Metabolic GHB precursor succinate binds to gamma-hydroxybutyrate receptors: characterization of human basal ganglia areas nucleus accumbens and globus pallidus

Binding of the metabolic gamma-hydroxybutyrate (GHB) precursor succinate to NCS-382-sensitive [3H]GHB-labeled sites in crude synaptosomal or purified synaptic membrane fractions prepared from the human nucleus accumbens (NA), globus pallidus (GP) and rat forebrain has been shown. This site can be characterized by binding of ethyl hemisuccinate and gap-junction blockers, including carbenoxolone hemisuccinate and beta-GRA. There was no significant binding interaction between GABAB receptor ligands (CGP 55845, (R)-baclofen) and these [3H]GHB-labeled sites. GHB, NCS-382 and succinate binding profile of [3H]GHB-labeled sites in rat forebrain, human NA or GP synaptic membranes were similar. The synaptic fraction isolated from the rat forebrain was characterized by GHB binding inhibition constants: Ki,NCS-382 = 1.2 +/- 0.2 microM, Ki,GHB = 1.6 +/- 0.3 microM and Ki,SUCCINATE = 212 +/- 66 microM. In crude membranes containing mainly extrasynaptic membranes, distinct GHB and GABAB receptor sites were found in the NA. By contrast, extrasynaptic GABAB receptor sites of rat forebrain and GP were GHB- and succinate-sensitive, respectively. The heterogeneity of GABAB sites found in native membranes indicates GABAB receptor-dependent differences in GHB action. Based on these findings, we suggest that succinate (and possibly drugs available as succinate salt derivatives) can mimic some of the actions of GHB.     

Quantitative autoradiographic distribution of gamma-hydroxybutyric acid binding sites in human and monkey brain

gamma-Hydroxybutyric acid (GHB), a naturally occurring metabolite of GABA, is present in micromolar concentrations in various areas of the mammalian brain. Specific GHB binding sites, uptake system, synthetic and metabolizing enzymes have been identified in CNS. The present study shows the anatomical distribution of GHB binding sites in sections of primate (squirrel monkey) and human brain by radioligand quantitative autoradiography. In both species the highest densities of binding sites were found in the hippocampus, high to moderate densities in cortical areas (frontal, temporal, insular, cingulate and entorhinal) and low densities in the striatum; no binding sites were detected in the cerebellum. High density of GHB binding was found in the monkey amygdala. In addition the binding characteristics of [(3)H]GHB to membrane preparations of human brain cortex were examined. Scatchard analysis and saturation curves revealed both a high (K(d1) 92+/-4.4 nM; B(max1) 1027+/-110 fmol/mg protein) and a low-affinity binding site (K(d2) 916+/-42 nM; B(max2) 8770+/-159 fmol/mg protein). The present study is the first report on the autoradiographic distribution of specific GHB binding sites in the primate and human brain: such distribution is in both species in good agreement with the distribution found in the rat brain.     

Specific gamma-hydroxybutyrate-binding sites but loss of pharmacological effects of gamma-hydroxybutyrate in GABA(B)(1)-deficient mice

gamma-Hydroxybutyrate (GHB), a metabolite of gamma-aminobutyric acid (GABA), is proposed to function as a neurotransmitter or neuromodulator. gamma-Hydroxybutyrate and its prodrug, gamma-butyrolactone (GBL), recently received increased public attention as they emerged as popular drugs of abuse. The actions of GHB/GBL are believed to be mediated by GABAB and/or specific GHB receptors, the latter corresponding to high-affinity [3H]GHB-binding sites coupled to G-proteins. To investigate the contribution of GABAB receptors to GHB actions we studied the effects of GHB in GABAB(1)-/- mice, which lack functional GABAB receptors. Autoradiography reveals a similar spatial distribution of [3H]GHB-binding sites in brains of GABAB(1)-/- and wild-type mice. The maximal number of binding sites and the KD values for the putative GHB antagonist [3H]6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene acetic acid (NCS-382) appear unchanged in GABAB(1)-/- compared with wild-type mice, demonstrating that GHB- are distinct from GABAB-binding sites. In the presence of the GABAB receptor positive modulator 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol GHB induced functional GTPgamma[35S] responses in brain membrane preparations from wild-type but not GABAB(1)-/- mice. The GTPgamma[35S] responses in wild-type mice were blocked by the GABAB antagonist [3-[[1-(S)-(3,4dichlorophenyl)ethyl]amino]-2-(S)-hydroxy-propyl]-cyclohexylmethyl phosphinic acid hydrochloride (CGP54626) but not by NCS-382. Altogether, these findings suggest that the GHB-induced GTPgamma[35S] responses are mediated by GABAB receptors. Following GHB or GBL application, GABAB(1)-/- mice showed neither the hypolocomotion, hypothermia, increase in striatal dopamine synthesis nor electroencephalogram delta-wave induction seen in wild-type mice. It, therefore, appears that all studied GHB effects are GABAB receptor dependent. The molecular nature and the signalling properties of the specific [3H]GHB-binding sites remain elusive.     

Modulation of Mg(2+)-dependent [3H]TCP binding by L-glutamate, glycine, and guanine nucleotides in rat cerebral cortex.

Biochemical and electrophysiological studies have demonstrated that phencyclidine (PCP) recognition site exists in the ion channel of the N-methyl-D-aspartate (NMDA) receptor ion channel complex. Using an extensively washed rat cortical membrane preparation, the effects of Mg2+ and guanylylimidodiphosphate (GppNHp) were examined on the binding of [3H]-N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP). Low concentrations of Mg2+ (EC50 = 11 microM) stimulated [3H]TCP binding under the basal condition and high concentrations of Mg2+ (IC50 = 1 mM) inhibited it. In the presence of 10 microM L-glutamate and 10 microM glycine, their EC50 values for Mg2+ enhancement of [3H]TCP binding were markedly reduced (to 1.9 microM or 8.4 microM), respectively. By contrast, the IC50 values for Mg2+ inhibition of [3H]TCP binding were reduced in the presence of L-glutamate, but not glycine. Furthermore, a stimulatory effect of Mg2+ on [3H]TCP binding was additional to the [3H]TCP binding stimulated by a maximally effective concentration of L-glutamate (10 microM) or glycine (10 microM). In the kinetic study, 300 microM Mg2+ produced an increase in the rates of both association and dissociation of [3H]TCP. Similar results were obtained with L-glutamate (10 microM) and glycine (10 microM); 10 mM Mg2+ also caused an acceleration of the association rate but strongly decreased [3H]TCP binding at equilibrium. Compared with [3H]TCP binding under the basal condition, K+ (10 mM) alone decreased the maximal binding without producing any change in the association rate; 10 mM K+ also significantly decreased Mg(2+)-stimulated [3H]TCP binding but caused no change in the acceleration of the association rate caused by Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterization of vanilloid receptor located in the brain

Specific [3H]resiniferatoxin (RTX) binding detects the vanilloid receptor type I (VR1). In the present study we demonstrate specific, high-affinity, saturable [3H]RTX binding in various areas of monkey brain not known to be innervated by primary afferent neurons as well as in spinal cord and dorsal root ganglion neurons of the same origin. Detailed pharmacological characterization and comparison revealed no major difference in binding affinities between the peripheral and the central sites as measured by K(d)/K(i) values. In general, lower receptor density was measured in selected brain areas than in the periphery. Areas with higher receptor density were detected in the locus ceruleus, preoptic area, and medial basal hypothalamus of the brain. Both capsaicin and the competitive antagonist capsazepine inhibited the specific binding of [3H]RTX to membrane preparations of the dorsal horn of the spinal cord and dorsal root ganglia with K(i) values of 4.3+/-0.32 microM and 2.7+/-0.33 microM, respectively. Inhibition was observed in the central areas (hypothalamus) with K(i) values of 0.95+/-0.1 microM for capsaicin and 0.86+/-0.11 microM for capsazepine. Previous biological and pharmacological evidence suggested that vanilloid receptors were present in the brain. Our results demonstrate that the pharmacological properties of both the peripheral and central receptor sites display appropriate pharmacological similarity to represent the same receptor class. The modest differences in ligand affinities for the vanilloid receptor expressed in the brain nuclei and the dorsal root ganglion neurons may correspond to differences in sequence, modification or associated proteins.     

m-Azido-phencyclidine covalently labels the rat brain PCP receptor, a putative K channel

Phencyclidine (PCP) is a schizophrenomimetic drug of abuse. PCP binds with high affinity (apparent dissociation constant, KD less than 10(-6) M) to rat brain membranes and blocks, selectively, a voltage-gated, noninactivating K channel found in rat brain synaptosomes (presynaptic nerve terminals). Thus, it has been proposed that the high-affinity PCP receptor in brain is this K channel. Consistent with this hypothesis, we now show that several K channel blockers displace 3H-PCP from the rat brain receptor. Additionally, we have used a photolabile analog of PCP, m-azido-PCP (Az-PCP), to identify the brain PCP receptor/putative K channel. In the dark, Az-PCP bound reversibly to 2 classes of sites on rat brain synaptic membranes [KD = 0.14 +/- 0.01 microM (n = 5) for high-affinity binding, and KD = 255 +/- 55 microM for low-affinity binding]. Competitive binding studies between Az-3H-PCP and nonlabeled PCP analogs, and between Az-PCP and several tritiated PCP analogs, indicated that the high-affinity Az-PCP binding site is the high-affinity PCP receptor. Several amino-pyridines (APs) and tetraalkylamines (TAAs), which are known to block K channels in excitable cells, were also found to displace 3H-PCP from its high-affinity binding site on rat brain synaptic membranes. The rank order of potency for displacement of 3H-PCP from this site for the APs was 4-AP approximately equal to 3,4-diAP greater than 2-AP much greater than 3-AP; for the TAAs it was TBA greater than TEA much greater than TMA (the tetra-butyl, ethyl, and methyl amines, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Histamine H1 receptors in rat dorsal raphe nucleus: pharmacological characterisation and linking to increased neuronal activity

In this work we studied the presence of histamine H(1) receptors in the rat dorsal raphe nucleus (DRN) and the effect of their activation on the activity of presumed serotonergic DRN neurones. [(3)H]-Mepyramine bound to DRN membranes with best-fit values of 107+/-13 fmol/mg protein for maximum binding (B(max)) and 1.2+/-0.4 nM for the equilibrium dissociation constant (K(d)). In DRN slices labelled with [(3)H]-inositol and in the presence of 10 mM LiCl, histamine stimulated the accumulation of [(3)H]-inositol phosphates ([(3)H]-IPs) with maximum effect 172+/-6% of basal and EC(50) 3.2+/-1.3 microM. [(3)H]-IPs accumulation induced by 100 microM histamine (162+/-5% of basal) was markedly, but not fully blocked by the selective H(1) antagonist mepyramine (300 nM; 64+/-6% inhibition). The simultaneous addition of mepyramine and the selective H(2) antagonist ranitidine (10 microM) abolished histamine-induced [(3)H]-IPs accumulation. The presence of H(2) receptors was confirmed by [(3)H]-tiotidine binding and by the determination of histamine-induced [(3)H]-cyclic AMP formation. Extracellular single-unit recording in brain stem slices showed that the exposure to histamine resulted in a marked increase in the firing rate of DRN presumed serotonergic neurones (471+/-10% of basal), that was dependent on the concentration of the agonist (EC(50) 4.5+/-0.3 microM). The action of histamine was not affected by the H(2) antagonist tiotidine (2 microM) but was fully prevented by 1 microM mepyramine. Taken together, our results indicate that histamine modulates the firing of DRN presumed serotonergic neurones through the activation of H(1) receptors coupled to phosphonositide hydrolysis.     

Direct radiolabeling by [3H]quisqualic acid of group I metabotropic glutamate receptor in rat brain synaptic membranes

[3H]Quisqualic acid (QA) was synthesized and used to label metabotropic glutamate receptor (mGluR) in rat brain synaptic membranes in the presence of three different ionotropic glutamate receptor agonists at respective saturating concentrations. Of several mGluR agonists tested, group I agonists were more potent in displacing [3H]QA binding than group II and group III agonists in the presence of the three ionotropic agonists. [3H]QA binding was markedly inhibited by guanine nucleotide analogues in a concentration-dependent manner at a concentration range of 10 nM to 1 mM. Scatchard analysis revealed that [3H]QA binding consisted of a single component with a K(d) of 50.9+/-5.3 nM and a B(max) of 431. 6+/-18.3 fmol/mg protein. These results suggest that [3H]QA indeed labels group I mGluR functionally coupled to GTP binding protein in rat brain synaptic membranes when determined under the experimental conditions employed.     

Presynaptic effect of 7-OH-DPAT on evoked [3H]-acetylcholine release in rat striatal synaptosomes

The objective of the present experiments was to study the presynaptic effect of 7-hydroxy-N,N-di-n-propyl-2-aminotetraline (7-OH-DPAT, a D(2)-like dopamine receptor agonist) on [3H]-acetylcholine ([3H]-ACh) release induced by potassium (15 mM, 25 mM and 60 mM), potassium channel-blockers (4-aminopyridine, 4-AP; tetraethylammonium, TEA and quinine) and veratridine to gain insight into the mechanisms involved in the activation of the D(2) dopamine-receptor subtype located at striatal cholinergic nerve terminals. 7-OH-DPAT (1 microM) inhibited the evoked [3H]-ACh release induced by K(+) 15 mM in a similar percentage than that obtained during basal conditions (30% and 27%, respectively). Nevertheless, in the presence of 25 mM and 60 mM of K(+) the inhibitory effect of 7-OH-DPAT was completely abolished. 4-AP (1-100 microM) and TEA (1 and 5 mM) significantly enhanced [3H]-ACh release, showing 69.32%+/-7.60% (P<0.001) and 52.27%+/-5.64% (P<0.001), respectively, at the highest concentrations tested. In these conditions, 7-OH-DPAT (1 microM) inhibited the release induced by potassium channel-blockers approximately 25-27%. Quinine (0.1-1 microM) did not alter [3H]-ACh release either in the presence or absence of 7-OH-DPAT. Veratridine 10 microM evoked [3H]-ACh release in the presence of a low-calcium medium, but in such conditions 7-OH-DPAT (1 microM) did not modify the neurotransmitter release in the absence or presence of veratridine. Present data indicate that activation of the presynaptic D(2) dopamine receptor inhibits the [3H]-ACh release by increasing K(+) conductance, as high K(+) concentrations abolished the inhibitory control of 7-OH-DPAT on [3H]-ACh release. This effect could be mediated by potassium channels different from those sensitive to 4-AP, TEA and quinine. In addition, the presynaptic D(2) dopamine-receptor activation seems to not involve changes in intracellular Ca(2+).     

Gamma-hydroxybutyrate and ethanol depress spontaneous excitatory postsynaptic currents in dopaminergic neurons of the substantia nigra

Gamma-hydroxybutyrate (GHB) has been shown to have therapeutical properties in various psychiatric disorders, especially in alcohol abuse, and to mimic different actions of ethanol at the cellular and system level. Using whole-cell patch-clamp recordings on brain slices of 21- to 25-day-old rats, the present study investigated the effects of GHB and ethanol on spontaneous excitatory postsynaptic currents (sEPSCs) in dopaminergic neurons of the substantia nigra pars compacta (SNc). sEPSCs are an index of glutamate release from the excitatory input to dopamine cells, which play a key role in different reward-related behaviors. We found that GHB and ethanol depressed both the frequency and the amplitude of sEPSCs. These effects were GABA(B)-independent and the GHB-induced depression was blocked by the GHB receptor antagonist 6,7,8,9-tetrahydro-5[H]benzocyclohepte-5-ol-4-ylidene acetic acid (NCS-382), pointing to a specific effect of this drug. The effects of ethanol were not affected by NCS-382. This study indicates that GHB and ethanol share the effect of reducing the efficacy of excitatory glutamatergic neurotransmission in the SNc by acting through different mechanisms.     

Binding and transport of [3H](2S,4R)- 4-methylglutamate, a new ligand for glutamate transporters, demonstrate labeling of EAAT1 in cultured murine astrocytes

Transporters for L-glutamate (excitatory amino acid transporters; EAATs), localized to astrocytes, are involved intimately in intermediary metabolism within the brain. Because (2S,4R)-4-methylglutamate (4MG) has affinity for glial EAATs, we employed [(3)H]4MG to define the characteristics of EAATs in cultured murine astrocytes and describe new approaches to analyze EAAT function. Specific binding of [(3)H]4MG in astrocytic membranes at 4 degrees C represented 90% of total binding. Binding was rapid (apparent t(1/2) approximately 7 min) and saturable. Saturation and Scatchard analyses indicated a single binding site (n(H) = 0.8) with a K(d) of 6.0 +/- 1.5 microM and B(max) = 9.7 +/- 2.9 pmol/mg protein. Binding of [(3)H]4MG to astrocytic homogenates was Na(+)-dependent and inhibited by K(+). Compounds acting at EAATs, such as L-glutamate (Glu), D-aspartate (D-Asp), L-(2S,3S,4R)-2-(carboxycyclopropyl)glycine and L-trans-pyrrolidine-2,4-dicarboxylate displaced binding to nonspecific levels. L-Serine-O-sulphate, an EAAT1-preferring ligand, fully displaced binding of [(3)H]4MG. In contrast, inhibitors having preferential affinity for EAAT2, L-threo-3-methylglutamate, dihydrokainate, and kainate, were relatively ineffective binding displacers. Agonists and antagonists for Glu receptors failed to significantly inhibit [(3)H]4MG binding. Studies with [(3)H]D-Asp reinforced evidence that [(3)H]4MG was binding to EAATs. These data were consistent with Western blot analyses, which indicated abundant expression of EAAT1 but not EAAT2. [(3)H]4MG was also accumulated rapidly (apparent t(1/2) approximately 4 min) into whole astrocytes by a sodium- and temperature-sensitive process (K(m) of 146 +/- 24 microM, V(max) = 336 +/- 27 nmol/mg protein/min), which possessed an EAAT1-like pharmacologic profile. These findings confirm that 4MG is a substrate for EAAT1 and that the binding assay developed using [(3)H]4MG can be utilized in various preparations including cultured astrocytes.     

Synaptosomal plasma membrane transport of excitatory sulphur amino acid transmitter candidates: kinetic characterisation and analysis of carrier specificity.

The transport kinetics of the excitatory sulphur-containing amino acid (SAA) transmitter candidates, L-cysteine sulphinate (L-CSA), L-cysteate (L-CA), L-homocysteine sulphinate (L-HCSA), and L-homocysteate (L-HCA), together with their plasma membrane carrier specificity, was studied in cerebrocortical synaptosome fractions by a sensitive high performance liquid chromatographic assay. A high affinity uptake system could be demonstrated for L-CSA (Km = 57 +/- 6 microM; Vmax = 1.2 +/- 0.1 nmol/min/mg protein) and L-CA (Km = 23 +/- 3 microM; Vmax = 3.6 +/- 0.1 nmol/min/mg protein), whereas L-HCSA (Km = 502 +/- 152 microM; Vmax = 6.1 +/- 1.3 nmol/min/mg protein) and L-HCA (Km = 1550 +/- 169 microM; Vmax = 10.3 +/- 1.1 nmol/min/mg protein) exhibited much lower affinity as transport substrates. In all cases, only a single, saturable Na(+)-dependent component of uptake could be identified, co-existing with a non-saturable, Na(+)-independent influx component. Plasma membrane carrier specificity of the SAAs was established following comparison with other high-affinity neurotransmitter systems. High-affinity L-CSA and L-CA transport and low-affinity L-HCSA and L-HCA transport demonstrate strong positive correlations in inhibition profiles when compared against each other or individually against the high-affinity transport of L-[3H]glutamate, L-[3H]aspartate, or D-[3H]aspartate. Moreover, the transport systems for the excitatory SAAs exhibited a negative correlation when compared in inhibition profiles with the high affinity transport of both [3H] gamma-aminobutyric acid (GABA) and [3H]taurine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of gamma-hydroxybutyrate on the reactivity of pial arteries before and after ischemia

The effect of gamma-hydroxybutyrate (GHB) on the reactivity of pial arteries to local metabolic factors was tested in chloralose-anesthetized cats before or after a period of transient ischemia induced by air embolism. The vascular reactions were determined during the perivascular microapplication of artificial CSFs with increasing concentrations of adenosine (10(-11)-10(-3) M), H+ (pH 5.1-7.6), or K+ (0-10 mM). During nonischemic conditions the pial arterial reactivity to adenosine and H+, but not to K+, was significantly increased by GHB (250 mg/kg i.v.) when compared with the control reactivity. After cerebral ischemia the reactivity to adenosine was abolished with and without the administration of GHB prior to air embolism. The reactivity to K+ was partly preserved but not increased by GHB when compared with previous results without GHB. In contrast GHB improved the postischemic reactivity to perivascular H+ that had been found to be abolished in previous experiments without GHB. The perivascular microapplication of GHB showed no influence of GHB on the vascular diameter. An important finding of the present study is the demonstration of an increase in cerebrovascular reactivity, which may give scope for therapeutic improvement of the regulation of CBF in pathophysiological conditions.     

Reduced High-Affinity Agonist Binding at the M1 Muscarinic Receptor in Alzheimer's Disease Brain: Differential Sensitivity to Agonists and Divalent Cations

M(1) muscarinic acetylcholine receptor (M(1)AchR)-G protein coupling, as measured by high-affinity agonist binding, was examined in membranes prepared from postmortem human temporal cortex (Brodmann area 38) from individuals with Alzheimer's disease (AD, n = 8) and age-matched controls (n = 6). Binding competitions between the M(1)AchR-selective antagonist [(3)H]pirenzepine ([(3)H]PZ) and muscarinic agonists carbachol, acetylcholine, oxotremorine, and oxotremorine M were conducted. In the presence of 1 mM MgCl(2), the inhibition of [(3)H]PZ binding by carbachol, acetylcholine, or oxotremorine M was best described by a two-affinity state model for control and AD cases, while oxotremorine binding affinity was best fit to a single-state model. Although both control and AD groups had similar K(D) values for the high- and low-affinity agonist binding sites, the proportion of M(1)AchRs exhibiting high affinity for carbachol and acetylcholine was reduced by 48 and 33%, respectively, in AD membranes relative to controls (P < 0.05). No changes in the binding of the oxotremorine M or oxotremorine were noted. The nonhydrolyzable guanine nucleotide GppNHp (100 microM) reduced the proportion of M(1)AchRs with high affinity for agonists in both control and AD membranes. Substitution of 1 mM MnCl(2) for MgCl(2) restored high-affinity carbachol binding at the M(1)AchR in AD membranes similar to that seen in controls. In the presence of 1 mM MnCl(2), agonist binding in controls did not differ from 1 mM MgCl(2). In the absence of cations (1 mM EDTA), no differences between control and AD M(1)AchR carbachol binding were observed. Thus, the loss of high-affinity agonist binding at the M(1)AchR in AD is dependent on the agonist and cation studied.     

Ca-dependent release of [H]GABA in cultured chick retina cells

Depolarization by K+ (50 mM) of cultured chick retina cells released 1.14 +/- 0.28% of the accumulated [3H] gamma-aminobutyric acid (GABA) in the absence of Ca2+, but when 1.0 mM Ca2+ was present, the internal free calcium ion concentration [Ca2+]i rose by about 750 nM and the [3H]GABA release about doubled to a value of 2.22 +/- 0.2% of the total [3H]GABA. Nitrendipine (0.1 microM), a blocker of the L-type Ca2+ channels, blocked the [Ca2+]i response to K+ depolarization by about 65%, and the omega-Conotoxin GVIA (omega-CgTx) (0.5 microM), a blocker of the N-type of Ca2+ channels, inhibited by 27% the [Ca2+]i rise due to K+ depolarization. Parallel experiments showed that nitrendipine inhibits [3H]GABA release to the level observed in the absence of Ca2+, whereas omega-CgTx did not inhibit significantly the release of [3H]GABA. The results also show that the release of [3H]GABA due to K(+)-depolarization in the absence of Ca2+ can be totally blocked by 1-(2-(((Diphenylmethylene) amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride (NNC-711), an inhibitor of the GABA carrier. However, in the presence of Ca2+, NNC-711 blocks the release only by about 66%, corresponding to the Ca(2+)-independent release. Thus, it is concluded that [3H]GABA is released in chick retina cells by the exocytotic mechanism, which is Ca(2+)-dependent, and by reversal of the carrier, which is Ca(2+)-independent, in much the same way as has been found for other GABAergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional coupling of the nociceptin/orphanin FQ receptor in dog brain membranes

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ receptor (NOP) which is yet to be functionally characterized in dog brain. Ligand binding data reports low NOP density (29 fmol mg(-1) protein) in dog. In this study using dog brain membranes, we have examined the effects of N/OFQ on [leucyl-(3)H]N/OFQ(1-17)OH ([leucyl-(3)H]N/OFQ) binding in the presence and absence of 120 mM NaCl and 100 microM GTPgammaS. Data from standard [(35)S]GTPgammaS binding and immunoprecipitation (G(alphai1-3)) assays are also presented, along with data from a limited number of control experiments with human NOP expressed in Chinese hamster ovary (CHO(hNOP)) cells. N/OFQ displaced [leucyl-(3)H]N/OFQ binding with pK(i) and slope values of 9.62+/-0.07 and 0.38+/-0.05, respectively. Addition of NaCl/GTPgammaS produced a steepening (slope 0.95+/-0.06, n=3) of the curve. N/OFQ stimulated [(35)S]GTPgammaS binding with pEC(50) and E(max) values of 8.21+/-0.17 and 1.17+/-0.01, respectively (in CHO(hNOP), pEC(50) and E(max) values were 8.47+/-0.01 and 7.01+/-0.63). N/OFQ stimulated [(35)S]GTPgammaS binding in dog and CHO(hNOP) cell membranes could be immunoprecipitated with an anti-G(alphai1-3) antibody, indicating coupling to a pertussis toxin (PTx)-sensitive G-protein. N/OFQ actions were competitively antagonized by the selective NOP antagonists, 100 nM J-113397, 1 microM [Nphe(1)]N/OFQ(1-13)NH(2) and 1 microM [Phe(1)Psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) (partial agonist) yielding pK(B) values of 8.58+/-0.21, 7.06+/-0.59 and 7.32+/-0.41, respectively (in CHO(hNOP), a pK(B) for J-113397 of 8.33+/-0.02 was obtained). Despite relatively low receptor density, we were able to detect functional activity of native dog NOP, with pharmacology consistent with reports for other species.     

The heterogeneity of central benzodiazepine receptor subtypes in the human hippocampal formation,frontal cortex and cerebellum using [3H]flumazenil and zolpidem

The ability of clonazepam and zolpidem to displace [3H]flumazenil binding was measured in the human hippocampal formation, frontal cortex (BA9) and the cerebellum using in situ radioligand binding and autoradiography. The use of high resolution phosphorimaging in all regions indicated the displacement of [3H]flumazenil by clonazepam was monophasic with K(i) values ranging from 2.73+/-0.17 to 6.49+/-0.21 nM. [3H]flumazenil binding that was not displaced by clonazepam ranged from 3.39+/-0.86 to 7.15+/-1.11%. The ability of zolpidem to displace [3H]flumazenil was also monophasic in the frontal cortex and cerebellum with K(i) values of 37.53+/-1.79 and 31.80+/-1.68 nM, respectively. In contrast, within all hippocampal regions, zolpidem displacement of [3H]flumazenil was biphasic, with K(i) values for the high affinity site ranging from 0.13+/-0.04 to 0.54+/-0.03 nM, whereas the low affinity site was between 84.98+/-1.58 and 98.84+/-1.89 nM. In addition, zolpidem insensitive [3H]flumazenil binding was observed to vary markedly between brain regions, ranging between 37.85+/-1.60 and 6.13+/-0.83%. In conclusion, the present results indicate that in situ radioligand binding and high-resolution phosphorimaging techniques can be utilized to measure the differential displacement of [3H]flumazenil by zolpidem and clonazepam. Moreover, our data suggests that the differential distribution of the zolpidem insensitive component of [3H]flumazenil binding is an indicator of GABA/BZ receptors assembled by different subunits within the human brain.     

Characterization of wild-type (R100R) and mutated (Q100Q) GABAA alpha 6 subunit in Sardinian alcohol non-preferring rats (sNP)

Sardinian alcohol non-preferring (sNP) rats, selected for their low ethanol preference and consumption, carry a point mutation (R100Q) in the gene coding for GABA(A) receptor alpha(6) subunit, which becomes more sensitive to diazepam-evoked GABA currents. We performed binding studies in the cerebellum of normal (RR) and mutated (QQ) sNP rats using [3H]Ro 15-4513, an inverse agonist for the benzodiazepine site which binds both diazepam insensitive and diazepam sensitive sites. Saturation curves performed on cerebellar membrane from genotyped rats indicated an higher affinity of [3H]Ro 15-4513 for GABA(A) receptors in QQ with respect to RR rats (K(d) values 4.0+/-0.67 and 6.24+/-0.95 nM, respectively), with similar B(max) values (3.5+/-0.25 and 3.9+/-0.39 pmol/mg protein, respectively). Diazepam displacement curves showed a two component model for both genotypes, with similar K(i1) values for QQ and RR (3.6+/-0.62 and 4.9+/-0.33 nM, respectively). In QQ rats diazepam is able to completely displace [3H]Ro 15-4513 (K(i2)=1.48+/-0.27 microM), while in RR rats the diazepam sensitive sites are still present (K(i2)>10 microM). The basal mRNA and protein expression level of the alpha(6) subunit were similar in RR and QQ rats. The electrophysiological profile of oocytes of Xenopus laevis injected with cerebellar synaptosomes showed that ethanol positively modulated GABA-evoked currents significantly more in QQ than in RR rats. These data contribute to the characterization of the function of GABA(A) alpha(6) subunit and its involvement in determining alcohol related behavior.     

High- and low-affinity [3H]desipramine-binding sites in human postmortem brain tissue

The binding of [3H]desipramine to human brain tissue was characterized. Competition studies in the frontal cortex and hypothalamus revealed a single-site binding model for noradrenaline (Ki 120-190 microM). The noradrenaline uptake inhibitors nisoxetine, nortriptyline and desipramine fitted two-site binding models and these compounds exhibited 10-80 times lower Ki values than the serotonin uptake inhibitor citalopram. The high-affinity component of the nisoxetine-sensitive [3H]desipramine binding (Ki 50-110 nM) approximated the binding sensitive to noradrenaline. This binding fraction was defined as that sensitive to 1 microM nisoxetine and showed a maximum binding capacity (Bmax) of 380 +/- 80 fmol/mg protein and an apparent Kd of 5.1 (4.5-5.7) nM in the hypothalamus. The binding was also investigated in 25 additional brain regions without finding detectable amounts of binding. However, when the specific binding was defined as that sensitive to 100 microM nisoxetine, low-affinity binding where Bmax and Kd were not possible to determine was obtained in all brain regions investigated. It is concluded that [3H]desipramine binding to human brain tissue represents multiple binding sites. Only when regarding binding sensitive to noradrenaline and to the high-affinity component of noradrenaline uptake inhibitors is the binding saturable and of high affinity. It is possible that this site represents the uptake site for noradrenaline.     

GABAA and GABAB sites in bovine adrenal medulla membranes

The effect of several ligands and Ca2+ ions on [3H]GABA binding to bovine adrenal medulla membranes was investigated. Without any blockade, the [3H]GABA binding showed two components, one of low affinity (Kd = 139 +/- 22 nM and Bmax = 3.2 +/- 0.4 pmol/mg protein) and the other of high affinity (Kd = 41 +/- 6 nM and Bmax = 0.35 +/- 0.26 pmol/mg protein). Muscimol specifically blocked low-affinity sites, and (-)baclofen blocked high-affinity components. Ca2+ ions were strictly necessary for maximum binding to high-affinity sites, whereas they did not significantly affect sites of the lower affinity. These results show that the bovine adrenal medulla has a GABAA receptor population of low affinity together with a GABAB receptor of high affinity.