Dopamine D4-like binding sites labeled by [3H]nemonapride include substantial serotonin 5-HT2A receptors in primate cerebral cortex

Dopamine D4-like binding sites are abundant in human cerebral cortex as detected by [3H]nemonapride. The extremely low density of D4 mRNA in human cerebral cortex is inconsistent with the high amount of D4-like binding sites. To investigate the nature of the D4-like receptors, [3H]nemonapride binding sites in the nonhuman primate cerebral cortex were characterized. Although [3H]nemonapride binding sites were D4-like, displaceable by clozapine but not raclopride, [3H]nemonapride binding was not displaced by selective D4 antagonists but was displaced by the selective 5-HT2A antagonist MDL100907. Using [3H]ketanserin as a 5-HT2A ligand, nemonapride showed high affinity for monkey (Ki = 10.4 nM) and cloned human (Ki = 9.4 nM) 5-HT2A receptors, while its affinity for rat receptors was lower (Ki = 140 nM). The present study demonstrates that cerebral cortical D4-like binding sites labeled by [3H]nemonapride in nonhuman primates consist of a very small portion of D4, but a substantial portion of 5-HT2A receptors. The unexpectedly high affinity of nemonapride for primate 5-HT2A receptor suggests reconsidering previous data from other studies using [3H]nemonapride, particularly those on D4-like receptors.     

Neuronal nicotinic receptor deficits in Alzheimer patients with the Swedish amyloid precursor protein 670/671 mutation

The influence of beta-amyloid on cholinergic neurotransmission was studied by measuring alterations in nicotinic acetylcholine receptors (nAChRs) in autopsy brain tissue from subjects carrying the Swedish amyloid precursor protein (APP) 670/671 mutation. Significant reductions in numbers of nAChRs were observed in various cortical regions of the Swedish 670/671 APP mutation family subjects (-73 to -87%) as well as in sporadic Alzheimer's disease (AD) cases (-37 to -57%) using the nicotinic agonists [3H]epibatidine and [3H]nicotine, which bind with high affinity to both alpha3 and alpha4 and to alpha4 nAChR subtypes, respectively. Saturation binding studies with [3H]epibatidine revealed two binding sites in the parietal cortex of AD subjects and controls. A significant decrease in Bmax (-82%) for the high-affinity site was observed in APP 670/671 subjects with no change in K(D) compared with controls (0.018 nM APP 670/671; 0.036 nM control). The highest load of neuronal plaques (NPs) was observed in the parietal cortex of APP 670/671 brains, whereas the number of [3H]nicotine binding sites was less impaired compared with other cortical brain regions. Except for a positive significant correlation between the number of [3H]nicotine binding sites and number of NPs in the parietal cortex, no strict correlation was observed between nAChR deficits and the presence of NPs and neurofibrillary tangles, suggesting that these different processes may be closely related but not strictly dependent on each other.     

Studies on Muscarinic Binding Sites in Human Brain Identified with [3H]Pirenzepine

Pirenzepine, a potent antimuscarinic agent with apparent selectivity for a subtype (M1) of muscarinic receptors, was used in tritiated form to characterize its binding to human brain tissue. Specific [3H]pirenzepine binding showed rapid association and dissociation. From kinetic and competitive binding experiments, its KD was 5.5 nM and 9 nM, respectively. Regional distribution of [3H]pirenzepine binding determined in parallel with [3H]quinuclidinyl benzilate binding, a nonselective muscarinic antagonist, indicated a significant correlation for the maximum number of binding sites for the two radioligands in 13 brain regions, with the highest amount of binding for each in the putamen and the least in the cerebellum. Binding for [3H]pirenzepine averaged 57% of that for [3H]quinuclidinyl benzilate, with a range of 20% (cerebellum) to 77% (frontal cortex). Most antidepressants and neuroleptics tested had affinities for [3H]pirenzepine binding sites that were not significantly different from their previously reported values obtained with the use of [3H]quinuclidinyl benzilate.     

Detection of a Novel Serotonin Receptor Subtype (5-HT1E) in Human Brain: Interaction with a GTP-Binding Protein

[3H]Serotonin (5-hydroxytryptamine, [3H]5-HT) was used as a radioligand probe of brain 5-HT receptors in homogenates of human cortical tissue. Two binding sites were detected in the presence of 1 microM pindolol (to block 5-HT1A and 5-HT1B receptors), and 100 nM mesulergine (to block 5-HT1C and 5-HT2 receptors). One of these sites demonstrated high affinity for 5-carboxyamidotryptamine (5-CT) and ergotamine, consistent with the known pharmacology of the 5-HT1D receptor; the second site demonstrated low affinity for 5-CT and ergotamine. Computer-assisted analyses indicated that both drugs displayed high affinities (Ki values of 1.1 nM and 0.3 nM for 5-CT and ergotamine, respectively) for 55% of the sites and low affinities (Ki values of 910 nM and 155 nM for 5-CT and ergotamine, respectively) for 45% of the sites. To investigate the non-5-HT1D component of the binding, 100 nM 5-CT (to block 5-HT1A, 5-HT1B, and 5-HT1D receptors) was coincubated with [3H]5-HT, membranes, and mesulergine. The remaining [3H]5-HT binding (hereafter referred to as "5-HT1E") displayed high affinity and saturability (KD, 5.3 nM; Bmax, 83 fmol/mg) in human cortical tissue. Competition studies with nonradioactive drugs indicated that, of the drugs tested, 5-CT and ergotamine displayed the highest selectivity for the 5-HT1D site versus the 5-HT1E site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serotonin 5-HT1D Receptors in Human Prefrontal Cortex and Caudate: Interaction with a GTP Binding Protein

Radioligand binding studies were performed to characterize serotonin 5-HT1D receptors in postmortem human prefrontal cortex and caudate homogenates. [3H]5-HT binding, in the presence of pindolol (to block 5-HT1A and 5-HT1B receptors) and mesulergine (to block 5-HT1C receptors), was specific, saturable, reversible, and of high affinity. Scatchard analyses of [3H]5-HT-labeled 5-HT1D sites in human prefrontal cortex produced a KD value of 4.2 nM and Bmax of 126 fmol/mg protein. In competition experiments, 8-hydroxydipropylaminotetralin, trifluoromethylphenylpiperazine, mesulergine, 4-bromo-2,5-dimethoxyphenylisopropylamine, and ICS 205-930 had low affinity for [3H]5-HT-labeled 5-HT1D sites, indicating that the pharmacology of the 5-HT1D site is distinct from that of previously identified 5-HT1A, 5-HT1B, 5-HT1C, 5-HT2, and 5-HT3 sites. 5-HT1D sites in human brain have a similar pharmacology to the 5-HT1D sites previously identified in rat, porcine and bovine brains. Guanyl nucleotides, guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) and guanosine 5'-(beta, gamma-imido)-triphosphate (Gpp(NH)p), modulated the binding of [3H]5-HT to 5-HT1D sites, whereas adenyl nucleotides had no effect. These findings are supportive of the presence of serotonin 5-HT1D receptors in human prefrontal cortex and caudate which appear to be coupled to a GTP binding protein.     

[3H]1-[2-(4-Aminophenyl)Ethyl]-4-(3-Trifluoromethylphenyl)Piperazine: A Selective Radioligand for 5-HT1A Receptors in Rat Brain

1-[2-(4-Aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (PAPP) inhibits [3H]5-hydroxytryptamine (5-HT, serotonin) binding to 5-HT1A and 5-HT1B sites in rat brain with apparent equilibrium dissociation constants (KD) of 2.9 and 328 nM, respectively. [3H]PAPP was synthesized, its binding to central serotonin receptors was examined, and its potential usefulness as a 5-HT1A receptor radioligand was evaluated. With either 10 microM 5-HT or 1 microM 8-hydroxy-2-(di-n-propylamino)tetralin to define nonspecific binding, [3H]PAPP bound to a single class of sites in rat cortical membranes with a KD of 1.6 nM and a maximal binding density (Bmax) of 162 fmol/mg of protein. d-Lysergic acid diethylamide and 5-HT, two nonselective inhibitors of [3H]5-HT binding, displaced 1 nM [3H]PAPP with a potency that matched their affinity for 5-HT1 receptors. Spiperone and 8-hydroxy-2-(di-n-propylamino)tetralin, two compounds that discriminate [3H]5-HT binding to 5-HT1A and 5-HT1B sites, inhibited [3H]PAPP binding in accordance with their much higher affinities for the 5-HT1A receptor subtype. Furthermore, the ability of N-(m-trifluoromethylphenyl)piperazine and ketanserin to inhibit [3H]PAPP binding reflected their low affinities for the 5-HT1A receptor. Several nonserotonergic compounds were also found to be relatively poor displacers of [3H]PAPP binding. The regional distribution of serotonin-sensitive [3H]PAPP sites correlated with the densities of 5-HT1A receptors in the cortex, hippocampus, corpus striatum, and cerebellum of the rat. These results indicate that [3H]PAPP binds selectively and with high affinity to 5-HT1A receptor sites in rat brain.     

Binding of [3H]Neurotensin in Human Brain: Properties and Distribution

The binding of [3H]neurotensin to membranes from human brain at 0 degrees C was specific, saturable, and reversible. In the frontal cortex, the equilibrium dissociation constant (KD) for [3H]neurotensin determined from the ratio of rate constants (k-1/k1), saturation isotherms, and inhibition binding experiments was 0.80, 2.0, and 2.0 nM, respectively, and the maximum number of binding sites (Bmax) from the saturation isotherms and the competitive binding experiments was 2.4 and 2.2 pmol/g of tissue, respectively. Hill coefficients for binding were equal to 1, indicating the presence of single, noncooperative binding sites. Inhibition of specific binding of [3H]-neurotensin by several analogs of neurotensin showed that [Gln4]neurotensin and neurotensin(8-13) had the highest affinities for these binding sites in human frontal cortex, with each analog being approximately 13-fold more potent than neurotensin. In addition, these data showed that the carboxy-terminal portion of neurotensin played an important part in the binding of this neuropeptide in human brain, a result described for other species. Regional distribution of binding sites was different from that reported for animal brains. Of the 33 different regions investigated, the uncus and substantia nigra showed the highest specific binding of [3H]neurotensin, whereas such areas as the pineal body, medulla, and corpus callosum had few binding sites.     

Identification of Multiple Binding Sites for [3H]5-Hydroxytryptamine in the Rat CNS

Recent studies indicate that there may be multiple subtypes of [3H]5-hydroxytryptamine ([3H]5-HT) binding sites. Mianserin and spiperone inhibited the specific binding of [3H]5-HT (2-3 nM) to rat brain cortical membranes with shallow displacement curves. The displacement data for spiperone were best described by the presence of three independent binding sites, for which spiperone had high, medium, and low affinities. The displacement data for mianserin were best fitted by two independent, high- and low-affinity sites. The inclusion of mianserin (250 nM) to inhibit [3H]5-HT binding to the mianserin-sensitive site selectively blocked one of the sites discriminated by spiperone. These results suggest the presence of three binding sites for [3H]5-HT, one blocked by low concentrations of spiperone (5-HT1A), one blocked by low concentrations of mianserin (5-HT1C), and one blocked only by high concentrations of both mianserin and spiperone (5-HT1B). Regional differences in the relative densities of the three sites were observed. The hippocampus was rich in 5-HT1A sites, whereas the striatum contained mainly 5-HT1B and 5-HT1C sites. Selective degeneration of 5-HT-containing nerve terminals induced by the neurotoxin 5,7-dihydroxytryptamine increased binding to all three sites in the cerebral cortex. Binding of [3H]5-HT to the three sites was differentially modulated by CaCl2 and guanylimidodiphosphate. The present data suggest the presence of three independent 5-HT1 binding sites having different affinities for mianserin and spiperone and having different regional distributions.     

Ascorbate Modulates 5-[3H]Hydroxytryptamine Binding to Central 5-HT3 Sites in Bovine Frontal Cortex

Ascorbate is present in millimolar concentrations in mammalian brain and can be released from cellular stores by membrane depolarization. We report here that physiologically relevant concentrations of ascorbate modulate 5-[3H]hydroxytryptamine ([3H]5-HT) binding to bovine frontal cortex membranes. Under conditions where [3H]5-HT binding is reversible and saturable, ascorbate causes a concentration-dependent increase in the affinity of [3H]5-HT for central 5-HT3 binding sites. At pH 7.4, increasing ascorbate from 0 to 5.7 mM changes the equilibrium affinity constant (KD) of binding to 5-HT3 sites from 125 nM to 30 nM, without affecting binding site number. These ascorbate-induced effects are pH dependent. At pH 7.1 binding to central 5-HT3 sites is essentially eliminated in the presence of ascorbate. These studies suggest that ascorbate and hydrogen ion concentration interactions may modulate serotonergic function.     

Pharmacological characterization of the binding of [3H]bremazocine in guinea-pig brain: evidence for multiplicity of the kappa-opioid receptors

In guinea-pig brain, [3H]bremazocine has a binding capacity of 27.2 pmol/g wet tissue, which is statistically different from that of [3H]ethylketazocine (14.7 pmol/g wet tissue) or the sum of the individual binding capacities of mu-, delta-, and kappa-selective ligands (15.0 pmol/g wet tissue). Saturation studies of [3H]bremazocine performed in the presence of unlabelled mu-, delta-, and kappa-blockers still reveal a homogeneous population of binding sites. [3H]Bremazocine under suppressed conditions displays at these sites a Kd of 2.51 nM with a binding capacity of 9.15 pmol/g wet tissue. We have performed the pharmacological characterization of these additional opioid binding sites. Displacement curves measured with a number of opioid substances were all best fitted to a one-site model. The stereoselectivity of these additional sites was demonstrated by using two groups of stereoisomers. Oripavine and benzomorphan opioids were among the most potent drugs at the [3H]bremazocine sites (mu + delta + kappa suppressed). Diprenorphine, bremazocine, cyclazocine, and ethylketazocine displayed apparent affinities constants (1/Ka) of 8.66, 7.57, 21.4, and 38.0 nM, respectively at those sites. The kappa-selective drugs U50488, U69593, PD117302, and tifluadom were inhibitors of the binding of [3H]bremazocine at these sites with apparent affinities of 113, 268, 76.9, and 47.9 nM. All mu- or delta-selective drugs tested in this study have caused weak or no inhibition of the binding. Correlation analyses were done between the different affinities measured at the [3H]bremazocine sites (mu + delta + kappa suppressed) and those observed at the known mu-, delta-, and kappa-sites of the guinea-pig brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Trifluoromethylphenyl Piperazine Derivative with High Affinity for 5-Hydroxytryptamine-1A Sites in Rat Brain

The inhibition of [3H]5-hydroxytryptamine [( 3H]5-HT) binding in rat brain by 1-[2-(3-bromoacetamidophenyl)ethyl]-4-(3-trifluoromethylphenyl) piperazine (BrAcTFMPP) and that by spiperone were compared. Spiperone inhibition of [3H]5-HT binding in cortex was consistent with displacement from two sites with dissociation constants (KD) of 24 nM (5-HT-1A site) and 19 microM (5-HT-1B site) for spiperone. BrAcTFMPP also discriminated two subpopulations of [3H]5-HT binding sites with dissociation constants of 0.5 nM and 146 nM for the compound. The proportion of high-affinity sites for each compound represented about 35% of the specific [3H]5-HT binding. In the presence of 1 microM spiperone, a concentration that saturates the 5-HT-1A sites while having a minimal effect on 5-HT-1B sites, BrAcTFMPP displaced [3H]5-HT from a single site with a KD for BrAcTFMPP of 145 nM. The inhibition of [3H]5-HT binding by spiperone in the presence of 30 nM BrAcTFMPP was best fit by a single-site model with a KD of 21 microM for spiperone. In corpus striatum, 5-HT-1A sites, as defined with spiperone, represented 15% of the specific [3H]5-HT binding and 30 nM BrAcTFMPP also blocked about 15% of the binding. A significant difference between spiperone and BrAcTFMPP was their affinity for 5-HT-2 receptors. BrAcTFMPP (KD = 41 nM) had an 80-fold lower affinity for these sites than spiperone (KD = 0.5 nM). Thus, BrAcTFMPP and spiperone discriminate the same two subpopulations of [3H]5-HT binding sites and BrAcTFMPP displays a high affinity and a selectivity for 5-HT-1A sites versus both 5-HT-1B and 5-HT-2 sites.     

3H]U69,593 binding in guinea-pig brain: comparison with [3H]ethylketazocine binding at the kappa-opioid sites

[3H]U69,593 and [3H]ethylketazocine (mu + delta suppressed) binding was measured in homogenates of guinea-pig brain. Both ligands bind with high affinity to a single class of opioid sites. The relative equilibrium dissociation constant (KD) for [3H]U69,593 is 1.15 nM, while [3H]ethylketazocine has a KD value of 0.33 nM. Their respective maximum binding capacities are 4.49 and 4.48 pmol/g of wet tissue. Various mu-selective, delta-selective, kappa-selective, and nonselective opioids were tested in competition studies against the binding of [3H]U69,593 or [3H]ethylketazocine (in the presence of mu- and delta-blockers) to measure their relative affinity. [D-Ala2, MePhe4,Gly5-ol]enkephalin (mu-selective) has low affinity (600-3000 nM) and [D-Pen2,D-Pen5]enkephalin and [D-Ser2, Leu5, Thr6]enkephalin (delta-selective) have very low affinities (greater than 20,000 nM) at the sites labelled with [3H]U69,593 or [3H]ethylketazocine. On the other hand, unlabelled U69,593, U50,488H, and tifluadom (all three kappa-selective substances) display high affinity (1-5 nM) at those sites. Nonselective opioids, such as bremazocine, levorphanol, and ethylketazocine show similar affinities at the sites labelled with [3H]U69,593 and at the sites labelled with [3H]ethylketazocine. These data indicate that [3H]U69,593 is a selective high-affinity ligand for the same sites that are labelled with [3H]ethylketazocine (in the presence of mu- and delta-blockers) and that these are kappa-sites.     

The binding of [3H]mepyramine to histamine H1 receptors in monkey brain

Several laboratories have reported ligand binding studies using radioactive histamine H1 antagonists to label the H1 receptors in mammalian brain. We have extended these studies to a detailed examination of the binding of [3H]mepyramine to monkey brain and have shown that the distribution is similar to that in man, with specific binding sites being concentrated in the frontal cortex with relatively low binding to the pons and basal ganglia. The binding shows a single saturable component with a KD of about 1 nM and a Hill plot slope close to unity. These observations are the same for all structures tested. Comparison with data from other laboratories suggests that in this species, the histamine receptor is the same as that in peripheral tissues. From Ki values for various ligands and comparison of KD estimates in other species, the receptor seems to be essentially identical to the H1 receptor in central and peripheral tissues of the guinea pig and also to that in human brain. The rat and possibly the dog have minor differences from the monkey in terms of KD values for [3H]mepyramine binding.     

Discrimination of Multiple [3H]5-Hydroxytryptamine Binding Sites by the Neuroleptic Spiperone in Rat Brain

Certain neuroleptic drugs, such as spiperone and (+) butaclamol, can discriminate between two populations of [³H]5-hydroxytryptamine ([³H]5-HT) binding sites in rat brain. The butyrophenone neuroleptic spiperone shows the greatest selectivity for these two binding sites, having at least a 3000-fold difference between its dissociation constants (2-12 nM versus 35,000 nM) for the high- and low-affinity sites, respectively. Inhibition of [³H]5-HT binding by spiperone in rat frontal cortex and corpus striatum yields distinctly biphasic inhibition curves with Hill slopes significantly less than unity. Results from nonlinear regression analysis of these inhibition studies were consistent with a two-site model in each brain region. In the frontal cortex the high-affinity neuroleptic sites comprised about 60% of the total [^3/H]5-HT binding sites whereas in the corpus striatum they accounted for only 20% of the sites. Furthermore, saturation studies of [³H]5-HT binding assayed in the absence or presence of 1 μM-spiperone (a concentration that completely blocks the high-affinity site while having minimal activity at the low-affinity site) reveal a parallel shift in the Scatchard plot with no change in the dissociation constant of [³H]5-HT, but a significant decrease (64% in frontal cortex or 28% in corpus striatum) in the number of specific binding sites. These observations are consistent with the existence of at least two populations of [³H]5-HT binding sites having a differential regional distribution in rat brain.     

Histamine H1-receptor in the retina: species differences

Histamine H1-receptors in membranes of the various mammalian retinas were studied by [3H]mepyramine binding assay. Specific [3H]mepyramine bindings to bovine, pig, dog and human retinas were observed with the dissociation constants (KD), 3.8 +/- 1.2 nM, 1.8 +/- 0.6 nM, 2.6 +/- 0.6 nM and 3.0 +/- 0.9 nM, respectively, which were similar to those found in brains. But there was no detectable specific binding in the guinea-pig and rabbit retinas. The number of binding sites (Bmax) ranged from negligible value to 290.7 +/- 51.7 fmole/mg protein(human retina). Some H1-antagonists acted as potent agents in competing with [3H]mepyramine binding to bovine and pig retinas. These results indicated that histamine H1-receptors exist in some mammalian retina and have similar characteristics to those in brain membranes, but they distributes in the wide difference of the binding capacities among the species, while in brain variations were smaller.     

Pharmacologic characterization of muscarinic receptors of insect brains.

Muscarinic receptors in brain membranes from honey bees, houseflies, and the American cockroach were identified by their specific binding of the non-selective muscarinic receptor antagonist [3H]quinuclidinyl benzilate ([3H]QNB) and the displacement of this binding by agonists as well as subtype-selective antagonists, using filtration assays. The binding parameters, obtained from Scatchard analysis, indicated that insect muscarinic receptors, like those of mammalian brains, had high affinities for [3H]QNB (KD = 0.47 nM in honey bees, 0.17 nM in houseflies and 0.13 nM in the cockroach). However, the receptor concentration was low (108, 64.7, and 108 fmol/mg protein for the three species, respectively). The association and dissociation rates of [3H]QNB binding to honey bee brain membranes, sensitivity of [3H]QNB binding to muscarinic agonists, and high affinity for atropine were also features generally similar to muscarinic receptors of mammalian brains. In order to further characterize the three insect brain muscarinic receptors, the displacement of [3H]QNB binding by subtype-selective antagonists was studied. The rank order of potency of pirenzepine (PZ), the M1 selective antagonist, 11-[2-[dimethylamino)-methyl)1-piperidinyl)acetyl)-5,11- dihydro-6H-pyrido(2,3-b)-(1,4)-benzodiazepin-6 one (AF-DX 116), the M2-selective antagonist, and 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) the M3-selective antagonist, was also the same as that of mammalian brains, i.e., 4-DAMP greater than PZ greater than AF-DX 116. The three insect brain receptors had 27-50-fold lower affinity for PZ (Ki 484-900 nM) than did the mammalian brain receptor (Ki 16 nM), but similar to that reported for the muscarinic receptor subtype cloned from Drosophila. Also, the affinity of insect receptors for 4-DAMP (Ki 18.9-56.6 nM) was much lower than that of the M3 receptor, which predominates in rat submaxillary gland (Ki of 0.37 nM on [3H]QNB binding). These drug specificities of muscarinic receptors of brains from three insect species suggest that insect brains may be predominantly of a unique subtype that is close to, though significantly different from, the mammalian M3 subtype.     

(+)-[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.     

Characterization of leukotriene C4 binding sites in the brain of the American bullfrog, Rana catesbeiana.

In this study, specific binding sites for [3H]-LTC4 on membrane preparations from American bullfrog (Rana catesbeiana) brain were characterized. Binding assays were done in the presence of serine (5mM) borate (10 mM) for 30 min at 23 degrees C. Under these conditions, no metabolism of LTC4 to LTD4 occurred. Specific binding of [3H]-LTC4 reached steady state within 10 min, remained constant for 60 min, and was reversible with the addition of 1,000-fold excess unlabelled LTC4. Scatchard analysis of the binding data indicated a single class of binding sites with an estimated Kd of 89.83 nM and Bmax of 43.79 pmol/mg protein. Competition binding studies demonstrated that LTD4 and LTE4 were ineffective in displacing [3H]-LTC4 from its binding site. The Ki for LTC4 was 51 nM. S-decylglutathione, glutathione and hematin had Ki values of 44, 312,602, and 25,576 nM, respectively. The mammalian cysteinyl leukotriene antagonist L-660,711 inhibited specific binding of [3H]-LTC4, with a Ki of 87,149 nM. Guanosine-5'-0-3-thiotriphosphate (GTP gamma S) did not affect specific binding of [3H]-LTC4 indicating that, like mammalian LTC4 receptors, a Gi protein is not involved in the transduction mechanism. The LTC4 binding site in bullfrog brain demonstrates both similarities and differences from its mammalian counterpart.     

n-[3H]Butyl-β-Carboline-3-Carboxylate, a Putative Endogenous Ligand, Binds Preferentially to Subtype 1 of Central Benzodiazepine Receptors

Synthetic n-butyl beta-carboline-3-carboxylate, an endogenous central benzodiazepine receptor inhibitor found in brain, was tritium-labeled from the butenyl ester. Binding of this [3H]beta-carboline was concentrated particularly in the synaptosomal membrane fraction of the cerebral cortex; this fraction showed a single type of high-affinity site (KD = 2.7 +/- 0.1 nM) with a Bmax of 1.16 +/- 0.08 pmol/mg of protein. The number of sites labeled was about half of that obtained with [3H]flunitrazepam binding (Bmax = 2.36 +/- 0.06 pmol/mg of protein). On the other hand, in the cerebellum, both ligands bound to practically the same number of sites. When [3H]flunitrazepam binding was done in the presence of 10(-11)-10(-5) M butyl beta-carboline, the differences between the two brain regions were more apparent. In cerebellar membranes the data fitted a straight line in the Eadie-Hofstee plot; this finding and a Hill number near unity suggest a single type of binding site. In the cortical membranes the data of binding fitted a concave curve, and the Hill number was 0.6. These are characteristics of two types of binding sites with different affinities (KD1 = 0.6-1.5 nM and KD2 = 12-18 nM). The differentiation of a high- and low-affinity site in the cerebral cortex was corroborated by experiments in which [3H]butyl beta-carboline binding was displaced by the triazolopyridazine CL 218,872. These results demonstrate that in the cerebral cortex there are two subtypes of sites (1 and 2) of central benzodiazepine receptors and that CL 218,872 binds preferentially to subtype 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional Distribution of α2A-and α2B-Adrenoceptor Subtypes in Postmortem Human Brain

The newly available and highly selective radiolabeled antagonist [3H]RX 821002 was used to examine the distribution of alpha 2 adrenoceptors in human brain. High densities of alpha 2 adrenoceptors were found in the hippocampus, frontal cortex, thalamus, amygdala, pons, and medulla oblongata. Intermediate densities were observed in the striatum (nucleus accumbens, nucleus caudatus, and putamen), globus pallidus, and substantia nigra. The KD values for [3H]RX 821002 were similar in all regions (ranging from 2.8 to 7.5 nM). On the basis of their different affinities for prazosin and oxymetazoline, the alpha 2 adrenoceptors have been divided into alpha 2A and alpha 2B subtypes. To examine the alpha 2A/alpha 2B-adrenoceptor ratio in the different brain regions, we performed oxymetazoline and prazosin/[3H]RX 821002 competition binding experiments. In frontal cortex membranes, the competition curves with prazosin were steep, indicating a single class of binding sites, whereas the competition curves with oxymetazoline were shallow and fitted by computer best to a two-site model. However, in the presence of GTP, the high-affinity sites for oxymetazoline were partially converted into low-affinity sites, indicating that this agonist interacts with high- and low-affinity states of the alpha 2 adrenoceptors. This implies that oxymetazoline is not very suitable for discriminating the alpha 2A- and alpha 2B-receptor subtypes in radioligand binding studies. Therefore, prazosin/[3H]RX 821002 competition binding experiments were used to investigate the distribution of the alpha 2-adrenoceptor subtypes in human brain. The alpha 2A-receptor subtype was detected in all brain regions examined. In contrast, alpha 2B receptors were only observed in striatum and globus pallidus.