Actions of avermectin B1a on the γ-aminobutyric AcidA receptor and chloride channels in rat brain

The interaction of avermectin B_1a (AVM) with the γ-aminobutyric acid (GABA) receptor of rat brain was studied using radioactive ligand binding and tracer ion flux assays. Avermectin potentiated the binding of [³H]flunitrazepam and inhibited the binding of both [³H]muscimol and [³⁵S]t-butylbicyclo-phosphorothionate to the GABA_A receptor. Inhibition of muscimol binding by AVM suggested competitive displacement. Two kinds of ³⁶chloride (Cl) flux were studied. The ³⁶Cl efflux from preloaded microsacs was potentiated by AVM and was highly inhibited by the Cl-channel blocker 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS). However, it was not potentiated by GABA nor was it sensitive to the convulsants picrotoxin or bicuculline. On the other hand, ³⁶Cl-influx measurement in a different microsac preparation of rat brain was very sensitive to GABA and other GABA-ergic drugs. Avermectin induced ³⁶Cl influx into these microsacs in a dose–dependent manner, but to only 35% of the maximal influx induced by GABA. The AVM-induced ³⁶Cl influx was totally blocked by bicuculline. It is suggested that AVM opens the GABA_A-receptor Cl channel by binding to the GABA recognition site and acting as a partial receptor agonist, and also opens a voltage–dependent Cl channel which is totally insensitive to GABA but is very sensitive to DIDS.     

[3H]Bicuculline Methochloride Binding to Low-Affinity γ-Aminobutyric Acid Receptor Sites

Abstract: The binding of [³H]bicuculline methochloride (BMC) to mammalian brain membranes was characterized and compared with that of [³H]γ-aminobutyric acid ([³H]GABA). The radiolabeled GABA receptor antagonist showed significant displaceable binding in Tris-citrate buffer that was improved by high concentrations of chloride, iodide, or thiocyanate, reaching >50% displacement in the presence of 0.1 M SCN^?. An apparent single class of binding sites for [³H]BMC (K_D= 30 nM) was observed in 0.1 M SCN^? for fresh or frozen rat cortex or several regions of frozen and thawed bovine brain. The B_max was about 2 pmol bound/mg of crude mitochondrial plus microsomal membranes from unfrozen washed and osmotically shocked rat cortex, similar to that for [³H]GABA. Frozen membranes, however, showed decreased levels of [³H]BMC binding with no decrease or an actual increase in [³H]GABA binding sites. [³H]BMC binding was inhibited by GABA receptor specific ligands, but showed a higher affinity for antagonists and lower affinity for agonists than did [³H]GABA binding. Kinetics experiments with [³H]GABA binding revealed that low- and high-affinity sites showed a similar pharmacological specificity for a series of GABA receptor ligands, but that whereas all agonists had a higher affinity for slowly dissociating high-affinity [³H]GABA sites, bicuculline had a higher affinity for rapidly dissociating low-affinity [³H]GABA sites. This reverse potency between agonists and antagonists during assay of radioactive antagonists or agonists supports the existence of agonist- and antagonist-preferring conformational states or subpopulations of GABA receptors. The differential affinities, as well as opposite effects on agonist and antagonist binding by anions, membrane freezing, and other treatments, suggest that [³H]BMC may relatively selectively label low-affinity GABA receptor agonist sites. This study, using a new commercially available preparation of [³H]bicuculline methochloride, confirms the report of bicuculline methiodide binding by Mohler and Okada (1978), and suggests that this radioactive GABA antagonist will be a valuable probe in analyzing various aspects of GABA receptors.     

Characterization of the Binding of the GABA Agonist [3H]Piperidine-4-Sulphonic Acid to Bovine Brain Synaptic Membranes

Abstract: The binding of radioactive piperidine-4-sulphonic acid ([³H]P4S) to thoroughly washed, frozen, and thawed membranes isolated from cow and rat brains has been studied. Quantitative computer analysis of the binding curves for four regions of bovine brain revealed the general presence of two binding sites. In these brain regions less satisfactory computer fits were obtained for receptor models showing one or three binding sites or negative cooperativity. With the use of Tris-citrate buffer at 0°C the two affinity classes for P4S in bovine cortex membranes revealed the following binding parameters: K_D= 17 ± 7 nM (B_max= 0.15 ± 0.07 pmol/mg protein) and K_D= 237 ± 100 nM (B_max= 0.80 ± 0.20 pmol/mg protein). Heterogeneity was also observed for association and dissociation rates of [³H]P4S. The slow binding component (k_on= 5.6 × 10⁷ or 8.8 × 10⁷ M^-1 min^-1, k_Off= 0.83 min^-1, and K_D= 14.7 or 9.4 nM, determined by two different methods in phosphate buffer containing potassium chloride) corresponds to the high-affinity component of the equilibrium binding curve (K_D= 11 nM, B_max= 0.12 pmol/mg protein in the same buffer system). The association and dissociation rates for the subpopulation of rapidly dissociating sites, apparently corresponding to the low-affinity sites, were too rapid to be measured accurately. The binding of [³H]P4S appears to involve the same two populations of sites with B_max values similar to those for [³H]GABA binding to the same tissue, although the kinetic parameters for the two ligands are somewhat different. Furthermore, comparative studies on the inhibition of [³H]P4S and [³H]GABA binding by various GABA analogues, strongly suggest that P4S binds to the GABA receptors. The different effects of P4S and GABA on benzodiazepine binding are discussed.     

Avermectin Bla Modulation of γ-Aminobutyric Acid/Benzodiazepine Receptor Binding in Mammalian Brain

The anthelminthic natural product avermectin B1a (AVM) modulates the binding of gamma-aminobutyric acid (GABA) and benzodiazepine (BZ) receptor ligands to membrane homogenates of mammalian brain. The potent (EC50 = 40 nM) enhancement by AVM of [3H]diazepam binding to rat or bovine brain membranes resembled that of barbiturates and pyrazolopyridines in being inhibited (partially) by the convulsants picrotoxin, bicuculline, and strychnine, and by the anticonvulsants phenobarbital and chlormethiazole. The maximal effect of AVM was not increased by pentobarbital or etazolate. However, AVM affected BZ receptor subpopulations or conformational states in a manner different from pentobarbital. Further, unlike pentobarbital and etazolate, AVM did not inhibit allosterically the binding of the BZ receptor inverse agonist [3H]beta-carboline-3-carboxylate methyl ester, nor did it inhibit, but rather enhanced, the binding of the cage convulsant [35S]t-butyl bicyclophosphorothionate to picrotoxin receptor sites. AVM at submicromolar concentrations had the opposite effect of pentobarbital and etazolate on GABA receptor binding, decreasing by half the high-affinity binding of [3H]GABA and related agonist ligands, and increasing by over twofold the binding of the antagonist [3H]bicuculline methochloride, an effect that was potentiated by picrotoxin. AVM also reversed the enhancement of GABA agonists and inhibition of GABA antagonist binding by barbiturates and pyrazolopyridines. These overall effects of AVM are unique and require the presence of another separate drug receptor site on the GABA/BZ receptor complex.     

Characterization and Localization of Adenosine A2 Receptors in Bovine Rod Outer Segments

Abstract: Previous work from this laboratory has shown that retinal adenosine A₂ binding sites are localized over outer and inner segments of photoreceptors in rabbit and mouse retinal sections. In the present study, adenosine receptor binding has been characterized and localized in membranes from bovine rod outer segments (ROS). Saturation studies with varying concentrations (10–150 nM) of 5′-(N-[2,8-³H]ethylcarboxamido)adenosine ([³H]NECA) and 100 μg of ROS membrane protein show a single site with a K_D of 103 nM and a B_max of 1.3 pM/mg of protein. Cold Scatchards, which used nonradiolabeled NECA (concentrations ranging from 10 nM to 250 nM) in competition with a fixed amount of [³H]NECA (30 nM), demonstrated the presence of a low-affinity site (K_D, 50 μM) in addition to the high-affinity site. To confirm the presence of A_2abinding sites, saturation analyses with 2-p-(2-[³H]-carboxyethyl)phenylamino-5′-N-ethylcarboxamido adenosine (0–80 nM) also revealed a single population of high-affinity A_2a receptors (K_D, 9.4 nM). The binding sites labeled by [³H]NECA appear to be A₂ receptor sites because binding was displaced by increasing concentrations of 5′-(N-methylcarboxamido)adenosine and 2-chloroadenosine. ROS were fractionated into plasma and disk membranes for localization studies. Receptor binding assays, used to determine specific binding, showed that the greatest concentration of A₂ receptors was on the plasma membranes. Therefore, adenosine A₂ receptors are in a position to respond to changes in the concentration of extracellular adenosine, which may exhibit a circadian rhythm.     

Effects of Nucleotides on [3H]Bradykinin Binding in Guinea Pig: Further Evidence for Multiple B2 Receptor Subtypes

Abstract: We have suggested recently the existence of three subtypes of B₂ bradykinin receptors in tissues of guinea pigs. We have classified these B₂ bradykinin receptors into B_2a, B_2b, and B_2c subtypes depending on their affinity for various bradykinin antagonists. Because the actions of bradykinin in different cell systems appear to be both dependent on and independent of G proteins, we sought to determine whether the binding of [³H]bradykinin to the B₂ subtypes is sensitive to guanine nucleotides and, therefore, possibly coupled to G proteins. In the ileum, where we have demonstrated B_2a and B_2b subtypes, specific [³H]bradykinin binding was reduced with GDP (100 μM) and the nonmetabolized analogue of GTP, guanyl-5′-yl-imidodiphosphate (GppNHp; 100 μM). Competition studies with bradykinin and with [Hyp³]-bradykinin, which shows approximately 20-fold greater selectivity for the B_2a subtype than bradykinin, were performed in the presence or absence of GppNHp (100 μM). The competition experiments demonstrated that binding to the B_2a subtype, which has higher affinity for [Hyp³]-bradykinin and bradykinin than the B_2b subtype, was lost in the presence of GppNHp, whereas binding to the B_2b subtype was unaffected. In contrast, GppNHp (100 μM) and GDP (100 μM) failed to alter specific [³H]bradykinin binding to B_2b and B_2c subtypes in lung. [³H]Bradykinin binding was unaffected by AMP, ADP, ATP, and GMP (100 μM each). Based on this evidence, we suggest that the B_2a bradykinin subtype is coupled to G proteins. The B_2b and B_2c subtypes are either not coupled to G proteins, or may be coupled to the G^o-type GTP binding proteins, which have been suggested to be less sensitive to guanine nucleotides. These data provide further evidence for three subtypes of B₂-type bradykinin receptors in guinea pig.     

In vitro neuropharmacological evaluation of penitrem-induced tremorgenic syndromes: importance of the GABAergic system

The effects of the fungal neurotoxin penitrem A on the GABAergic and glutamatergic systems in rat brain were evaluated. Penitrem A inhibited binding of the GABA_A-receptor ligand [³H]TBOB to rat forebrain and cerebellar membrane preparations with IC₅₀ (half maximal inhibitory concentration) values of 11 and 9 μM, respectively. Furthermore, penitrem A caused a concentration-dependent increase of [³H]flunitrazepam and [³H]muscimol binding in rat forebrain, but not in cerebellar preparations. The stimulation of [³H]flunitrazepam binding by penitrem A was abolished by the addition of GABA. In cerebellar preparations, a different pharmacological profile was found, with penitrem A allosterically inhibiting [³H]TBOB binding by interacting with a bicuculline-sensitive site. Moreover, penitrem A inhibited the high affinity uptake of GABA and glutamate into cerebellar synaptosomes with IC₅₀ values of 20 and 47 μM, respectively. The toxin showed no effect on NMDA or AMPA glutamate receptor binding. In conclusion, our results suggest that penitrem A exerts region-specific effects in the brain, leading to positive modulation of GABA_A-receptor function in forebrain. Conversely, penitrem A may act as a bicuculline-like convulsant in cerebellum.     

Binding of [3H] γ-Aminobutyric Acid and [3H]Muscimol in Purified Rat Brain Synaptic Plasma Membranes and the Effects of Bicuculline

Abstract: The binding of [³H] γ-aminobutyric acid ([³H]GABA) and [³H]muscimol has been studied in purified synaptic plasma membrane (SPM) preparations from rat brain. Scatchard analysis of specific binding (defined as that displaced by 100 μMγ-aminobutyrate) indicated that the binding of both radiolabelled ligands was best described by a two component Langmuir adsorption isotherm. The apparent K_D and B_max values for [³H]GABA at 4°C were K_D1, 20 nM; K_D2,165 nM; B_max1, 0.48 pmol;B_max2, 6.0 pmol. mg^?1; for [³H]muscimol at 4°C they were: K_D1, 1.75 nM; K_D2, 17.5 nM; B_maxl, 0.84 pmol. mg^?1; B_max2, 4.8 pmol.mg^?1; and for [³H]muscimol at 37°C they were: K_D1, 7.0 nM; K_m, 60 nM; B_max], 0.5 pmol-mg^?1; B_max2, 7.2 pmol-mg¹. Under the experimental conditions used, the similar B_milx values for [³H]GABA and [³H]muscimol binding to the SPM preparations suggests that the high- and low-affinity components for the two radiolabeled ligands are identical. The effects of the GAB A antagonist bicuculline on the binding of [³H]muscimol at 4^CC and 37°C were studied. At 4°C, antagonism of muscimol binding appeared to be competitive at the high-affinity site but noncompetitive at the low-affinity site. At 37°C, antagonism was again competitive at the high-affinity site but was of a mixed competitive/noncompetitive nature at the low-affinity site. Assuming that binding to the high-affinity site is associated with the pharmacological actions of bicuculline, the apparent K_D values obtained suggest a pA₂ value of 5.3 against [³H]muscimol at 4°C and 37°C. This figure is in good agreement with several estimates of the potency of bicuculline based on pharmacological measurements. Results from displacement studies using [³H]GABA and [³H]muscimol suggest that [³H]GABA might be a more satisfactory ligand than [³H]muscimol in GABA radioreceptor assays.     

Characterisation of the Binding of [3H]WAY-100635, a Novel 5-Hydroxytryptamine1A Receptor Antagonist, to Rat Brain

Abstract: The specific binding of [³H]WAY-100635 {N-[2-[4-(2-[O-methyl-³H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride} to rat hippocampal membrane preparations was time, temperature, and tissue concentration dependent. The rates of [³H]WAY-100635 association (k₊₁ = 0.069 ± 0.015 nM^?1 min^?1) and dissociation (k_?1 = 0.023 ± 0.001 min^?1) followed monoexponential kinetics. Saturation binding isotherms of [³H]WAY-100635 exhibited a single class of recognition site with an affinity of 0.37 ± 0.051 nM and a maximal binding capacity (B_max) of 312 ± 12 fmol/mg of protein. The maximal number of binding sites labelled by [³H]WAY-100635 was ～36% higher compared with that of 8-hydroxy-2-(di-n-[³H]-propylamino)tetralin ([³H]8-OH-DPAT). The binding affinity of [³H]WAY-100635 was significantly lowered by the divalent cations CaCl₂ (2.5-fold; p < 0.02) and MnCl₂ (3.6-fold; p < 0.05), with no effect on B_max. Guanyl nucleotides failed to influence the K_D and B_max parameters of [³H]WAY-100635 binding to 5-HT_1A receptors. The pharmacological binding profile of [³H]WAY-100635 was closely correlated with that of [³H]8-OH-DPAT, which is consistent with the labelling of 5-hydroxytryptamine_1A (5-HT_1A) sites in rat hippocampus. [³H]WAY-100635 competition curves with 5-HT_1A agonists and partial agonists were best resolved into high- and low-affinity binding components, whereas antagonists were best described by a one-site binding model. In the presence of 50 µM guanosine 5′-O-(3-thiotriphosphate) (GTPγS), competition curves for the antagonists remained unaltered, whereas the agonist and partial agonist curves were shifted to the right, reflecting an influence of G protein coupling on agonist versus antagonist binding to the 5-HT_1A receptor. However, a residual (16 ± 2%) high-affinity agonist binding component was still apparent in the presence of GTPγS, indicating the existence of GTP-insensitive sites.     

GABAA agonists: Resolution and pharmacology of (+)- and (−)-isoguvacine oxide

(3SR,4RS)-3,4-Epoxypiperidine-4-carboxylic acid (isoguvacine oxide) is a potent and specific GABA_A receptor agonist. Isoguvacine oxide, originally designed as a potentially alkylating agonist, turned out to interact with the GABA_A receptor in a fully reversible manner. The protected form of isoguvacine oxide, benzyl (3SR,4RS)-1-(benzyloxycarbonyl)-3,4-epoxypiperidine-4-carboxylate ( 1 ) (Scheme 1), has now been resolved by chiral chromatography using cellulose triacetate as a chiral stationary phase. The enantiomers of 1 (ee ≥ 98.8%) were subsequently deprotected by hydrogenolysis. Whereas both enantiomers of isoguvacine oxide were inactive as inhibitors of the binding of [³H]GABA to GABA_B receptor sites (IC₅₀ > 100 μM), (+)-isoguvacine oxide (IC₅₀ = 0.20 ± 0.03 μM) and (?)-isoguvacine oxide (IC₅₀ = 0.32 ± 0.05 μM) showed comparable potencies as inhibitors of the binding of [³H]GABA to GABA_A receptor sites. Furthermore, (+)-isoguvacine oxide (EC₅₀ = 6 μM; 33% relative efficacy) and (?)-isoguvacine oxide (EC₅₀ = 5 μM; 38% efficacy relative to 10 μM muscimol) were approximately equipotent and equiefficacious as stimulators of the binding of [³H]diazepam to the GABA_A receptor-associated benzodiazepine site. This latter effect is an in vitro estimate of GABA_A agonist efficacy. These pharmacological data for isoguvacine oxide and its enantiomers do not seem to support our earlier conception of the topography of the GABA_A recognition site(s), derived from extensive structure—activity studies on GABA_A agonists. Thus, the model of the GABA_A recognition site(s) comprising a narrow cleft or pocket, in which the anionic moiety of the zwitterionic GABA_A agonists is assumed to be embedded during receptor activation, may have to be revised. © 1995 Wiley-Liss, Inc.     

Decreased GABA<Subscript>A</Subscript> Receptor Function in the Brain Stem during Pancreatic Regeneration in Rats

Gamma amino butyric acid is a major inhibitory neurotransmitter in the central nervous system. In the present study we have investigated the alteration of GABA receptors in the brain stem of rats during pancreatic regeneration. Three groups of rats were used for the study: sham operated, 72 h and 7 days partially pancreatectomised. GABA was quantified by [³H]GABA receptor displacement method. GABA receptor kinetic parameters were studied by using the binding of [³H]GABA as ligand to the Triton X-100 treated membranes and displacement with unlabelled GABA. GABA_A receptor activity was studied by using the [³H]bicuculline and displacement with unlabelled bicuculline. GABA content significantly decreased (P < 0.001) in the brain stem during the regeneration of pancreas. The high affinity GABA receptor binding showed a significant decrease in B _max (P < 0.01) and K _d (P < 0.05) in 72 h and 7 days after partial pancreatectomy. [³H]bicuculline binding showed a significant decrease in B _max and K _d (P < 0.001) in 72 h pancreatectomised rats when compared with sham where as B _max and K _d reversed to near sham after 7 days of pancreatectomy. The results suggest that GABA through GABA receptors in brain stem has a regulatory role during active regeneration of pancreas which will have immense clinical significance in the treatment of diabetes.     

Evidence for Presynaptic Adenosine A2a Receptors Associated with Norepinephrine Release and Their Desensitization in the Rat Nucleus Tractus Solitarius

Abstract: Rat medullary brain segments containing primarily nucleus tractus solitarius (NTS) were used for superfusion studies of evoked transmitter release and for isotherm receptor binding assays. Isotherm binding assays with [³H]CGS-21680 on membranes prepared from NTS tissue blocks indicated a single high-affinity binding site with a K_D of 5.1 ± 1.4 nM and a B_max of 20.6 ± 2.4 fmol/mg of protein. The binding density for [³H]CGS-21680 on NTS membranes was 23 times less than comparable binding on membranes from striatal tissue. Electrically stimulated (1 min at 25 mA, 2 ms, 3 Hz) release of [³H]norepinephrine ([³H]NE) from 400-µm-thick NTS tissue slices resulted in an S₂/S₁ ratio of 0.96 ± 0.02. Superfusion of single tissue slices with 0.1–100 nM CGS-21680, a selective adenosine A_2a receptor agonist, for 5 min before the S₂ stimulus produced a significant concentration-dependent increase in the S₂/S₁ fractional release ratio that was maximal (31.3% increase) at 1.0 nM. However, superfusion of tissue slices with CGS-21680 over the same concentration range for 20 min before the S₂ stimulus did not alter the S₂/S₁ ratio significantly from control release ratios. The augmented release of [³H]NE mediated by 1.0 nM CGS-21680 with a 5-min tissue exposure was abolished by 1.0 and 10 nM CGS-15943 as well as by 100 nM 8-(3-chlorostyryl)caffeine, both A_2a receptor antagonists, but not by 1.0 nM 8-cyclopentyl-1,3-dipropylxanthine, the A₁ receptor antagonist. Taken together, these results suggest that CGS-21680 augmented the evoked release of [³H]NE in the NTS via activation of presynaptic A_2a receptors within the same concentration range as the binding affinity observed for [³H]CGS-21680. It was also apparent that this population of presynaptic adenosine A_2a receptors in the NTS desensitized within 20 min because the augmenting action of CGS-21680 on evoked transmitter release was not evident at the longer interval.     

Interacting Presynaptic k-Opioid and GABAA Receptors Modulate Dopamine Release from Rat Striatal Synaptosomes

Abstract— The presynaptic regulation of stimulated dopa-mine release from superfused rat striatal synaptosomes by opioids and γ-aminobutyric acid (GABA) was studied. It was found that in addition to dopamine D₂ autoreceptors, calcium-dependent K⁺-stimulated [³H]dopamine release was inhibited through activation of a homogeneous population of k -opioid receptors in view of the potent inhibitory effect of the k -selective agonist U69.593 (EC₅₀ 0.2 nM) and its antagonism by norbinaltorphimine. Neither μ-nor δ-selective receptor agonists affected release of [³H]-dopamine. In addition, GABA potently inhibited the evoked [³H]dopamine release (EC₅₀ 0.4 nM) through activation of GABA_A receptors in view of the GABA-mimicking effect of muscimol, the sensitivity of its inhibitory effect to picro-toxin and bicuculline, and the absence of an effect of the GABA_B receptor agonist baclofen. In the presence of a maximally effective concentration of GABA, U69,593 did not induce an additional release-inhibitory effect, indicating that these receptors and the presynaptic D₂ receptor are colocalized on the striatal dopaminergic nerve terminals. The excitatory amino acid agonists N-methyl-d -aspartate and kainate, as well as the cholinergic agonist carbachol, stimulated [³H]dopamine release, which was subject to k -opioid receptor-mediated inhibition. In conclusion, striatal dopamine release is under regulatory control of multiple excitatory and inhibitory neurotransmitter by activation of colocalized presynaptic receptors for excitatory amino acids, acetylcholine, dopamine, dynorphins, and GABA within the dopaminergic nerve terminals. Together, these receptors locally control ongoing dopamine neurotransmission.     

Autoradiography of Serotonin 5-HT1A Receptor-Activated G Proteins in Guinea Pig Brain Sections by Agonist-Stimulated [35S]GTPγS Binding

Abstract: G protein activation mediated by serotonin 5-HT_1A and 5-HT_1B/D receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist-stimulated [³⁵S]GTPγS binding to brain sections. [³⁵S]GTPγS binding was stimulated by the mixed 5-HT_1A/5-HT_1B/D agonist L694247 in brain structures enriched in 5-HT_1A binding sites, i.e., hippocampus (+140 ± 14%), dorsal raphe (+70 ± 8%), lateral septum (+52 ± 12%), cingulate (+36 ± 8%), and entorhinal cortex (+34 ± 5%). L694247 caused little or no stimulation of [³⁵S]GTPγS binding in brain regions with high densities of 5-HT_1B/D binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [³⁵S]GTPγS binding response was antagonized by WAY100635 (10 µM) and methiothepin (10 µM). In contrast, the 5-HT_1B inverse agonist SB224289 (10 µM) did not affect the L694247-mediated [³⁵S]GTPγS binding response, and the mixed 5-HT_1B/D antagonist GR127935 (10 µM) yielded a partial blockade. The distribution pattern of the [³⁵S]GTPγS binding response and the antagonist profile suggest the L694247-mediated response in guinea pig brain to be mediated by 5-HT_1A receptors. In addition to L694247, 8-hydroxy-2-(di-n-propylamino)tetralin, and flesinoxan also stimulated [³⁵S]GTPγS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 µM) stimulated [³⁵S]GTPγS binding in the hippocampus by 20–50%. Naratriptan, CP122638, and dihydroergotamine stimulated [³⁵S]GTPγS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5-HT_1A but not 5-HT_1B/D receptors can be measured in guinea pig brain sections.     

Heterogeneity of Cortical and Hippocampal 5-HT1A Receptors: A Reappraisal of Homogenate Binding with 8-[3H]Hydroxydipropylaminotetralin

Abstract: The selective serotonin (5-HT) agonist 8-hydroxydipropylaminotetralin (8-OH-DPAT) has been extensively used to characterize the physiological, biochemical, and behavioral features of the 5-HT_1A receptor. A further characterization of this receptor subtype was conducted with membrane preparations from rat cerebral cortex and hippocampus. The saturation binding isotherms of [³H]8- OH-DPAT (free ligand from 200 pM to 160 nM) revealed high-affinity 5-HT_1A receptors (K_H= 0.7–0.8 nM) and lowaffinity (K_L= 22–36 nM) binding sites. The kinetics of [³H]8-OH-DPAT binding were examined at two ligand concentrations, i.e., 1 and 10 nM, and in each case revealed two dissociation rate constants supporting the existence of high- and low-affinity binding sites. When the high-affinity sites were labeled with a 1 nM concentration of [³H]8- OH-DPAT, the competition curves of agonist and antagonist drugs were best fit to a two-site model, indicating the presence of two different 5-HT_1A binding sites or, alternatively, two affinity states, tentatively designated as 5-HT_1AHIGH and 5-HT_1A^LOW. However, the low correlation between the affinities of various drugs for these sites indicates the existence of different and independent binding sites. To determine whether 5-HT_1A sites are modulated by 5′-guanylylimidodiphosphate, inhibition experiments with 5-HT were performed in the presence or in the absence of 100 μM 5′-guanylylimidodiphosphate. The binding of 1 nM [³H]8-OH-DPAT to the 5-HT_1A^HIGH site was dramatically (80%) reduced by 5′-guanylylimidodiphosphate; in contrast, the low-affinity site, or 5-HT_1A^LOW, was seemingly insensitive to the guanine nucleotide. The findings suggest that the high-affinity 5-HT_1A^HIGH site corresponds to the classic 5-HT_1A receptor, whereas the novel 5-HT_1A^LOW binding site, labeled by 1 nM [³H]8-OH-DPAT and having a micromolar affinity for 5-HT, may not belong to the G protein family of receptors. To further investigate the relationship of 5-HT_1A sites and the 5-HT innervation, rats were treated with p-chlorophenylalanine or with the neurotoxin p-chloroamphetamine. The inhibition of 5-HT synthesis by p-chlorophenylalanine did not alter either of the two 5-HT_1A sites, but deafferentation by p-chloroamphetamine caused a loss of the low-affinity [³H]8-OH- DPAT binding sites, indicating-that these novel binding sites may be located presynaptically on 5-HT fibers and/or nerve terminals.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS: γ-AMINOBUTYRIC ACID BINDING IN THE RAT CEREBRAL CORTEX

—The binding of [¹⁴C]GABA to nerve-ending membranes isolated from rat cerebral cortex follows a hyperbolic curve saturating at 0·4pmol/μg protein. This binding is about 60% inhibited by chloropromazine, and about 40%, inhibited by bicuculline. A hydrophobic protein fraction binding [¹⁴C]GABA was separated from the total. lipid extract of nerve-ending membranes. The binding follows a hyperbolic curve that saturates at 10·5 pmol of [¹⁴C]GABA/μg of protein, with an apparent K_d= 30 μm . The binding is competitively inhibited by bicuculline with a K_i= 273 μm . These results are compared with those previously obtained on a GABA binding protein from crustacean muscle.     

Characterization and Subcellular Localization of l-[3H]Carnitine Binding Sites in Rat Brain

Abstract: In the present study, we investigated the existence of a binding site for l -carnitine in the rat brain. In crude synaptic membranes, l -[³H]carnitine bound with relatively high affinity (K_D = 281 nM) and in a saturable manner to a finite number (apparent B_max value = 7.3 pmol/mg of protein) of binding sites. Binding was reversible and dependent on protein concentration, pH, ionic strength, and temperature. Kinetic studies revealed a K_off of 0.018 min^?1 and a K_on of 0.187 × 10^?3 min^?1 nM^?1. Binding was highest in spinal cord, followed by medulla oblongata-pons ≥ corpus striatum ≥ cerebellum = cerebral cortex = hippocampus = hypothalamus = olfactory bulb. l -[³H]Carnitine binding was stereoselective for the l -isomers of carnitine, propionylcarnitine, and acetylcarnitine. The most potent inhibitor of l -[³H]carnitine binding was l -carnitine followed by propionyl-l -carnitine. Acetyl-l -carnitine and isobutyryl-l -carnitine showed an affinity ～500-fold lower than that obtained for l -carnitine. The precursor γ-butyrobetaine had negligible activity at 0.1 mM. l -Carnitine binding to rat crude synaptic membrane preparation was not inhibited by neurotransmitters (GABA, glycine, glutamate, aspartate, acetylcholine, dopamine, norepinephrine, epinephrine, 5-hydroxytryptamine, histamine) at a final concentration of 0.1 mM. In addition, the binding of these neuroactive compounds to their receptors was not influenced by the presence of 0.1 mMl -carnitine. Finally, a subcellular fractionation study showed that synaptic vesicles contained the highest density of l -carnitine membrane binding sites whereas l -carnitine palmitoyltransferase activity was undetectable, thus excluding the possibility of the presence of an active site for carnitine palmitoyltransferase. This finding indicated that the localization of the l -[³H]carnitine binding site should be essentially presynaptic.     

Actions of avermectin B1a on the gamma-aminobutyric acidA receptor and chloride channels in rat brain

The interaction of avermectin B1a (AVM) with the gamma-aminobutyric acid (GABA) receptor of rat brain was studied using radioactive ligand binding and tracer ion flux assays. Avermectin potentiated the binding of [3H]flunitrazepam and inhibited the binding of both [3H]muscimol and [35S]t-butylbicyclophosphorothionate to the GABAA receptor. Inhibition of muscimol binding by AVM suggested competitive displacement. Two kinds of 36chloride (Cl) flux were studied. The 36Cl efflux from preloaded microsacs was potentiated by AVM and was highly inhibited by the Cl-channel blocker 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS). However, it was not potentiated by GABA nor was it sensitive to the convulsants picrotoxin or bicuculline. On the other hand, 36Cl-influx measurement in a different microsac preparation of rat brain was very sensitive to GABA and other GABA-ergic drugs. Avermectin induced 36Cl influx into these microsacs in a dose-dependent manner, but to only 35% of the maximal influx induced by GABA. The AVM-induced 36Cl influx was totally blocked by bicuculline. It is suggested that AVM opens the GABAA-receptor Cl channel by binding to the GABA recognition site and acting as a partial receptor agonist, and also opens a voltage-dependent Cl channel which is totally insensitive to GABA but is very sensitive to DIDS.     

Effect of baclofen on in vitro noradrenaline release from rat hippocampus and cerebellum: an action at an α2-adrenoceptor

The release of [³H]noradrenaline from rat hippocampal synaptosomes by 25 mM K⁺ and 5 μM veratridine, but not by the Ca²⁺ ionophore A23187 was depressed by baclofen. This depression was reversed by 8-Bromo-cAMP. This action of baclofen was stereospecific and mimicked both that of GABA in the presence of bicuculline and that of clonidine. The α₂-adrenoceptor antagonists yohimbine and Wy25309 antagonised the action of clonidine and baclofen but not that of GABA. Specific binding of [³H]clonidine was displaced by Wy25309 and baclofen, but not by GABA. Specific binding of [³H]GABA in the presence of Ca²⁺ was displaced by baclofen but not by Wy25309. It is concluded that baclofen is not a specific agonist at GABA_B receptors in the brain.     

Substrate Specificity of [3H] Muscimol Binding to a Particulate Fraction of a Neuron-enriched Culture of Embryonic Rat Brain

Abstract: The effects of some GABA analogues and some drugs on the binding of [³H]muscimol (3.08 nM) to thoroughly washed subcellular particles prepared from a neuron-enriched culture of embryonic rat brain were examined using Na+-free Tris-citrate medium and a centrifugation method. Competition for [³H]muscimol binding sites by excess(10^?5 M) unlabelled GABA provided estimates of “specific” binding. In accord with in vivo neuropharmacological studies on GABA receptors and with in vitro studies on cerebral membrane preparations, [³H]muscimol binding was potently inhibited by muscimol itself (IC₅₀, 2.5 nM), GABA (1C₅₀, 43 nM), isoguvacine (IC₅₀, 61 nM), and 3-aminopropanesulphonic acid (IC₅₀, 160 nM), and less potently inhibited by the GABA antagonist bicuculline methobromide (IC₅₀, 800 nM). δ- Aminovaleric acid (IC₅₀, 2.6 μM), the glycinelp-alanine antagonist strychnine (IC₅₀, 6.6 μM), and the predominantly glial GABA uptake inhibitors β-alanine (IC₅₀, 23 μM) and p-proline (IC₅₀, 66 μM) also inhibited [³H]muscimol binding. Other inhibitors of Na+-dependent GABA uptake, (±)-nipecotic acid, L- 2,4-diaminobutyric acid, and guvacine, as well as picrotoxinin, were relatively inactive as inhibitors of [³H]muscimol binding (IC₅₀≥ 1 mM). In addition to revealing that GABA receptors are present on neuronal membranes before the formation of most synapses, this binding of [³H]muscimol that occurs to neuronal, but not to glial, membranes might be useful as a “neuronal marker” and for the further characterization and isolation of GABA receptors.