(+)-[3H]MK-801 Binding Sites in Postmortem Human Brain

The pharmacological specificity and the regional distribution of the N-methyl-D-aspartate receptor-associated 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) binding sites in human postmortem brain tissue were determined by binding studies using (+)-[3H]MK-801. Scatchard analysis revealed a high-affinity (KD = 0.9 +/- 0.2 nM, Bmax = 499 +/- 33 fmol/mg of protein) and a low-affinity (KD = 3.6 +/- 0.9 nM, Bmax = 194 +/- 44 fmol/mg of protein) binding site. The high-affinity site showed a different regional distribution of receptor density (cortex greater than hippocampus greater than striatum) compared to the low-affinity binding site (cerebellum greater than brainstem). The rank order pharmacological specificity and stereoselectivity of the high-(cortex) and low-(cerebellar) affinity binding sites were identical. However, all compounds tested showed greater potency at the high-affinity site in cortex. The results indicate that (+)-[3H]MK-801 binding in human postmortem brain tissue shows pharmacological and regional specificity.     

Solubilization of an Adenosine Uptake Site in Brain

Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat brain tissue. Using [3H]nitrobenzylthioinosine [( 3H]NBI) the solubilized site is characterized both kinetically and pharmacologically. The binding is dependent on protein concentration and is saturable, reversible, specific, and high affinity in nature. The KD and Bmax of guinea pig extracts are 0.13 +/- 0.02 nM and 133 +/- 18 fmol/mg protein, respectively, with linear Scatchard plots obtained routinely. Similar kinetic parameters are observed in rat brain. Adenosine uptake inhibitors are the most potent inhibitors of [3H]NBI binding with the following order of potency, dilazep greater than hexobendine greater than dipyridamole. Adenosine receptor ligands are much less potent inhibitors of binding, and caffeine is without effect. The solubilized adenosine uptake site is, therefore, shown to have virtually identical properties to the native membrane site. The binding of the adenosine A1 receptor agonist [3H]cyclohexyladenosine [( 3H]CHA) to the solubilized brain extract was also studied and compared with that of [3H]NBI. In contrast to the [3H]NBI binding site [3H]CHA binds to two apparent populations of adenosine receptor, a high-affinity site with a KD of 0.32 +/- 0.06 nM and a Bmax of 105 +/- 30 fmol/mg protein and a lower-affinity site with a KD of 5.50 +/- 0.52 nM and Bmax of 300 +/- 55 fmol/mg protein. The pharmacology of the [3H]CHA binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake [( 3H]NBI binding) site. Therefore, we show that the adenosine uptake site can be solubilized and that it retains both its binding and pharmacologic properties in the solubilized state.     

A Comparison of [3H]MK-801 and N-[1-(2-Thienyl)cyclohexyl]-3,4-[3H]Piperidine Binding to the N-Methyl-D-Aspartate Receptor Complex in Human Brain

The binding of (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate ([3H]MK-801) and N-[1-(2-thienyl)cyclohexyl]-3,4-[3H]piperidine ([3H]TCP) to the N-methyl-D-aspartate (NMDA) receptor complex of human brain has been investigated. Significant differences were noted between the binding of the two ligands in the same tissue samples. Binding of both ligands was stimulated by addition of glutamic acid or glycine. However, addition of both compounds resulted in an additional effect with [3H]MK-801 but not [3H]TCP binding. Saturation analysis revealed approximately twice as many high-affinity sites for [3H]MK-801 (Bmax, 1,500 +/- 300 fmol/mg of protein) than for [3H]TCP (Bmax, 660 +/- 170 fmol/mg of protein). In addition, a low-affinity site was detected for [3H]MK-801 binding but not [3H]TCP binding. The pharmacology of the high-affinity [3H]MK-801 and [3H]TCP binding sites was similar with rank order of potency of inhibitors being MK801 greater than TCP greater than phencyclidine greater than N-allylnormetazocine (SKF 10047). 2-Amino-5-phosphonopentanoate inhibited binding of both ligands with comparable potency whereas both 7-chlorokynurenic acid and ZnCl2 were more potent inhibitors of [3H]MK-801 than of [3H]TCP binding. All compounds examined exhibited Hill coefficients of significantly less than unity. Saturation analysis performed in the striatum revealed that the number of binding sites was the same for both [3H]MK-801 (Bmax, 1,403 +/- 394 fmol/mg) and [3H]TCP (Bmax, 1,292 +/- 305 fmol/mg). Addition of glutamate or glycine stimulated striatal binding but there was no further increase on addition of both together.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cooperative Modulation of [3H]MK-801 Binding to the N-Methyl-d-Aspartate Receptor-Ion Channel Complex by l-Glutamate, Glycine, and Polyamines

In extensively washed rat cortical membranes [3H](+)-5-methyl-10,11-dihydro-5 H-dibenzo [a,d]cyclohepten-5,10-imine ([3H]MK-801) labeled a homogeneous set of sites (Bmax = 1.86 pmol/mg protein) with relatively low affinity (KD = 45 nM). L-Glutamate, glycine, and spermidine produced concentration-dependent increases in specific [3H]MK-801 binding due to a reduction in the KD of the radioligand. In the presence of high concentrations of L-glutamate, glycine, or spermidine, the KD values for [3H]MK-801 were reduced to 11 nM, 18 nM, and 15 nM, respectively. Maximally effective concentrations of combinations of the three compounds further increased [3H]MK-801 binding affinity as follows: L-glutamate + glycine, KD = 6.2 nM; L-glutamate + spermidine, KD = 2.2 nM; glycine + spermidine, KD = 8.3 nM. High concentrations of spermidine did not inhibit either [3H]glycine orf [3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid binding to the N-methyl-D-aspartate (NMDA) receptor complex. The concentration of L-glutamate required to produce half-maximal enhancement (EC50) of [3H]MK-801 binding was reduced from 218 nM to 52 nM in the presence of 30 microM glycine and to 41 nM in the presence of 50 microM spermidine. The EC50 value for glycine enhancement of [3H]MK-801 binding was 184 nM. This was lowered to 47 nM in the presence of L-glutamate and to 59 nM in the presence of spermidine. Spermidine enhanced [3H]MK-801 binding with an EC50 value of 19.4 microM which was significantly reduced by high concentrations of L-glutamate (EC50 = 3.9 microM) or glycine (EC50 = 6.2 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

[3H]MK-801 Labels a Site on the N-Methyl-D-Aspartate Receptor Channel Complex in Rat Brain Membranes

The potent noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist [3H]MK-801 bound with nanomolar affinity to rat brain membranes in a reversible, saturable, and stereospecific manner. The affinity of [3H]MK-801 was considerably higher in 5 mM Tris-HCl (pH 7.4) than in previous studies using Krebs-Henseleit buffer. [3H]MK-801 labels a homogeneous population of sites in rat cerebral cortical membranes with KD of 6.3 nM and Bmax of 2.37 pmol/mg of protein. This binding was unevenly distributed among brain regions, with hippocampus greater than cortex greater than olfactory bulb = striatum greater than medulla-pons, and the cerebellum failing to show significant binding. Detailed pharmacological characterization indicated [3H]MK-801 binding to a site which was competitively and potently inhibited by known noncompetitive NMDA receptor antagonists, such as phencyclidine, thienylcyclohexylpiperidine (TCP), ketamine, N-allylnormetazocine (SKF 10,047), cyclazocine, and etoxadrol, a specificity similar to sites labelled by [3H]TCP. These sites were distinct from the high-affinity sites labelled by the sigma receptor ligand (+)-[3H]SKF 10,047. [3H]MK-801 binding was allosterically modulated by the endogenous NMDA receptor antagonist Mg2+ and by other active divalent cations. These data suggest that [3H]MK-801 labels a high-affinity site on the NMDA receptor channel complex, distinct from the NMDA recognition site, which is responsible for the blocking action of MK-801 and other noncompetitive NMDA receptor antagonists.     

Solubilized Rat Brain Adenosine Receptors Have Two High-Affinity Binding Sites for l, 3-Dipropyl-8-Cyclopentylxanthine

The specific binding of L-N6-[3H]phenylisopropyladenosine (L-[3H]PIA) to solubilized receptors from rat brain membranes was studied. The interaction of these receptors with relatively low concentrations of L-[3H]PIA (0.5-12.0 nM) in the presence of Mg2+ showed the existence of two binding sites for this agonist, with respective dissociation constant (KD) values of 0.24 and 3.56 nM and respective receptor number (Bmax) values of 0.28 +/- 0.03 and 0.66 +/- 0.05 pmol/mg of protein. In the presence of GTP, the binding of L-[3H]PIA also showed two sites with KD values of 24.7 and 811.5 nM and Bmax values of 0.27 +/- 0.09 and 0.93 +/- 0.28 pmol/mg of protein for the first and the second binding site, respectively. Inhibition of specific L-[3H]PIA binding by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (0.1-300 nM) performed with the same preparations revealed two DPCPX binding sites with Ki values of 0.29 and 13.5 nM, respectively. [3H]DPCPX saturation binding experiments also showed two binding sites with respective KD values of 0.81 and 10.7 nM and respective Bmax values of 0.19 +/- 0.02 and 0.74 +/- 0.06 pmol/mg of protein. The results suggest that solubilized membranes from rat brain possess two adenosine receptor subtypes: one of high affinity with characteristics of the A1 subtype and another with lower affinity with characteristics of the A3 subtype of adenosine receptor.     

Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Analysis of the equilibrium binding of [3H]-neurotensin(1-13) at 25 degrees C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (KD) of 3.3 nM and a density (Bmax) of 350 fmol/mg protein (Hill coefficient nH = 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising less than 10% of the total. KD values of 0.3 and 2.0 nM were obtained for the higher and lower affinity classes of sites, respectively, from association-dissociation kinetic studies. The binding of [3H]neurotensin was decreased by cations (monovalent and divalent) and by a nonhydrolysable guanine nucleotide analogue. Competition studies gave a potency ranking of [Gln4]neurotensin greater than neurotensin(8-13) greater than neurotensin(1-13). Smaller neurotensin analogues and neurotensin-like peptides were unable to compete with [3H]neurotensin. Stable binding activity for [3H]neurotensin in detergent solution (Kd = 5.5 nM, Bmax = 250 fmol/mg protein, nH = 1.0) was obtained in 2% digitonin/1 mM Mg2+ extracts of membranes which had been preincubated (25 degrees C, 1 h) with 1 mM Mg2+ prior to solubilization. Association-dissociation kinetic studies then revealed the presence of two classes of sites (KD1 = 0.5 nM, KD2 = 3.6 nM) in a similar proportion to that found in the membranes. The solubilized [3H]-neurotensin activity retained its sensitivity to cations and guanine nucleotide.     

Solubilization of Calcium Channel Antagonist Binding Sites from Rat Brain

Calcium antagonist binding sites were solubilized from rat brain membranes using the detergent 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulfonate (CHAPS). CHAPS-solubilized [3H]nitrendipine binding sites are saturable over a range of 0.05-4 nM and Scatchard analysis reveals a single, high-affinity (KD = 0.49 +/- 0.10 nM), low-capacity (Bmax = 56 +/- 4 fmol/mg of protein) binding site. Reversible ligand competition experiments using solubilized binding sites demonstrated appropriate pharmacologic specificity, with dihydropyridines (nifedipine = nitrendipine greater than Bay K 8644) completely displacing binding, verapamil partially displacing binding, and diltiazem enhancing binding, as previously described in membrane preparations. Lyophilized Crotalus atrox venom was purified by ion exchange chromatography followed by gel filtration to a single peptide band on sodium dodecyl sulfatepolyacrylamide gel electrophoresis. This fraction of molecular weight 60,000 competitively inhibits [3H]nitrendipine binding to both membrane and soluble preparations with an IC50 of 5 micrograms/ml. This polypeptide should serve as a useful ligand for future efforts in purifying the dihydropyridine calcium channel binding site in brain.     

Characterization of Opioid Receptors in Cultured Neurons

The appearance of mu-, delta-, and kappa-opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high-affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (-)-[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax = 210 +/- 40 fmol/mg protein); this density is approximately two-thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the mu-specific ligand [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 +/- 0.1 nM and a density (Bmax) of 160 +/- 20 fmol/mg of protein. Specific binding of (-)-[3H]bremazocine under conditions in which mu- and delta-receptor binding was suppressed (kappa-receptor labeling conditions) occurred to an apparent single class of sites (KD = 2 +/- 1 nM; Bmax = 40 +/- 15 fmol/mg of protein). There was no detectable binding of the selective delta-ligand [3H]D-Pen2,D-Pen5-enkephalin. Thus, cultured striatal neurons expressed mu- and kappa-receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not delta-receptors.     

Characterization of the non-competitive antagonist binding site of the NMDA receptor in dark Agouti rats

The ability of non-competitive NMDA antagonists and other selected compounds to inhibit [3H]MK-801 binding to the NMDA receptor in brain membranes was evaluated in female, dark Agouti rats. In homologous competition binding studies the average apparent affinity (KD) of [3H]MK-801 for its binding site was 5.5 nM and the binding site density (Bmax) was 1.83 pmol/mg protein. Inhibition of [3H]MK-801 binding by non-competitive NMDA antagonists was best described with a one-site competition model and the average Hill coefficients were -1. A series of eight non-competitive NMDA antagonists inhibited [3H]MK-801 binding with the following rank order of affinity (K(i), nM): MK-801 (5.5) > dexoxadrol (21.5) > or = TCP (24.2) > phencyclidine (100.8) > (+)-SKF 10,047 (357.7) > dextrorphan (405.2) > ketamine (922.2) > dextromethorphan (2913). These inhibition binding constants determined in dark Agouti rat brain membranes were significantly correlated (P = 0.0002; r2 = 0.95) with previously reported values determined in Sprague-Dawley rats [Wong et al., 1988, J. Neurochem. 50, 274-281]. Despite significant differences in metabolic capability between these strains, the central nervous system NMDA receptor ion channel shares similar characteristics.     

Characterization of a Digitonin-Solubilized Bovine Brain H3 Histamine Receptor Coupled to a Guanine Nucleotide-Binding Protein

The H3 receptor is a high-affinity histamine receptor that inhibits release of several neurotransmitters, including histamine. We have characterized H3 receptor binding in bovine brain and developed conditions for its solubilization. Particulate [3H]histamine binding showed an apparently single class of sites (KD = 4.6 nM; Bmax = 78 fmol/mg of protein). Of the detergents tested, digitonin at a detergent/protein ratio of 1:1 (wt/wt) yielded the greatest amount of solubilized receptors, typically 15-30% of particulate binding. Neither equilibrium binding of [3H]histamine to receptors (KD = 6.1 nM; Bmax = 92 fmol/mg of protein) nor the inhibitor profile was substantially altered by digitonin solubilization. However, solubilization did increase the rate of [3H]histamine association with and dissociation from the receptor. Size-exclusion chromatography indicated an apparent molecular weight of 220,000 for the solubilized receptor, and peak binding from this column retained its guanine nucleotide sensitivity. These last two observations are consistent with the solubilized receptor occurring in complex with a guanine nucleotide-binding protein.     

Solubilization of phencyclidine receptors from rat cerebral cortex in an active ligand binding state

A phencyclidine (PCP) receptor binding site has been solubilized in an active ligand-binding state from rat cerebral cortical membranes with sodium deoxycholate. Optimal receptor solubilization occurs at a detergent/protein ratio of 0.5 (w/w); for 5 mg protein/ml solubilized with 0.25% sodium deoxycholate, about 60% of the protein and 25% of the receptor is solubilized. Specific binding of either [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) or [3H]MK-801 is measurable by filtration through Sephadex G-50 columns or glass fiber filters; more than 60% of the binding activity is stable after 48 h at 4 degrees C. In the presence of detergent, [3H]TCP binding exhibits a Kd of 250 nM, a Bmax of 0.56 pmol/mg protein, and a pharmacological profile consistent with that of the membrane-bound PCP receptor, although most drugs bind with affinities 2 to 8 fold lower than in membranes. Upon reduction of detergent concentration, binding parameters approximate those for the membrane-bound receptor ([3H]TCP binding: Kd = 48 nM, Bmax = 1.13 pmol/mg protein).     

Characterization and Regional Distribution of α2-Adrenoceptors in Postmortem Human Brain Using the Full Agonist [3H]UK 14304

The full agonist [3H]UK 14304 [5-bromo-6-(2-imidazolin-2-yl-amino)-quinoxaline] was used to characterize alpha 2-adrenoceptors in postmortem human brain. The binding at 25 degrees C was rapid (t1/2, 4.6 min) and reversible (t1/2, 14.1 min), and the KD determined from the kinetic studies was 0.48 nM. In frontal cortex, the rank order of potency of adrenergic drugs competing with [3H]UK 14304 or [3H]clonidine showed the specificity for an alpha 2A-adrenoceptor: UK 14304 approximately equal to yohimbine approximately equal to oxymetazoline approximately equal to clonidine greater than phentolamine approximately equal to (-)-adrenaline greater than idazoxan approximately equal to (-)-noradrenaline greater than phenylephrine greater than (+/-)-adrenaline much greater than corynanthine greater than prazosin much greater than (+/-)-propranolol. GTP induced a threefold decrease in the affinity of [3H]UK 14304, with no alteration in the maximum number of binding sites, suggesting that the radioligand labelled the high-affinity state of the alpha 2-adrenoceptor. In the frontal cortex, analyses of saturation curves indicated the existence of a single population of noninteracting sites for [3H]UK 14304 (KD = 0.35 +/- 0.13 nM; Bmax = 74 +/- 9 fmol/mg of protein). In other brain regions (hypothalamus, hippocampus, cerebellum, brainstem, caudate nucleus, and amygdala) the Bmax ranged from 68 +/- 7 to 28 +/- 4 fmol/mg of protein. No significant changes in the KD values were found in the different regions examined. The binding of [3H]UK 14304 was not affected by age, sex or postmortem delay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilisation of the 5-Hydroxytryptamine3 Receptor from Pooled Rat Cortical and Hippocampal Membranes

5-Hydroxytryptamine3 (5-HT3) receptors have been identified in the rat brain using the radioligand [3H]Q ICS 205-930. We report here that these sites have been solubilised from membranes prepared from pooled rat cerebral cortex and hippocampus using various detergents. Of the six detergents tested (1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate, 0.5% deoxycholate, 1% Lubrol, 0.5% digitonin, 1% Triton X-100, and 1% octyl glucoside), deoxycholate (0.5%) yielded the best solubilisation (54.6 +/- 6% of receptor, 70.5 +/- 4% of protein; n = 3). However, most detergents inhibited binding of [3H]Q ICS 205-930 in solution. Binding was found to be optimal after the receptor had been exchanged by gel filtration through Sephadex G-25 into the detergent Lubrol PX (0.05%). Binding of [3H]Q ICS 205-930 to these soluble sites was saturable and specific (Bmax = 46.1 +/- 6 fmol/mg of protein; KD = 0.33 +/- 0.09 nM; n = 4) and was similar to that observed in membranes. Kinetic studies of [3H]Q ICS 205-930 binding demonstrated it to be rapid, with equilibrium being achieved within 15 min at 4 degrees C. The KD determined from the rates of association and dissociation (0.38 nM) agreed well with that determined by saturation analysis. Various antagonists completed for the soluble receptors with a rank order of potency typical for binding at a 5-HT3 receptor site: zacopride (Ki = 0.26 nM) greater than quipazine (0.37 nM) = Q ICS 205-930 (0.33 nM) greater than ICS 205-930 (0.93 nM) greater than GR 38032F (2.2 nM) greater than BRL 24924 (4.1 nM) greater than MDL 72222 (23.4 nM) greater than ketanserin (6,000 nM). The agonists 5-HT and 2-methyl-5-HT also competed for [3H]Q ICS 205-930 binding with high affinity (39.6 and 55.6 nM, respectively). Therefore, we conclude that the 5-HT3 receptor of rat brain has been successfully solubilised, and this should provide a good starting point for purification of the receptor.     

Immunological Detection of the NMDAR1 Glutamate Receptor Subunit Expressed in Human Embryonic Kidney 293 Cells and in Rat Brain

The rat NMDAR1 (N-methyl-D-aspartate receptor) was expressed transiently in human embryonic kidney cells. Transfected cell homogenates showed saturable [3H]MK-801 binding activity that was best fit by a single high-affinity site with a KD of 9 nM and a Bmax of 113 fmol of binding sites/mg of protein. Antibodies raised against the peptide sequence NMDAR1 (929-938) coupled to keyhole limpet haemocyanin specifically recognised a single band with M(r) 117,000 in immunoblots from adult rat brain. In the transfected cells, the antibody recognised two bands: one with M(r) 117,000, which was coincident with that from brain membranes, and one with M(r) 97,000, which was identified as nonglycosylated NMDAR1 subunit. These results identify the NMDAR1 of rat brain and further show that the homooligomer binds MK-801, albeit at low efficiency.     

Differential Binding Properties of Adenosine Receptor Agonists and Antagonists in Brain

The binding properties of N6-cyclohexyl [3H]adenosine ( [3H]CHA) and 1,3-diethyl-8-[3H]phenylxanthine ( [3H]DPX) in rat forebrain membrane are compared. The kinetic parameters of binding for each ligand are quite distinct, with [3H]CHA displaying two populations of binding sites (KD = 0.4 +/- 0.05 nM and 4.2 +/- 0.3 nM; Bmax = 159 +/- 17 and 326 +/- 21 fmol/mg protein), whereas [3H]DPX yielded monophasic Scatchard plots (KD = 13.9 +/- 1.1 nM; Bmax = 634 +/- 27 fmol/mg protein). The metals copper, zinc, and cadmium are potent inhibitors of [3H]CHA binding, with respective IC50 concentrations of 36 microM, 250 microM, and 70 microM. Copper is a much less potent inhibitor of [3H]DPX binding (IC50 = 350 microM). The inhibitory effect of copper on both [3H]CHA and [3H]DPX binding is apparently irreversible, as membranes pretreated with copper cannot be washed free of its inhibitory effect. The inhibitory effect of both copper and zinc on [3H]CHA binding was reversed by the guanine nucleotide Gpp(NH)p. [3H]DPX binding is only partially inhibited by zinc and cadmium (60% of specific binding remains unaffected), suggesting that this adenosine receptor ligand binds to two separate sites. Guanine nucleotides had no effect on the inhibition of [3H]DPX binding by either copper or zinc. Differential thermal and proteolytic denaturation profiles are also observed for [3H]CHA and [3H]DPX binding, with the former ligand binding site being more labile in both cases. Stereospecificity is observed in the inhibition of both [3H]CHA and [3H]DPX binding, with L-N-phenylisopropyladenosine (PIA) being 50-fold more potent than D-PIA in both cases. Evidence is therefore provided that adenosine receptor agonists and antagonists have markedly different binding properties to brain adenosine receptors.     

(+)-PN200-l 10 and Ouabain Binding Sites in Purified Bovine Adrenomedullary Plasma Membranes and Chromaffin Cells

Bovine adrenal medulla plasma membranes were purified by a differential centrifugation procedure using sucrose and Urografin discontinuous density gradients; the membranes were enriched 10-12-fold in acetylcholinesterase activity and [3H]ouabain binding sites. Specific (+)-[3H]PN200-110 binding to these membranes amounted to 90% of total binding and was saturable and of high affinity (KD = 41 pM; Bmax = 119 fmol/mg of protein) with a Hill coefficient close to 1, a result suggesting the presence of a single, homogeneous population of dihydropyridine receptors. The association and dissociation rate constants were, respectively, 7.5 X 108 M-1 min-1 and 0.023 min-1. Unlabeled (+)-PN200-110 displaced (+)-[3H]PN200-110 binding with a potency 100-fold higher than (-)-PN200-110 (IC50,0.5 and 45nM, respectively). Although the two enantiomers of BAY K 8644 completely displaced (+)-[3H]PN200-110 binding, they exhibited no stereoselectivity (IC50, 69 and 83 nM,respectively). Whereas ( +/- )-nitrendipine very potently displaced (+)-[3H]PN200-110 binding (IC50 = 1.3 nM) verapamil and cinnarizine displaced the binding by only 30 and 40% at 1 microM, and diltiazem increased it by 20% at 10 microM. [3H]Ouabain bound to plasma membranes with a KD of 34 nM and a Bmax of 9.75 pmol/mg of protein, a figure 80-fold higher than the Bmax for (+)-PN200-110. [3H]Ouabain also bound to intact chromaffin cells with a Bmax of 244 fmol/10(6) cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of chronic nicotine treatment on nicotinic receptors in the rabbit urinary bladder

Nicotine induced a phasic contraction in the rabbit urinary bladder. The response was abolished by hexamethonium and partially reduced by atropine and capsaicin. Simultaneous atropine and capsaicin treatment did not abolish the contraction. These findings suggest that the response to nicotine is due to acetylcholine, tachykinins, and unknown mediator release. In contrast, nicotine-induced contraction diminished following the chronic nicotine treatment without a change of its pharmacological properties. These results suggest the possibility that chronic nicotine treatment causes a decrease in nicotinic receptor numbers. Therefore, the binding properties of (-)-[3H]nicotine on rabbit urinary detrusor muscle membrane fractions were studied to evaluate the effects of chronic nicotine treatment on nicotinic receptors. Specific (-)-[3H]nicotine binding reached saturation and Scatchard plots were curvilinear, suggesting the existence of two different affinity sites for (-)-[3H]nicotine. Dissociation constants (KD) and maximum binding sites (Bmax) were KD1 = 4.91 +/- 1.88 nM, Bmax1 = 2.42 +/- 0.22 fmol/mg protein and KD2 = 263 +/- 56 nM, Bmax2 = 25.0 +/- 4.3 fmol/mg protein. In urinary bladder membrane fractions from chronic nicotine-treated rabbits, KD and Bmax values were KD1 = 3.96 +/- 0.38 nM, Bmax1 = 1.07 +/- 0.25 fmol/mg protein and KD2 = 249 +/- 12 nM, Bmax2 = 10.8 +/- 1.5 fmol/mg protein. Dissociation constants for both sites following chronic nicotine treatment did not change but maximum binding site numbers for both sites significantly decreased (p less than 0.05). These results suggest that the decrease in contractile response evoked by nicotine after chronic nicotine treatment in rabbit urinary bladder is due to a decrease in numbers of nicotinic receptors.     

Benzodiazepine Binding Characteristics of Embryonic Rat Brain Neurons Grown in Culture

The binding of [3H]diazepam to cell homogenates of embryonic rat brain neurons grown in culture was examined. Under the conditions used to prepare and maintain these neurons, only a single, saturable, high-affinity binding site was observed. The binding of [3H]diazepam was potently inhibited by the CNS-specific benzodiazepine clonazepam (Ki = 0.56 +/- 0.08 nM) but was not affected by the peripheral-type receptor ligand Ro5-4864. The KD for [3H]diazepam bound specifically to cell homogenates was 2.64 +/- 0.24 nM, and the Bmax was 952 +/- 43 fmol/mg of protein. [3H]Diazepam binding to cell membranes washed three times was stimulated dose-dependently by gamma-aminobutyric acid (GABA), reaching 112 +/- 7.5% above control values at 10(-4) M. The rank order for potency of drug binding to the benzodiazepine receptor site in cultured neurons was clonazepam greater than diazepam greater than beta-carboline-3-carboxylate ethyl ester greater than Ro15-1788 greater than CL218,872 much greater than Ro5-4864. The binding characteristics of this site are very similar to those of the Type II benzodiazepine receptors present in rat brain. These data demonstrate that part, if not all, of the benzodiazepine-GABA-chloride ionophore receptor complex is being expressed by cultured embryonic rat brain neurons in the absence of accompanying glial cells and suggest that these cultures may serve as a model system for the study of Type II benzodiazepine receptor function.     

Characterization of Two Classes of Opioid Binding Sites in Drosophila melanogaster Head Membranes

Opioid receptors have been characterized in Drosophila neural tissue. [3H]Etorphine (universal opioid ligand) bound stereospecifically, saturably, and with high affinity (KD = 8.8 +/- 1.7 nM; Bmax = 2.3 +/- 0.2 pmol/mg of protein) to Drosophila head membranes. Binding analyses with more specific ligands showed the presence of two distinct opioid sites in this tissue. One site was labeled by [3H]dihydromorphine ([3H]DHM), a mu-selective ligand: KD = 150 +/- 34 nM; Bmax = 3.0 +/- 0.6 pmol/mg of protein. Trypsin or heat treatment (100 degrees C for 15 min) of the Drosophila extract reduced specific [3H]DHM binding by greater than 80%. The rank order of potency of drugs at this site was levorphanol greater than DHM greater than normorphine greater than naloxone much greater than dextrorphan; the mu-specific peptide [D-Ala2,Gly-ol5]-enkephalin and delta-, kappa-, and sigma-ligands were inactive at this site. The other site was labeled by (-)-[3H]ethylketocyclazocine ((-)-[3H]EKC), a kappa-opioid, which bound stereospecifically, saturably, and with relatively high affinity to an apparent single class of receptors (KD = 212 +/- 25 nM; Bmax = 1.9 +/- 0.2 pmol/mg of protein). (-)-[3H]EKC binding could be displaced by kappa-opioids but not by mu-, delta-, or sigma-opioids or by the kappa-peptide dynorphin. Specific binding constituted approximately 70% of total binding at 1 nM and approximately 50% at 800 nM for all three radioligands ([3H]etorphine, [3H]EKC, and [3H]DHM). Specific binding of the delta-ligands [3H][D-Ala2,D-Leu5]-enkephalin and [3H][D-Pen2,D-Pen5]-enkephalin was undetectable in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)