABA, Ro5-4864 enhanced binding to a maximum value of 170% of control. However, the same absolute amount of binding was observed with both of these effects at micromolar concentrations of Ro5-4864. Also, Ro5-4864 caused a rightward shift in GABA dose-response curves, increasing the IC50 value for GABA more than 6 fold. These results indicate the reciprocal allosteric interactions between a binding site for pyrethroids, a binding site for Ro5-4864, the GABA recognition moiety and the binding site for TBPS in the brain of trout. The similarity of these findings to previous results in preparations of rodent brain highlight the conservation of the modulation of GABAA receptor function during the evolution of vertebrates.

Differential binding properties of the peripheral-type benzodiazepine ligands [3H]PK 11195 and [3H]Ro 5-4864 in trout and mouse brain membranes.

High-affinity binding sites for [3H]PK 11195 have been detected in brain membranes of rainbow trout (Salmo gairdneri) and mouse forebrain, where the densities of receptors were 1,030 and 445 fmol/mg of protein, respectively. Ro 5-4864 (4'-chlorodiazepam) was 2,200-fold less potent as a competitor of [3H]PK 11195 binding in the piscine than the murine membranes. Investigation of the regional distribution of these sites in trout yielded a rank order of density of spinal cord greater than olfactory bulb = optic tectum = rhombencephalon greater than cerebellum greater than telencephalon. This site in trout shared some of the characteristics of the peripheral-type benzodiazepine receptor (PTBR) (also known as the mitochondrial benzodiazepine receptor) in rodents, i.e., high affinity for PK 11195 and the endogenous ligand protoporphyrin IX, but was unique in the low affinity of Ro 5-4864 (41 microM) and diazepam and the relatively high affinity of the calcium channel ligand diltiazem and two central benzodiazepine ligands, CGS 8216 and CGS 9896. The differential affinity for the two prototypic PTBR ligands in trout is similar to that previously observed in calf and human brain membranes. Structural differences for the trout sites are indicated by the relative inability of diethyl pyrocarbonate to modify histidine residues of the binding site in trout as compared with mouse membranes. Heterogeneity of binding of the two prototypic PTBR ligands in mouse brain membranes was indicated by additivity studies, equilibrium competition experiments, and saturation isotherms, which together support the hypothesis that Ro 5-4864 discriminates between two [3H]PK 11195 binding sites having high (nanomolar) and low (micromolar) affinity, respectively.