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.     

Subclasses of Adenosine Receptors in Brain Membranes from Adult Tissue and from Primary Cultures of Chick Embryo

Abstract: Membranes from adult chicken brain have high-affinity binding sites for N⁶-cyclohexyl[³H]adenosine (CHA) (K_D= 4 nM, Bmax = 0.6 pmol/mg protein). This CHA binding could be attributed to adenosine receptors of the A₁ type, since substituted adenosine analogs, e.g. N⁶-(l -2-phenylisopropyl)adeno sine (IC50 = 60 nM), were very potent displacers. Binding sites for 1,3-diethyl- 8-[³H]phenylxanthine (DPX) in adult brain membranes have a moderate affinity (K_D= 50 nM, Bmax = 1.5 pmol/mg). The association of DPX with these sites could be completely displaced by 8-phenyltheophylline (IC₅₀= 300 nM) and other xanthines, but only 45% of specific DPX binding could be displaced by phenylisopropyladenosine. This suggests that about half of DPX sites are putative A₁ receptors and the other half are of the A₂ type. Primary cultures of pure glial and neuronal cells from chick embryo brain were also examined for adenosine receptors. Specific binding of CHA could not be detected in these preparations, but both glial and neuronal membranes have specific sites for DPX. At a [³H]DPX concentration of 20 nM, specific binding was 50% higher (per mg protein) in glial than in neuronal membranes. The maximum binding of DPX to glial membranes (B_max= 1.6 pmol/mg) was comparable to values for adult brain, but the glial affinity (K_D= 90 nM) was somewhat less. Phenylisopropyladenosine was able to displace less than 20% of the total glial sites for DPX. This finding was in accord with the lack of CHA sites and demonstrates that A₁ receptors make little contribution to DPX binding in glial membranes. In decreasing order of potency, 8-phenyltheophylline, CHA, theophylline, caffeine, and 3-isobutyl-I-methylxanthine completely displace DPX association with glia. DPX binding to glial membranes thus appears due to a single class of receptors, which may prove to be of the A₂ type.     

Differentiating Adenosine Receptors and Adenosine Uptake Sites in Brain

Abstract

The characteristics of adenosine receptors and adenosine uptake sites in brain are presented. High affinity adenosine receptors of the A₁ type bind [³H]cyclohexyladenosine ([³H]CHA) and [³ H]diethyl-phenyl-xanthine ([³H]DPX) with 10^?9 potency while adenosine uptake sites are labeled 10^?10 potency with [³ H]nitrobenzyl-thioinosine ([³H]NBI). NBI does not inhibit either [³H]CHA (agonist) or [³H]DPX (antagonist) binding to adenosine receptors in brain cortical membranes and conversely CHA and other adenosine receptor ligands are very poor inhibitors of [³H]NBI binding to adenosine uptake sites. A number of other differences between the receptor and uptake site are discussed which provide rather strong evidence that these two sites are quite distinct and that the labeled ligands used represent specific probes for each site.     

Characterization of Inhibitor-Sensitive and -Resistant Adenosine Transporters in Cultured Human Fetal Astrocytes

Abstract: The kinetic characteristics of [³H]adenosine uptake, the extent to which accumulated [³H]adenosine was metabolized, the effects such metabolism had on measurements of apparent Michaelis-Menten kinetic values of K_T and V_max, and the sensitivities with which nucleoside transport inhibitors blocked [³H]adenosine accumulations were determined in cultured human fetal astrocytes. K_T and V_max values for accumulations of [³H]-labeled purines using 15-s incubations in the absence of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and the adenosine kinase inhibitor 5′-iodotubercidin (ITU) were 6.2 µM and 0.15 nmol/min/mg of protein for the high-affinity and 2.6 mM and 21 nmol/min/mg of protein for the low-affinity components respectively. In the presence of EHNA and ITU, where <4% of accumulated [³H]adenosine was metabolized, transport per se was measured, and kinetic values for K_T and V_max were 179 µM and 5.2 nmol/min/mg of protein, respectively. In the absence of EHNA and ITU, accumulated [³H]adenosine was rapidly metabolized to AMP, ADP, and ATP, and caused an appearance of “concentrative” uptake in that the intracellular levels of [³H]-labeled purines (adenosine plus its metabolites) were 1.4-fold higher than in the medium. No apparent concentrative accumulations of [³H]adenosine were found when assays were conducted using short incubation times in the absence or presence of EHNA and ITU. The nucleoside transport inhibitors dipyridamole (DPR), nitrobenzylthioinosine (NBI), and dilazep biphasically inhibited [³H]adenosine transport; for the inhibitor-sensitive components the IC₅₀ values were 0.7 nM for NBI, 1.3 nM for DPR, and 3.3 nM for dilazep, and for the inhibitor-resistant component the IC₅₀ values were 2.5 µM for NBI, 5.1 µM for dilazep, and 39.0 µM for DPR. These findings, in cultured human fetal astrocytes, represent the first demonstration of inhibitor-sensitive and -resistant adenosine transporters in nontransformed human cells.     

Excitatory and Inhibitory Effects of A1 and A2A Adenosine Receptor Activation on the Electrically Evoked [3H]Acetylcholine Release from Different Areas of the Rat Hippocampus

Abstract: The modulation by adenosine analogues and endogenous adenosine of the electrically evoked release of [³H]acetylcholine ([³H]ACh) was compared in subslices of the three areas of the rat hippocampus (CA1, CA3, and dentate gyrus). The mixed A₁/A₂ agonist 2-chloroadenosine (CADO; 2–10 µM) inhibited, in a concentration-dependent manner, the release of [³H]ACh from the three hippocampal areas, being more potent in the CA1 and CA3 areas than in the dentate gyrus. The inhibitory effect of CADO (5 µM) on [³H]ACh release was prevented by the A₁ antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 nM) in the three hippocampal areas and was converted in an excitatory effect in the CA3 and dentate gyrus areas. The A_2A agonist CGS-21680 (30 nM) produced a greater increase of the evoked release of [³H]ACh in the CA3 than in the dentate gyrus areas, whereas no consistent effect was found in the CA1 area or in the whole hippocampal slice. The excitatory effect of CGS-21680 (30 nM) in the CA3 area was prevented by the adenosine receptor antagonist 3,7-dimethyl-1-propargylxanthine (10 µM). Both adenosine deaminase (2 U/ml) and DPCPX (250 nM) increased the evoked release of [³H]ACh in the CA1 and CA3 areas but not in the dentate gyrus. The amplitude of the effect of DPCPX and adenosine deaminase was similar in the CA1 area, but in the CA3 area DPCPX produced a greater effect than adenosine deaminase. It is concluded that the electrically evoked release of [³H]ACh in the three areas of the rat hippocampus can be differentially modulated by adenosine. In the CA1 area, only A₁ inhibitory receptors modulate ACh release, whereas in the CA3 area, both A_2A excitatory and A₁ inhibitory adenosine receptors modulate ACh release. In the dentate gyrus, both A₁ inhibitory and A_2A excitatory adenosine receptors are present, but endogenous adenosine does not activate them.     

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.     

[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.     

Autoradiographic Distribution and Characteristics of High- and Low-Affinity Polyamine-Sensitive [3H]Ifenprodil Sites in the Rat Brain: Possible Relationship to NMDAR2B Receptors and Calmodulin

Abstract: We have studied the regional distribution and characteristics of polyamine-sensitive [³H]ifenprodil binding sites by quantitative autoradiography in the rat brain. In forebrain areas ifenprodil displaced [³H]ifenprodil (40 nM) in a biphasic manner with IC₅₀ values ranging from 42 to 352 nM and 401 to 974 µM. In hindbrain regions, including the cerebellum, ifenprodil displacement curves were monophasic with IC₅₀ values in the high micromolar range. Wiping studies using forebrain slices (containing both high- and low-affinity sites) or cerebellar slices (containing only the low-affinity site) showed that high- and low-affinity ifenprodil sites are sensitive to spermine and spermidine, to the aminoglycoside antibiotics neomycin, gentamicin, and kanamycin, and to zinc. Two calmodulin antagonists, W7 and calmidazolium, also displaced [³H]ifenprodil from both sites. Other calmodulin antagonists, including trifluoperazine, prenylamine, and chlorpromazine, selectively displaced [³H]ifenprodil from its low-affinity site in hindbrain and forebrain regions. High-affinity [³H]ifenprodil sites, defined either by ifenprodil displacement curves or by [³H]ifenprodil binding in the presence of 1 mM trifluoperazine, were concentrated in the cortex, hippocampus, striatum, and thalamus with little or no labeling of hindbrain or cerebellar regions. This distribution matches that of NMDAR2B mRNA, supporting data showing that ifenprodil has a preferential action at NMDA receptors containing this subunit. Low-affinity [³H]ifenprodil sites have a more ubiquitous distribution but are especially concentrated in the molecular layer of the cerebellum. [³H]Ifenprodil was found to bind to calmodulin-agarose with very low affinity (IC₅₀ of ifenprodil = 516 µM). This binding was displaced by calmodulin antagonists and by polyamines, with a potency that matched their displacement of [³H]ifenprodil from its low-affinity site in brain sections. However, the localization of the low-affinity [³H]ifenprodil site does not strictly correspond to that of calmodulin, and its identity remains to be further characterized. The restricted localization of high-affinity [³H]ifenprodil binding sites to regions rich in NMDAR2B subunit mRNA may explain the atypical nature of this NMDA antagonist.     

[3H]Desipramine Binding to Rat Brain Tissue: Binding to Both Noradrenaline Uptake Sites and Sites Not Related to Noradrenaline Neurons

Abstract The pharmacological and biochemical characteristics of [³H]desipramine binding to rat brain tissue were investigated. Competition studies with noradrenaline, nisoxetine, nortriptyline, and desipramine suggested the presence of more than one [³H]desipramine binding site. Most of the noradrenaline-sensitive binding represented a high-affinity site, and this site appeared to be the same as the high-affinity site of nisoxetine-sensitive binding. The [³H]desipramine binding sites were abolished by protease treatment, a result suggesting that the binding sites are protein in nature. When specific binding was defined by 0.1 μM nisoxetine, the binding was saturable and fitted a single-site binding model with a binding affinity of ～1 nM. This binding fraction was abolished by lesioning of the noradrenaline neurons with the noradrenaline neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromo-benzylamine (DSP4). In contrast, when 10 μM nisoxetine was used to define the specific binding, the binding was not saturable over the nanomolar range, but the binding fitted a two-site binding model with K_D values of 0.5 and >100 nM for the high- and low-affinity components, respectively. The high-affinity site was abolished after DSP4 lesioning, whereas the low-affinity site remained. The binding capacity (B_max) for binding defined by 0.1 μM nisoxetine varied between brain regions, with very low density in the striatum (B_max not possible to determine), 60-90 fmol/mg of protein in cortical areas and cerebellum, and 120 fmol/mg of protein in the hypothalamus. The binding capacities of these high-affinity sites correlated significantly with the regional distribution of [³H]noradrenaline uptake but not with 5-[³H]hydroxytryptamine uptake. The low-affinity sites did not correlate with the regional distribution of [³H]noradrenaline uptake. Drug inhibition studies showed that noradrenaline inhibits the binding defined by 0.1 μM nisoxetine in a competitive manner. Together, these findings suggest that only a small fraction of the [³H]desipramine binding can be regarded as “specific” binding, and this binding fraction may represent the substrate recognition site for noradrenaline uptake. Assuming that one molecule of desipramine binds to each carrier molecule, the turnover number for the noradrenaline carrier was calculated to be 20/min, i.e., the duration of one transport cycle was 3 s.     

Identification of D3 and σ Receptors in the Rat Striatum and Nucleus Accumbens Using (±)-7-Hydroxy-N,N-Di-n-[3H]Propyl-2-Aminotetralin and Carbetapentane

Abstract: Cross-reactions between dopamine D₃ and σ receptor ligands were investigated using (±)-7-hydroxy-N,N-di-n-[³H]propyl-2-aminotetralin [(±)-7-OH-[³H]DPAT], a putative D₃-selective radioligand, in conjunction with the unlabeled σ ligands 1,3-di(2-tolyl)guanidine (DTG), carbetapentane, and R(?)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane [R(?)-PPAP]. In transfected CCL1.3 mouse fibroblasts expressing the human D₃ receptor, neither DTG nor carbetapentane (0.1 µM) displaced (±)-7-OH-[³H]DPAT binding. R(?)-PPAP (0.1 µM) displaced 39.6 ± 1.0% of total (±)-7-OH-[³H]DPAT binding. In striatal and nucleus accumbens homogenates, (±)-7-OH-[³H]DPAT labeled a single site (15–20 fmol/mg of protein) with high (1 nM) affinity. Competition analysis with carbetapentane defined both high- and low-affinity sites in striatal (35 and 65%, respectively) and nucleus accumbens (59 and 41%, respectively) tissue, yet R(?)-PPAP identified two sites in equal proportion. Carbetapentane and R(?)-PPAP (0.1 µM) displaced ～20–50% of total (±)-7-OH-[³H]DPAT binding in striatum, nucleus accumbens, and olfactory tubercle in autoradiographic studies, with the nucleus accumbens shell subregion exhibiting the greatest displacement. To determine directly (+)-7-OH-[³H]DPAT binding to σ receptors, saturation analysis was performed in the cerebellum while masking D₃ receptors with 1 µM dopamine. Under these conditions (+)-7-OH-[³H]DPAT labeled σ receptors with an affinity of 24 nM. These results suggest that (a) (±)-7-OH-[³H]DPAT binds D₃ receptors with high affinity in rat brain and (b) a significant proportion of (±)-7-OH-[³H]DPAT binding consists of σ₁ sites and the percentages of these sites differ among the subregions of the striatum and nucleus accumbens.     

Comparison of the binding properties of A1 adenosine receptors in brain membranes of two congeneric marine fishes living at different depths

Summary The binding properties of A₁ adenosine receptors in brain membranes were compared in two congeneric marine teleost fishes which differ in their depths of distribution. Adenosine receptors were labeled using the A₁ selective radioligand [³H]cyclohexyladenosine ([³H]CHA). The A₁ receptor agonist [³H]CHA bound saturably, reversibly and with high affinity to brain membranes prepared fromSebastolobus altivelis andS. alascanus; however, the meanK _d values differed significantly (Figs. 1–3, Table 1). Saturation data fit to a one site model indicated that the A₁ receptor inS. alascanus exhibited a higher affinity (K _d=1.49 nM) for [³H]CHA whereas A₁ receptors inS. altivelis exhibited a significantly lower affinity (K _d=3.1 nM). Moreover,S. altivelis, but notS. alascanus, parameter estimates for [³H]CHA binding to two sites of receptor were obtained (Fig. 3, Table 1). The mean dissociation constant values for the high and low affinity sites for [³H]CHA inS. altivelis were 0.43 nM and 16.3 nM, respectively. In equilibrium competition experiments the adenosine analogs R-phenylisopropyladenosine (R-PIA), N-ethylcarboxamidoadenosine (NECA) and S-phenylisopropyladenosine (S-PIA) all displayed higher affinities for A₁ receptors inS. alascanus as compared toS. altivelis brain membranes (Table 2, Fig. 6). The specific binding of [³H]CHA was significantly increased by 0.1 and 1.0 mM MgCl₂ in both fishes; however, the sensitivity (95–131% increase) ofS. altivelis to this effect was significantly greater than that ofS. alascanus (48–91% increase) (Fig. 5). The results of kinetic, equilibrium saturation and equilibrium competition experiments all suggest that A₁ adenosine receptors ofS. altivelis andS. alascanus brain membranes differ with respect to their affinities for selected adenosine agonists.Abbreviations CHA cyclohexyladenosine - R-PIA R-phenylisopropyladenosine - S-PIA S-phenylisopropyladenosine - NECA N-ethylcarboxamidoadenosine - NEM N-ethylmaleimide - 2-ClAdo 2-chloroadenosine - GTP guanosine triphosphate - N protein guanine nucleotide binding protein - _{n H} Hill slope     

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.     

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.     

[3H]Dipyridamole Binding to Guinea Pig Brain Membranes: Possible Heterogeneity of Central Adenosine Uptake Sites

The binding of [3H]dipyridamole ([3H]DPR) to guinea pig brain membranes is described and compared to that of [3H]nitrobenzylthioinosine ([3H]NBI). The binding of [3H]DPR is saturable, reversible, and specific with pharmacologic evidence indicating that this ligand is binding to the adenosine uptake site. Compared to [3H]NBI the binding of [3H]DPR is of higher capacity (Bmax = 208 +/- 16 fmol/mg protein for [3H]NBI and 530 +/- 40 fmol/mg protein for [3H]DPR) and lower affinity (KD = 0.35 +/- 0.02 nM for [3H]NBI and 7.6 +/- 0.7 nM for [3H]DPR). The adenosine uptake inhibitors are the most potent inhibitors of binding (Ki of 10(-8)-10(-7) M) whereas adenosine receptor ligands such as cyclohexyladenosine, 2-chloroadenosine, and various methylxanthines are several orders of magnitude less potent (Ki 10(-5)-10(-2). The inhibition of [3H]DPR binding by NBI is biphasic, with only 40% of binding being susceptible to inhibition of NBI concentrations less than 10(-5) M. The tissue distribution of [3H]DPR binding parallels that of [3H]NBI although in most cases significantly more sites are observed with [3H]DPR. Calcium channel blocking agents such as nifedipine, nimodipine, and verapamil are also inhibitors of [3H]DPR binding with potencies in the micromolar range. The data are consistent with [3H]DPR being a useful additional ligand for the adenosine uptake site and provide evidence that multiple uptake binding sites exist of which only about 40% are NBI-sensitive.     

Binding of Aminoalkylindoles to Noncannabinoid Binding Sites in NG108-15 Cells

1. Aminoalkylindoles, typified by WIN 55212-2, bind to G protein-coupled cannabinoid receptors in brain. Although cannabinoids inhibit adenylyl cyclase in NG108-15 neuroblastoma × glioma hybrid cells, cannabinoid receptor binding in these cells has not been described previously. This study compares pharamcological characteristics of [³H]WIN 55212-2 binding sites in rat cerebellar membranes and in NG108-15 membranes.2. Although the K _D of specifid [³H]WIN 55212-2 binding was similar in brain and NG108-15 membranes, the B _max was 10 times lower in NG108-15 than in cerebellar membranes. In both brain and NG108-15 membranes, aminoalkylindole analogues were relatively potent in displacing [³H]WIN 55212-2 binding.However, IC₅₀ values for more traditional cannabinoids were significantly higher in NG108-15 membranes than in brain, e.g., the K _i values for CP55,940 were1.2nM in brain and >5000nM in NG108-15 membranes. Moreover, sodium and GTP--S decreased [³H]WIN 55212-2 binding in brain but not in NG108-15membranes.3. These data suggest that WIN 55212-2 does not label traditional cannabinoid receptors in NG108-15 cells and that these novel aminoalkylindolebinding sites are not coupled to G proteins.     

Characterization of an Adenylate Cyclase-Linked Serotonin (5-HT1) Receptor in a Neuroblastoma × Brain Explant Hybrid Cell Line (NCB-20)

Clonal cell line NCB-20 (a hybrid of mouse neuroblastoma N18TG2 and Chinese hamster 18-day embryonic brain expiant) expressed both high- (K_D 180 nM) and low-affinity (>3000 nM) binding sites for [³H]serotonin (5-HT) which were absent from the parent neuroblastoma. The low-affinity binding site was eliminated by 1 μM spiperone. The order of drug potency for inhibition of high-affinity [³H]5-HT binding was consistent with a 5-HT₁ receptor (5,6 - dihydroxytryptamine = 5-HT = methysergide = 5-methoxytryptamine > cyproheptadine = clozapine = mianserin > spiperone > dopamine = morphine = ketanserin = norepinephrine). [³H]5-HT binding was inhibited by guanine nucleotides (e.g., GTP and Gpp(NH)p), whereas antagonist binding was not; as-corbate was also inhibitory. A 30-min exposure of cells to 1—2 μM 5-HT or other agonists produced a three- to fivefold stimulation of cyclic AMP levels. The order of potency for 5-HT agonist stimulation of basal cyclic AMP levels and 5-HT antagonist reversal of agonist-stimulated levels was the same as the order of drug potency for inhibition of high-affinity [³H]5-HT binding, suggesting linkage of the 5-HT₁ receptor to adenylate cyclase in NCB-20 cells.     

High- and Low-Affinity α-[3H]Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid ([3H]AMPA) Binding Sites Represent Immature and Mature Forms of AMPA Receptors and Are Composed of Differentially Glycosylated Subunits

Abstract: Quantitative α-[³H]amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid ([³H]AMPA) binding autoradiography was performed on frozen-thawed sections from rat brain after preincubation at 0 or 35°C for 1 h. Preincubation at 35°C instead of 0°C resulted in a selective decrease of [³H]AMPA binding assayed at a low concentration of [³H]-AMPA (50 nM) and an enhancement of binding at a high concentration (500 nM). The decrease in [³H]AMPA binding after preincubation at 35°C was accompanied with the loss of the lighter organelles of P3 (microsomal) fractions. These organelles were found to contain a small subpopulation of AMPA/GluR receptors exhibiting a high affinity for [³H]AMPA(K_D～14 nM), whereas heavier organelles exhibited lower affinity for AMPA (K_D～190 nM). This small subpopulation of AMPA/GluR receptors contained almost exclusively a structurally distinct species of GluR2/3 subunits with an apparent molecular mass of 103.5 kDa (assessed with anti-GluR2/3, C-terminal antibodies). Experiments using two deglycosylating enzymes, N-glycopeptidase F and endoglycosidase H, clearly indicated that the 103.5-kDa species represented a partially unglycosylated form of GluR2/3 subunits containing the high-mannose type of oligosaccharide moiety, whereas receptors present in synaptosomal fractions were composed of subunits with complex oligosaccharides. A similar result was obtained by using an antibody recognizing the N-terminal domain of GluR2(4). The same enzymatic treatment indicated that GluR1 subunits also exhibited a partially glycosylated form. These data indicate that high-affinity [³H]AMPA binding sites represent nonsynaptic, intracellular membrane-bound AMPA receptors that differ from synaptic receptors by at least the glycosylation state of GluR2 (and GluR1) subunits. In addition, our results provide a relatively simple way of assessing changes in two spatially and structurally distinct [³H]AMPA binding/GluR sites.     

Support for Radiolabeling of a Glycine Recognition Domain on the N-Methyl-d-Aspartate Receptor Ionophore Complex by 5,7-[3H]Dichlorokynurenate in Rat Brain

Abstract: Pretreatment with Triton X-100 more than doubled the binding of radiolabeled 5,7-dichlorokynurenic acid (DCKA), a proposed antagonist at a glycine (Gly) recognition domain on the N-methyl-d -aspartate (NMDA) receptor ionophore complex, in rat brain synaptic membranes. The binding exhibited an inverse temperature dependency, reversibility, and saturability, the binding sites consisting of a single component with a high affinity (27.5 nM) and a relatively low density (2.87 pmol/mg of protein). The binding of both [³H]DCKA and [³H]Gly was similarly displaced by numerous putative agonists and antagonists at the Gly domain in a concentration-dependent manner at a concentration range of 100 nM to 0.1 mM. Among the 24 putative ligands tested, DCKA was the second most potent displacer of the binding of both radioligands with no intrinsic affinity for the binding of [³H]kainic acid and α-amino-3-hydroxy-5-[³H]methylisoxazole-4-propionic acid (AMPA) to the non-NMDA receptors. In contrast, the other proposed potent Gly antagonist, 5,7-dinitroquinoxaline-2,3-dione, was active in displacing the binding of [³H]glutamic ([³H]Glu) and D,L-(E)-2-amino-4-[³H]propyl-5-phosphono-3-pentenoic acids to the NMDA recognition domain with a relatively high affinity for the non-NMDA receptors. In addition, the proposed antagonist at the AMPA-sensitive receptor, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline, not only displaced weakly the binding of both [³H]- Gly and [³H]DCKA, but also inhibited the binding of (+)-5-[³H]methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([³H]MK-801) to an ion channel associated with the NMDA-sensitive receptor in the presence of added Glu alone in a manner sensitive to antagonism by further added Gly. Clear correlations were seen between potencies of the displacers to displace [³H]DCKA binding and [³H]Gly binding, in addition to between the potencies to displace [³H]-DCKA or [³H]Gly binding and to potentiate or inhibit [³H]MK-801 binding. All quinoxalines tested were invariably more potent displacers of [³H]DCKA binding than [³H]Gly binding, whereas kynurenines were similarly effective in displacing the binding of both [³H]Gly and [³H]-DCKA. These results undoubtedly give support to the proposal that [³H]DCKA is one useful radioligand available in terms of its high selectivity and affinity for the Gly domain in the brain. Possible multiplicity of the Gly domain is suggested by the differential pharmacological profiles between the binding of [³H]Gly and [³H]DCKA.     

[3H]Adenosine Transport in Synaptoneurosomes of Postmortem Human Brain

Abstract: Abstract: [³H]Adenosine transport was characterized in cerebral cortical synaptoneurosomes prepared from postmortem human brain using an inhibitor-stop/centrifugation method. The adenosine transport inhibitors dipyridamole and dilazep completely and rapidly blocked transmembrane fluxes of [³H]adenosine. For 5-s incubations, two kinetically distinguishable processes were identified, i.e., a high-affinity adenosine transport system with K_t and V_max values of 89 μM and 0.98 nmol/min/mg of protein, respectively, and a low-affinity adenosine transport system that did not appear to be saturable. For incubations with 1 μM [³H]adenosine as substrate, intrasynaptoneurosomal concentrations of [³H]adenosine were 0.26 μM at 5 s and 1 μM at 600 s. Metabolism of accumulated [³H]adenosine to adenine nucleotides was 15% for 5-s, 23% for 15-s, 34% for 30-s, 43% for 60-s, and 80% for 600-s incubations. The concentrations (μM) of total accumulated ³H-purines ([³H]-adenosine plus metabolites) at these times were 0.3, 0.5, 1.0, 1.3 and 5.6, respectively. These results indicate that in the presence of extensive metabolism, the intrasynaptoneurosomal accumulation of ³H-purines was higher than the initial concentration of 1 μM [³H]adenosine in the reaction medium. For 5-, 15-, 30-, 60-, and 600-s incubations in the presence of the adenosine deaminase inhibitor EHNA and the adenosine kinase inhibitor 5′-iodotubercidin, metabolism of the transported [³H]adenosine was 14, 14, 16, 14, and 38%, respectively. During these times, total ³H-purine accumulation was 0.3, 0.5, 0.5, 0.7, and 1.8 μM, respectively. Thus, the apparently “concentrative'’accumulation of ³H-purines can be prevented by inhibition of adenosine metabolism and, taken together, these results suggest that adenosine transport in at least synaptoneurosomes prepared from postmortem human brain is via a nonconcentrative and equilibrative system.     

Interconvertible Kinetic States of t-Butylbicycloorthobenzoate Binding Sites of the γ-Aminobutyric AcidA Ionophores

The kinetics of t-[³H]butylbicycloorthobenzoate (TBOB) binding to the convulsant sites of the γ-aminobutyric acid_A (GABA_A) receptor-ionophore complex were examined in synaptosomal membrane preparations of rat brain. On and off rates of TBOB binding were accelerated by 1 μM GABA and decelerated by 1 μM bicuculline methochloride, a GABA_A antagonist. The presence of GABA and bicuculline methochloride created rapid and slow phases of dissociation, respectively. The three groups of rate constants distinguished for the dissociation of 4 nM and 30 nM [³H]TBOB represent multiaffinity states of the convulsant sites depending on the presence of GABA or bicuculline methochloride. Apparent association rate constants do not obey the equation k_app=k_off±k_on [TBOB] without assuming interconvertibility of the kinetic states during binding. Avermectin B_1a (AVM B_1a), a chloride channel opening agent, accelerated the association and dissociation of TBOB and resulted in a biphasic effect on TBOB binding, i.e., enhancement at low concentrations (EC₅₀, 7.8 nM) followed by displacement at high concentrations (IC₅₀ 6.3 μM) of AVM B_1a. AVM B_1a resulted in similar biphasic effects on t- [³⁵S]butylbicyclophosphorothionate binding. DIDS, an isothiocyanatostilbene derivative with irreversible anion channel blocking effect, selectively inhibited basal [³H]TBOB binding (IC₅₀ 125 μM DIDS) leaving the enhancement by AVM B_1a unaffected.