Ca2+/Calmodulin-Mediated Regulation of Agonist-Induced Sequestration of Gq Protein-Coupled Histamine H1 Receptors in Human U373 MG Astrocytoma Cells

Abstract: We investigated the regulation by intracellular Ca²⁺ of agonist-induced sequestration of G_q protein-coupled histamine H₁ receptors in human U373 MG astrocytoma cells. Histamine-induced sequestration of H₁ receptors from the cell surface membrane was detected as the loss of [³H]mepyramine binding sites on intact cells accessible to the hydrophilic H₁-receptor antagonist pirdonium. The changes in the pirdonium-sensitive binding of [³H]mepyramine were mirrored by changes in the subcellular distribution of H₁ receptors detected by sucrose density gradient centrifugation. The histamine-induced sequestration of H₁ receptors did not occur in hypertonic medium, in which clathrin-mediated endocytosis is known to be inhibited, but was significantly accelerated in the absence of extracellular Ca²⁺ or in the presence of the calmodulin antagonists W-7 and calmidazolium. Inhibitors of protein kinase C (H-7 and GF109203X), Ca²⁺/calmodulin-dependent protein kinase II (KN-62), or protein phosphatase 2B (FK506) did not alter the time course of H₁-receptor sequestration. These results provide the first evidence that agonist-induced, clathrin-mediated sequestration of G_q protein-coupled receptors is transiently inhibited by Ca²⁺/calmodulin, with the result that receptors remain on the cell surface membrane during the early stage of agonist stimulation.     

Histamine H1-Receptor Binding Sites in Guinea Pig Brain Membranes: Regulation of Agonist Interactions by Guanine Nucleotides and Cations

Abstract: Agonist, but not antagonist, interactions with histamine H₂-receptors labeled by [³H]mepyramine are regulated selectively by sodium, divalent cations, and guanine nucleotides. Sodium decreases the affinity of histamine and the agonist 2-amino-ethylpyridine for [³H]mepyramine sites in guinea pig brain membranes up to 10-fold. The effect of sodium is exerted to a lesser extent by lithium, while potassium and rubidium are much weaker. Guanine nucleotides also decrease the affinity of histamine for H₁ binding sites about twofold. GTP and its nonmetabolized analogue GMP-PNP as well as GDP exert similar effects, while GMP, ATP, ADP, and AMP are inactive. The effects of GTP and sodium on histamine interactions with H₁-receptors are additive. By contrast, certain divalent cations enhance the potency of histamine at H₁-receptors. Manganese is most potent, while magnesium is almost as active as manganese and calcium is essentially inactive. Sodium, divalent cations, and guanine nucleotides have negligible effects on the interactions of antihistamines with H₁-receptors.     

Histamine H1 and Endothelin ETB Receptors Mediate Phospholipase D Stimulation in Rat Brain Hippocampal Slices

Abstract: Different neurotransmitter receptor agonists [carbachol, serotonin, noradrenaline, histamine, endothelin-1, and trans -(1 S ,3 R )-aminocyclopentyl-1,3-dicarboxylic acid ( trans -ACPD)], known as stimuli of phospholipase C in brain tissue, were tested for phospholipase D stimulation in [³²P]P_i-prelabeled rat brain cortical and hippocampal slices. The accumulation of [³²P]phosphatidylethanol was measured as an index of phospholipase D-catalyzed transphosphatidylation in the presence of ethanol. Among the six neurotransmitter receptor agonists tested, only noradrenaline, histamine, endothelin-1, and trans -ACPD stimulated phospholipase D in hippocampus and cortex, an effect that was strictly dependent of the presence of millimolar extracellular calcium concentrations. The effect of histamine (EC₅₀ 18 µ M ) was inhibited by the H₁ receptor antagonist mepyramine with a K _i constant of 0.7 n M and was resistant to H₂ and H₃ receptor antagonists (ranitidine and tioperamide, respectively). Endothelin-1-stimulated phospholipase D (EC₅₀ 44 n M ) was not blocked by BQ-123, a specific antagonist of the ET_A receptor. Endothelin-3 and the specific ET_B receptor agonist safarotoxin 6c were also able to stimulate phospholipase D with efficacies similar to that of endothelin-1, and EC₅₀ values of 16 and 3 n M , respectively. These results show that histamine and endothelin-1 stimulate phospholipase D in rat brain through H₁ and ET_B receptors, respectively.     

Modulation of Histamine Release and Synthesis in the Brain Mediated by α2-Adrenoceptors

Abstract: The adrenergic regulation of histamine release was studied in rat brain slices labeled with L-[³H]histidine. Noradrenaline in increasing concentrations progressively inhibited K⁺-evoked [³H]histamine release from cortical slices, whereas phenylephrine and isoprenaline were ineffective. Yohimbine, a preferential α2-adrenoceptor antagonist, reversed the noradrenaline effect in an apparently competitive manner and with a mean K i value of 30 n M . Phentolamine reversed the noradrenaline effect with a similar potency, whereas propranolol was ineffective. The imidazolines clo-nidine and oxymetazoline acted as partial agonists, oxymeta-zoline even behaving as an apparent antagonist. In vivo clo-nidine also inhibited [³H]histamine formation in cerebral cortex, an effect reversed by the administration of yohimbine. However, yohimbine failed to increase significantly [³H]histamine release in vitro and [³H]histamine formation in vivo, suggesting that adrenergic receptors are not activated by endogenous noradrenaline released under basal conditions. It is concluded that adrenergic α₂-adrenoceptors presumably located on histaminergic axons control release and synthesis of histamine in the brain.     

Astroglial Cells Express Large Amounts of GABAA Receptor Proteins in Mature Brain

Abstract: GABA_A receptors were characterized in cellular fractions isolated from adult bovine brain. The fraction enriched in cortical astrocytes is very rich in high-affinity binding sites for [³H]flunitrazepam and other "central-type" benzodiazepine ligands. The amount of specific [³H]flunitrazepam binding was more than five times higher in the glial fraction than in synaptosomal and perikaryal fractions. [³H]Flunitrazepam was displaced by low concentrations of clonazepam and other specific ligands for central GABA_A receptors. Specific binding sites for GABA, flunitrazepam, barbiturates, and picrotoxin-like convulsants were characterized. Allosteric interactions between the different sites were typical of central-type GABA_A receptors. The presence of α-subunit(s), as revealed by [³H]flunitrazepam photoaffinity labeling, was demonstrated in all brain fractions at molecular mass 51–53 kDa. Photoaffinity labeling was highest in the glial fraction. However, in primary cultured astrocytes from neonate rat cortex, no photoaffinity labeling was detected. Information obtained from astrocytes in culture should thus be taken with caution when extrapolated to differentiated astroglial cells. Our results actually show that, in mature brain, most of the fully pharmacologically active GABA_A receptors are extrasynaptic and expressed in astroglia.     

AMPA Receptor Development in Rat Telencephalon: [3H]AMPA Binding and Western Blot Studies

Abstract: Telencephalic membranes from rats of different embryonic (E16, E19) and postnatal (P2, P7, P14, adult) ages were assessed for α-[³H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([³H]AMPA) binding and for immunoreactivity levels of AMPA receptor subunits (GluR1, GluR2/3, and GluR4). In addition, the synaptic markers synaptophysin and NCAM₁₄₀ (a neural cell adhesion molecule isoform) were examined by immunoblot. The density of [³H]AMPA binding sites increased steadily with advancing age. This increase was due mainly to the development of the large low-affinity component ( K _D = 400 n M ) that dominates the [³H]AMPA binding profile of adult rat brain membranes. As resolved by two-site regression analysis, the high-affinity component ( K _D = 15 n M ) of the [³H]AMPA binding increased by approximately twofold from E16 to adult, whereas the low-affinity component increased by 25-fold. Staining for GluR1 and GluR2/3 increased steadily with increasing age at all time points examined; synaptophysin and NCAM₁₄₀ exhibited similar ontogenic immunostaining profiles. GluR4 immunoreactivity was first evident at P14 and increased by adulthood. These results indicate that AMPA receptor density increases steadily during development and that this increase is coincident with the ontogenic expression of other synaptic components. Furthermore, there is a shift toward a preponderance of low-affinity [³H]AMPA binding, which occurs during the period when AMPA receptors are being sorted into postsynaptic regions, suggesting that some element of the postsynaptic membrane environment modulates AMPA receptor properties.     

Distribution and Ontogeny of 1S,3R-1-Aminocyclopentane-1,3-Dicarboxylic Acid-Sensitive and Quisqualate-Insensitive [3H]Glutamate Binding Sites in the Rat Brain

Abstract: Displacement of [³H]glutamate by 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid and quisqualate (in the presence of saturating concentrations of ionotropic glutamate receptor agonists) was used to characterize optimal ionic conditions, distribution, and the ontogeny of glutamate receptor binding sites in rat brain. Using rat forebrain membranes or receptor autoradiography, optimal 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid-sensitive [³H]glutamate binding was found in the presence of 100 m M bromide ions and in the absence of calcium ions. Under these conditions, [³H]glutamate binding was relatively quisqualate insensitive. In regions of the neonatal (11-day-old) and adult rat brain, this [³H]glutamate binding was highest in forebrain (striatum, cerebral cortex, and hippocampus) and hypothalamus/midbrain but was lower in the cerebellum, olfactory bulb, and pons/medulla regions. 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid-sensitive and quisqualate-insensitive [³H]glutamate binding was present in the rat forebrain at 1 day of age and gradually increased more than twofold by day 50 (adult). Thus, in the presence of bromide ions and in the absence of calcium ions, [³H]glutamate labels a subpopulation of metabotropic glutamate receptors that are sensitive to 1 S ,3 R -1-aminocyclopentane-1,3-dicarboxylic acid but insensitive to quisqualate. Expression of [³H]glutamate binding under these conditions was both regionally and developmentally regulated in rat brain, suggesting that [³H]glutamate is labeling a distinct population of metabotropic glutamate receptors.     

[3H]Metergoline: A New Ligand of Serotonin Receptors in the Rat Brain

Abstract: A specific binding site for [³H]metergoline characterized by a K_D of 0.5–1.0 nM was detected in microsomal and synaptic plasma membranes from various areas of the adult rat brain. Experiments with 5,7-dihydroxy-tryptamine- and kainic acid-induced lesions indicated that this specific binding site was localized post-synaptically with respect to serotoninergic neurons. The pharmacological characteristics of [³H]metergoline binding to microsomal membranes from the whole forebrain strongly suggest that this ligand labels a class of serotonin receptors. This was particularly obvious in the hippocampus in which serotonin was about 400 times more potent than dopamine and norad-renaline for displacing bound [³H]metergoline. In the striatum, serotonin was only 10 times as potent as dopamine in inhibiting [³H]metergoline binding, suggesting that this ligand may also bind to dopamine receptors. Striking similarities between the binding sites for [³H]metergoline and [³H]serotonin were observed in the hippocampus. Thus, not only the total numbers of binding sites for these two ligands in control rats but also their respective increases following intracerebral 5,7-dihydroxytryptamine treatment were very similar. Therefore, at least in the hippocampus, [³H]metergoline might well be the appropriate ligand for studying the characteristics of the 'antagonist form' of serotonin receptors postulated by Bennett and Snyder.     

VITAMIN B6 TRANSPORT IN THE CENTRAL NERVOUS SYSTEM: IN VIVO STUDIES

Abstract— The total concentrations of vitamin B₆ (B₆) in plasma, choroid plexus, CSF and brain of adult New Zealand white rabbits, measured fluorometrically, were 0.30, 15.10, 0.39 and 8.90 μ mol/l or kg respectively. The mechanisms by which B₆ enters and leaves brain, choroid plexus and CSF were investigated by injecting [³H]pyridoxine (PIN) intravenously, intraventricularly and intraarterially. [³H]PIN, with or without unlabelled PIN, was infused intravenously at a constant rate into conscious rabbits. At 150 min, [³H]B₆ readily entered CSF, choroid plexus and brain. The addition of 0.5 mmol/kg carrier PIN to the infusion solution depressed the relative entry of [³H]B₆ into CSF, choroid plexus and brain by about 80%. After intraventricular injection, [³H]PIN readily entered brain from CSF. The intraventricular injection of carrier PIN with [³H]PIN decreased the amount of [³H]B₆ in brain and also decreased the percentage of [³H]B₆ in CSF and brain that was phosphorylated. During one pass through the cerebral circulation, [³H]PIN (1 μ m ) was cleared from the circulation no more rapidly than mannitol. These results were interpreted as showing that the entry of B₆ from blood into CSF and presumably the extracellular space of brain and thence into brain cells involves one or more saturable transport and/or metabolic steps.     

Regulation of DOPA Decarboxylase Activity in Brain of Living Rat

Abstract: To test the hypothesis that l -DOPA decarboxylase (DDC) is a regulated enzyme in the synthesis of dopamine (DA), we developed a model of the cerebral uptake and metabolism of [³H]DOPA. The unidirectional blood-brain clearance of [³H]DOPA ( K ^D₁) was 0.049 ml g⁻¹ min⁻¹. The relative DDC activity ( k ^D₃) was 0.26 min⁻¹ in striatum, 0.04 min⁻¹ in hypothalamus, and 0.02 min⁻¹ in hippocampus. In striatum, 3,4-[³H]dihydroxyphenylacetic acid ([³H]DOPAC) was formed from [³H]DA with a rate constant of 0.013 min⁻¹, [³H]homovanillic acid ([³H]HVA) was formed from [³H]DOPAC at a rate constant of 0.020 min⁻¹, and [³H]HVA was eliminated from brain at a rate constant of 0.037 min⁻¹. Together, these rate constants predicted the ratios of endogenous DOPAC and HVA to DA in rat striatum. Pargyline, an inhibitor of DA catabolism, substantially reduced the contrast between striatum and cortex, in comparison with the contrast seen in autoradiograms of control rats. At 30 min and at 4 h after pargyline, k ^D₃ was reduced by 50% in striatum and olfactory tubercle but was unaffected in hypothalamus, indicating that DDC activity is reduced in specific brain regions after monoamine oxidase inhibition. Thus, DDC activity may be a regulated step in the synthesis of DA.     

Pharmacological and Regional Characterization of [3H]LY278584 Binding Sites in Human Brain

Abstract: Binding of [³H]LY278584, which has been previously shown to label 5-hydroxytryptamine₃ (5-HT₃) receptors in rat cortex, was studied in human brain. Saturation experiments revealed a homogeneous population of saturable binding sites in amygdala ( K _D= 3.08 ± 0.67 n M, B _max= 11.86 ± 1.87 fmol/mg of protein) as well as in hippocampus, caudate, and putamen. Specific binding was also high in nucleus accumbens and entorhinal cortex. Specific binding was negligible in neocortical areas. Kinetic studies conducted in human hippocampus revealed a K _on of 0.025 ± 0.009 n M ⁻¹ min⁻¹ and a K _off of 0.010 ± 0.002 min⁻¹. The kinetics of [³H]LY278584 binding were similar in the caudate. Pharmacological characterization of [³H]LY278584 specific binding in caudate and amygdala indicated the compound was binding to 5-HT₃ receptors. We conclude that 5-HT₃ receptors labeled by [³H]LY278584 are present in both limbic and striatal areas in human brain, suggesting that 5-HT₃ receptor antagonists may be able to influence the dopamine system in humans, similarly to their effects in rodent studies.     

[3H]SCH 58261, a Selective Adenosine A2A Receptor Antagonist, Is a Useful Ligand in Autoradiographic Studies

Abstract: We have characterized the new potent and selective nonxanthine adenosine A_2A receptor antagonist SCH 58261 as a new radioligand for receptor autoradiography. In autoradiographic studies using agonist radioligands for A_2A receptors ([³H]CGS 21680) or A₁ receptors ( N ⁶-[³H]cyclohexyladenosine), it was found that SCH 58261 is close to 800-fold selective for rat brain A_2A versus A₁ receptors ( K _i values of 1.2 n M versus 0.8 µ M ). Moreover, receptor autoradiography showed that [³H]SCH 58261, in concentrations below 2 n M , binds only to the dopamine-rich regions of the rat brain, with a K _D value of 1.4 (0.8–1.8) n M . The maximal number of binding sites was 310 fmol/mg of protein in the striatum. Below concentrations of 3 n M , the nonspecific binding was <15%. Three adenosine analogues displaced all specific binding of [³H]SCH 58261 with the following estimated K _i values (n M ): 2-hex-1-ynyl-5'- N -ethylcarboxamidoadenosine, 3.9 (1.8–8.4); CGS 21680, 130 (42–405); N ⁶-cyclohexyladenosine, 9,985 (3,169–31,462). The binding of low concentrations of SCH 58261 was not influenced by either GTP (100 µ M ) or Mg²⁺ (10 m M ). The present results show that in its tritium-labeled form, SCH 58261 appears to be a good radioligand for autoradiographic studies, because it does not suffer from some of the problems encountered with the currently used agonist radioligand [³H]CGS 21680.     

Allosteric Interactions Among Agonists and Antagonists at 5-Hydroxytryptamine3 Receptors

Abstract: Cooperation in the action of agonists suggests that there are multiple binding sites on 5-hydroxytryptamine₃ (5-HT₃) receptors. The purpose of this study was to characterize these binding sites and their interactions on both native and cloned 5-HT₃ receptors. The affinities of competitive 5-HT₃ receptor antagonists were similar regardless of whether the receptors were labeled with [³H]RS-42358, [³H]granisetron, or 1-( m -[³H]chlorophenyl)biguanide ([³H]mCPG). By contrast, the affinities of the agonists 5-HT, mCPG, and phenylbiguanide were approximately 10-fold higher when the receptors were labeled with [³H]mCPG. The dissociation of [³H]mCPG, [³H]RS-42358, and [³H]RS-25259, but not [³H]granisetron, from both cloned and native 5-HT₃ receptors was markedly slower in the presence of 5-HT or 2-methyl-5-HT than in the presence of antagonists such as RS-42358. This suggests that the binding of these agonists to unoccupied sites on the receptor can increase the receptor's affinity for prebound ligands and thereby slow their dissociation. These data support previous indications of positive cooperation among multiple binding sites on both native and cloned 5-HT₃ receptors, and they extend this idea by demonstrating that agonists can modify the interaction of some, but not all, antagonists with the receptor.     

Pharmacology and Regional Distribution of the Binding of 6-[3H]Nitro-7-Sulphamoylbenzo[f]-Quinoxaline-2,3-Dione to Rat Brain

Abstract: 6-Nitro-7-sulphamoylbenzo[ f ]quinoxaline-2,3-dione (NBQX) is a competitive antagonist selective for α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. Here we report the pharmacological characteristics and anatomical distribution of [³H]NBQX binding to rat brain. The association rate of [³H]NBQX to rat cerebrocortical membranes was rapid, with peak binding occurring within 10 min at 0°C. The off-rate was also rapid, with near-complete dissociation of the radioligand within 5 min of addition of 1 m M unlabelled l -glutamate. [³H]NBQX bound to a single class of sites with K _D and B _max values of 47 n M and 2.6 pmol mg⁻¹ of protein, respectively. The rank order of inhibition of [³H]NBQX binding by AMPA receptor ligands was NBQX ≫ 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) ≥ ( S )-5-fluorowillardiine ≥ AMPA ≫ l -glutamate. The chaotrope KSCN had no effect on the IC₅₀ value of unlabelled NBQX displacement of [³H]NBQX binding. The kainate receptor-selective ligands NS102 and kainate were only very weak displacers. It is interesting that NBQX and CNQX displaced significantly more [³H]NBQX than any of the agonists tested. Autoradiographic analysis of the binding of [³H]NBQX to coronal sections showed a distribution compatible with that of [³H]AMPA binding. These data indicate that [³H]NBQX provides a useful novel tool to characterise the antagonist binding properties of AMPA receptors.     

Platelet-Activating Factor: Diminished Acetylcholine Release from Rat Brain Slices Is Mediated by a Gi Protein

Abstract: The effect of platelet-activating factor (PAF) on neurotransmitter release from rat brain slices prelabeled with [³H]acetylcholine ([³H]ACh), [³H]norepinephrine ([³H]NE), or [³H]serotonin ([³H]5-HT) was studied. PAF inhibited K⁺ depolarization-induced [³H]ACh release in slices of brain cortex and hippocampus by up to 59% at 10 n M but did not inhibit [³H]ACh release in striatal slices. PAF did not affect 5-HT or NE release from cortical brain slices. The inhibition of K⁺-evoked [³H]ACh release induced by PAF was prevented by pretreating tissues with several structurally different PAF receptor antagonists. The effect of PAF was reversible and was not affected by pretreating brain slices with tetrodotoxin. PAF-induced inhibition of [³H]ACh release was blocked 90 ± 3 and 86 ± 2% by pertussis toxin and by anti-Gαi_1/2 antiserum incorporated into cortical synaptosomes, respectively. The results suggest that PAF inhibits depolarization-induced ACh release in brain slices via a Gαi_1/2 protein-mediated action and that PAF may serve as a neuromodulator of brain cholinergic system.     

Involvement of Presynaptic Histamine H3 Receptors in the Modulation of Somatostatin Binding and Its Effects on Adenylyl Cyclase Activity in the Rat Frontoparietal Cortex

Abstract: Thioperamide (2 mg/kg, i.p.), a histamine H₃-receptor antagonist, increased the number of somatostatin (SS) receptors, with no change in the affinity constant, in the rat frontoparietal cortex. This effect was prevented by treatment with ( R )-α-methylhistamine (3.2 mg/kg, i.p.), a histamine H₃-receptor agonist. Thioperamide also induced an increase in SS binding in rats pretreated with mepyramine, a histamine H₁-receptor antagonist, or cimetidine, a histamine H₂-receptor antagonist. Pretreatment with mepyramine plus cimetidine administered simultaneously antagonized the thioperamide effect on SS binding. The increase in the number of SS receptors was accompanied by a greater SS-mediated inhibition of basal and forskolin-stimulated adenylyl cyclase (AC) activity in frontoparietal cortical membranes in the thioperamide group. Furthermore, the functional activity of the guanine nucleotide-binding inhibitory protein (G_i protein) was not altered by thioperamide or ( R )-α-methylhistamine administration in frontoparietal cortical membranes. In rats treated with mepyramine plus thioperamide or cimetidine plus thioperamide, the increase in the number of SS receptors was also accompanied by an increased SS inhibition of AC activity. Thioperamide induced a significant increase in SS-like immunoreactivity content in the frontoparietal cortex. Altogether, these results suggest that frontoparietal cortical histamine may play, at least in part, a role in the regulation of the somatostatinergic system.     

[3H]Nemonapride and [3H]Spiperone Label Equivalent Numbers of D2 and D3 Dopamine Receptors in a Range of Tissues and Under Different Conditions

Abstract: [³H]Nemonapride and [³H]spiperone are very widely used to study dopaminergic systems in vitro and in vivo, but it has been reported that [³H]nemonapride and [³H]spiperone give markedly different B _max values for preparations of D₂ dopamine receptors from recombinant cell lines or animal tissues. We have used the two radioligands in parallel to study a range of dopamine receptors [D_2(short), D_2(long), and D₃] in different buffers. B _max values derived using either radioligand differ by an average of <20%, independent of receptor type or buffer conditions. All competition experiments show that the two ligands compete at a single site. It seems that [³H]spiperone and [³H]nemonapride do not differentiate between different forms or populations of D₂-like receptors.     

Lindane Administration to the Rat Induces Modifications in the Regional Cerebral Binding of [3H]Muscimol, [3H]-Flunitrazepam, and t-[35S]Butylbicyclophosphorothionate: An Autoradiographic Study

Abstract: The effect of lindane administration on the specific binding of ligands to different sites on the GABA_A receptor-ionophore complex was studied in the rat brain by receptor mapping autoradiography. [³H]Muscimol (Mus), [³H]flunitrazepam (Flu), and t -[³⁵S]butylbicyclophosphorothionate (TBPS) were used as specific ligands of GABA, benzodiazepine, and picrotoxinin binding sites, respectively. Rats received a single oral dose of 30 mg/kg lindane and they were classified into two groups according to the absence or presence of convulsions. Vehicle-treated groups acted as controls. The effect of the xenobiotic on ligand binding was measured in different brain areas and nuclei 12 min or 5 h after its administration. Lindane induced a generalized decrease in [³⁵S]TBPS binding, which was present shortly after dosing. In addition, [³H]Flu binding was increased in lindane-treated animals, this modification also appearing shortly after administration but diminishing during the studied time. Finally, lindane induced a decrease in [³H]Mus binding, which became more evident over time. These modifications were observed both in the presence and in the absence of convulsions. However, an increase in [³H]-Mus binding was detected shortly after lindane-induced convulsions. The observed decrease in [³⁵S]TBPS binding is in agreement with the postulated action of lindane at the picrotoxinin binding site of the GABA_A receptor chloride channel. The effects observed on the binding of [³H]Flu and [³H]Mus may be secondary to the action of lindane as an allosteric antagonist of the GABA_A receptor.     

Inhibition of GABAB Receptor Binding by Guanyl Nucleotides

Abstract: GTP and GDP decreased the saturable binding of [³H]baclofen or [³H]γ-aminobutyric acid ([³H]GABA) to GABA_B but not GABA_A receptors whereas GMP displayed negligible activity. This effect was specific to guanyl nucleotides and was not mimicked by high concentrations of ATP. The inhibition of ligand binding was the result of a diminished receptor affinity with no change in receptor number. The use of a complete physiological saline solution rather than Tris buffer plus Ca²⁺ or Mg²⁺ increased the potency of GTP at the GABA_B receptor. The results are discussed in relation to the effects of GABA and GTP on adenylate cyclase activity in the brain.     

Guinea pig hippocampal 5-HT1E receptors: a tool for selective drug development

Recent studies have indicated that the serotonin [5-hydroxytryptamine (5-HT)] 1E receptor, originally discovered in human brain tissue, is not expressed in rat or mouse brain. Thus, there have been few reports on 5-HT_1E receptor drug development. However, expression of 5-HT_1E receptor mRNA has been shown in guinea pig brain. To establish this species as an animal model for 5-HT_1E drug development, we identified brain regions that exhibit 5-carboxyamidotryptamine, ritanserin, and LY344864 – insensitive [³H]5-HT binding (characteristic of the 5-HT_1E receptor). In hippocampal homogenates, where 5-HT_1E receptor density was sufficiently high for radioligand binding analysis, 100 nM 5-carboxyamidotryptamine, 30 nM ritanserin, and 100 nM LY344864 were used to mask [³H]5-HT binding at non-5-HT_1E receptors. The K _d of [³H]5-HT was 5.7 ± 0.7 nM and is indistinguishable from the cloned receptor K _d of 6.5 ± 0.6 nM. The affinities of 16 drugs for the cloned and hippocampal-expressed guinea pig 5-HT_1E receptors are essentially identical ( R ² = 0.97). These findings indicate that using these conditions autoradiographical distribution and signal transduction studies of the 5-HT_1E receptor in guinea pig brain are feasible. Using the guinea pig as an animal model should provide important insights into possible functions of this receptor and the therapeutic potential of selective human 5-HT_1E drugs.