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.     

Characterization of thromboxane A2/prostaglandin H2 receptors and histamine H1 receptors in cultured guinea-pig tracheal smooth-muscle cells.

We characterized thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors and histamine H1 receptors in Guinea-pig cultured tracheal smooth-muscle cells (TSMC). [3H]SQ 29,548 (a TXA2 antagonist)-binding sites were saturable and a high affinity with a dissociation constant of 6.2 +/- 0.60 nM (mean +/- S.E.) and a receptor density of 46 +/- 4.6 fmol/10(6) cells. [3H]SQ 29548 binding was completely inhibited by TXA2 mimetics or antagonists. Intracellular calcium concentration ([Ca2+]i) in TSMC was increased with U46619 stimulation and the increase was attenuated by TXA2 antagonists, the potencies of which correlated with those inhibiting the activities of the [3H]SQ 29548 binding. [3H]Mepyramine (a H1 antagonist)-binding sites were also present in TSMC. [3H]Mepyramine had a single class of low-affinity-binding sites with a dissociation constant of 2.6 +/- 0.081 microM and a receptor density of 10.6 +/- 0.11 nmol/mg protein. [3H]Mepyramine binding in TSMC membrane was inhibited by H1 antagonists, but not by H2 antagonists. The inhibition constants of mepyramine in TSMC were 910-times lower than those in tracheal membranes. In contrast, the histamine-induced increase in [Ca2+]i in TSMC was inhibited in the presence of low concentrations of H1 antagonists. All these observations provide evidence that TXA2/PGH2 receptors, mepyramine-binding sites and/or H1 receptors are expressed in cultured TSMC.     

Binding of 4(5)-[2-(4-azido-2-nitroanilino)ethyl]imidazole to histamine receptors in guinea pig cerebral cortical membranes

The interaction of 4(5)-[2-(4-azido-2-nitroanilino)ethyl]imidazole (AAH), a photolabile histamine receptor antagonist, with the binding of histamine, mepyramine, and tiotidine to guinea pig cerebral cortical membranes was examined to evaluate the specificity of AAH for histamine H1 and H2 receptors. Saturable, specific binding of [3H]histamine, [3H]mepyramine, and [3H]tiotidine to the membranes was observed. Competition assays were used to assess the relative affinity of AAH for H1- and H2-receptors. The rank order of IC50 values obtained was (most to least potent) (i) for competing with [3H]histamine binding: histamine greater than AAH much greater than mepyramine approximately equal to tiotidine; (ii) for competing with [3H]mepyramine binding: mepyramine much greater than AAH greater than histamine greater than tiotidine; and (III) for competing with [3H]tiotidine binding: tiotidine much greater than mepyramine greater than histamine approximately equal to AAH. The affinity of AAH for H1 receptors was ca. 14-fold greater than for H2 receptors. These findings support previous evidence obtained in isolated smooth muscle preparations that AAH shows H1-receptor selectivity as an antagonist.     

Equilibrium binding of [3H]tubocurarine and [3H]acetylcholine by Torpedo postsynaptic membranes: stoichiometry and ligand interactions

Studies are presented of the equilibrium binding of [3H]-d-tubocurarine (dTC) and [3H]acetylcholine (AcCh) to Torpedo postsynaptic membranes. The saturable binding of [3H]dTC is characterized by two affinities: Kd1 = 33 +/- 6 nM and Kd2 = 7.7 +/- 4.6 microM, with equal numbers of binding sites. Both components are completely inhibited by pretreatment with excess alpha-bungarotoxin or 100 microM nonradioactive dTC and competitively inhibited by carbamylcholine with a KI = 100 nM, but not affected by the local anesthetics dimethisoquin, proadifen, and meproadifen. The biphasic nature of [3H]dTC binding was unaltered in solutions of low ionic strength and by preparation of Torpedo membranes in the presence of N-ethylmaleimide, a treatment which yields dimeric AcCJ receptors. dTC competitively inhibits the binding of [3H]AcCH and decreases the fluorescence of 1-(5-dimethylaminonaphthalene-1-sulfonamido)ethane-2-trimethylammonium (Dns-Chol) in a manner quantitatively consistent with its directly measured binding properties. It decreases the initial rate of 3H-labeled Naja nigricollis alpha-toxin binding by 50% at 60 nM with an apparent Hill coefficient of 0.58. The stoichiometry of total dTC, AcCh, and alpha-neurotoxin binding sites in Torpedo membranes was determined by radiochemical techniques and by a novel fluorescence assay utilizing Dns-Chol as an indicator, yielding ratios of 0.9 +/- 0.1:0.9 +/- 0.2:1, respectively. The biphasic equilibrium binding function is not unique to dTC since other ligands inhibited [3h]acCh binding in a biphasic manner with apparent inhibition constants as follows: gallamine triethiodide (K11 = 2 microM, K12 = 1 mM); Me2dTC (K11 = 500 nM, K12 = 10 microM); decamethonium (K11 = 100 nM, K12 = 1.6 microM). Carbamylcholine, however, inhibited [3H]AcCh binding with a single KI = 100 nM. The observed competition between those ligands and [3H] AcCh cannot be completely accounted for by competitive interaction with two different affinities, and the deviations are discussed in terms of the positive cooperativity of the [3H] AcCh binding function itself. It is concluded that dTC binds only to the AcCh sites in Torpedo membranes and that those sites display two affinities for dTC but only one for AcCh.     

Characterization of binding sites for [3H]-DTG, a selective sigma receptor ligand, in the sheep pineal gland

Specific binding sites for [3H]-1,3 di-ortho-tolylguanidine ([3H]-DTG), a selective radiolabeled sigma receptor ligand, were detected and characterized in sheep pineal gland membranes. The binding of [3H]-DTG to sheep pineal membranes was rapid and reversible with a rate constant for association (K+1) at 25 degrees C of 0.0052 nM-1.min-1 and rate constant for dissociation (K-1) 0.0515 min-1, giving a Kd (K-1/K+1) of 9.9 nM. Saturation studies demonstrated that [3H]-DTG binds to a single class of sites with an affinity constant (Kd) of 27 +/- 3.4 nM, and a total binding capacity (Bmax) of 1.39 +/- 0.03 pmol/mg protein. Competition experiments showed that the relative order of potency of compounds for inhibition of [3H]-DTG binding to sheep pineal membranes was as follows: trifluoperazine = DTG greater than haloperidol greater than pentazocine greater than (+)-3-PPP greater than (+/-)SKF 10,047. Some steroids (testosterone, progesterone, deoxycorticosterone) previously reported to bind to the sigma site in brain membranes were very weak inhibitors of [3H]-DTG binding in the present study. The results indicate that [3H]-DTG binding sites having the characteristics of sigma receptors are present in sheep pineal gland. The physiological importance of these sites in regulating the synthesis of the pineal hormone melatonin awaits further study.     

Synaptic and extra-synaptic distribution of histamine H1-receptors in rat and guinea pig brains

Localization of histamine H1-receptors in subcellular fractions from rat and guinea pig brains was examined in a [3H]mepyramine binding study. Major [3H]mepyramine binding sites with increased specific activities [( 3H]mepyramine binding vs. protein amount) were recovered from P2 fractions from both rat and guinea pig brains by differential centrifugation. Further subfractionation of both rat and guinea pig P2 fractions by a discontinuous sucrose density gradient centrifugation showed the highest recovery of [3H]mepyramine binding with further increased specific activities found in synaptic plasma membrane (SPM) fractions. Minor [3H]mepyramine binding sites with increased specific activities were detected in both rat and guinea pig P3 fractions. [3H]Mepyramine binding sites in SPM and P3 fractions showed identical Kd values in each species. These results indicate that histamine H1-receptors are located not only in synaptic but also in extra-synaptic membranes of both rat and guinea pig brains.     

Evidence for Distinct 5-Hydroxytryptamine2 Binding Site Subtypes in Cortical Membrane Preparations

5-Hydroxytryptamine (5-HT) displays a sixfold higher affinity for 5-HT2 binding sites labeled by [3H]ketanserin in rat (IC50 = 200 +/- 40 nM) and human (IC50 = 190 +/- 50 nM) cortex than for 5-HT2 sites in bovine cortex (IC50 = 1,200 +/- 130 nM). The Hill slopes of the 5-HT competition curves are 0.67 +/- 0.04 in rat, 0.69 +/- 0.08 in human, and 0.96 +/- 0.02 in bovine cortex. Scatchard analysis of (+/-)-[3H]4-bromo-2,5-dimethoxyamphetamine ([3H]DOB) binding in the rat indicates a population of binding sites with a KD of 0.38 +/- 0.04 nM and a Bmax of 1.5 +/- 0.05 pmol/g tissue. In contrast, specific [3H]DOB binding cannot be detected in bovine cortical membranes. These data indicate that species variations exist in 5-HT2 binding site subtypes and that [3H]ketanserin appears to label a homogeneous population of 5-HT2 binding site subtypes in bovine cortex.     

Characterization of binding of a specific antagonist, [3H]-SQ 29,548, to soluble thromboxane A2/prostaglandin H2 (TP) receptors in human platelet membranes.

Binding of [3H]-SQ 29,548 was characterized to soluble thromboxane A2/prostaglandin H2 (TP) receptors from human platelet membranes as a means of examining ligand-receptor interactions outside the lipophilic environment of the cell membrane. Kinetic determination revealed a rate of ligand-receptor association of 1.4 x 10(7) +/- 0.2 M-1 x min-1 and a rate of dissociation of 0.5 +/- 0.07 min-1. The resultant equilibrium affinity constant was 36.3 +/- 5.8 nM. Saturation binding analysis revealed a single class of [3H]-SQ 29,548 binding sites with an affinity constant of 39.7 +/- 4.3 nM and a B(max) of 1735.7 +/- 69.1 fmol/mg protein. Specific [3H]-SQ 29,548 binding was inhibited by specific TP receptor antagonists and agonists in a rank order of potency similar to that seen in platelet membranes: SQ 33,961 much greater than SQ 29,548 greater than BM 13,505 greater than or equal to U 46619 greater than BM 13,177. PGD2, PGE2 and PGI2 did not appreciably inhibit the specific binding of [3H]-SQ 29,548. These data indicate that [3H]-SQ 29,548 binding to soluble human platelet TP receptors was specific, saturable, and reversible.     

Identification of specific [3H]adenine-binding sites in rat brain membranes

We recently reported that adenine acts as a neurotrophic factor independent of adenosine or P2 receptors in cultured Purkinje cells [Watanabe S. et al. (2003) J. Neurosci. Res. 74, 754-759], suggesting the presence of specific receptors for adenine in the brain. In this study, the characterization of adenine-binding activity in the rat brain was performed to further characterize the receptor-like adenine-binding sites. Specific binding sites for [(3)H]adenine were detected in membrane fractions prepared from rat brains. The kinetics of [(3)H]adenine binding to membranes was described by the association and dissociation rate constants, 8.6 x 10(5) M(-1) min(-1) and 0.118 +/- 0.045 min(-1), respectively. A single binding site for [(3)H]adenine with a K (D) of 157.1 +/- 20.8 nM and a B (max) of 16.3 +/- 1.1 pmol/mg protein (n = 6) was demonstrated in saturation experiments. A displacement study involving various related compounds showed that the [(3)H]adenine binding was highly specific for adenine. It was also found that [(3)H]adenine-binding activity was inhibited by adenosine, although other adenosine receptor ligands were ineffective as to [(3)H]adenine binding. The brain, especially the cerebellum and spinal cord, showed the highest [(3)H]adenine-binding activity of the tissues examined. These results are consistent with the presence of a novel adenine receptor in rat brain membranes.     

Estimation of platelet-activating factor receptors in the endometrium of the pregnant rabbit: regulation of ligand availability and catabolism by bovine serum albumin.

High affinity receptors have been demonstrated for the potent phospholipid autacoid, platelet-activating factor (PAF C18:0; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) in a variety of tissues, including the endometrium. Because of the relative instability of PAF and our previous demonstration that lyso-PAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphorylcholine), the major metabolite of PAF, displaced [3H]PAF from endometrial PAF receptor sites, we have examined the ability of bovine serum albumin (BSA) to prevent degradation of PAF and have characterized PAF and lyso-PAF binding sites in purified rabbit endometrial membranes isolated on Day 6 of pregnancy. In buffer containing the phospholipase A2 inhibitors, quinacrine (10 microM) and dibromoacetophenone (2 microM), and 0.25% BSA, 87.4 +/- 3.2% of added [3H]PAF C18:0 remained intact after incubation at 25 degrees C for 150 min. The metabolic products, lyso-PAF and 1-O-alkyl-2-acyl-sn-glycero-3-phosphorylcholine (alkylacyl-GPC), only amounted to 5.2 +/- 3.2 and 3.3 +/- 1.1, respectively. At the same concentration, rabbit serum albumin (RSA) also significantly protected [3H]PAF C18:0 from metabolism, but bovine gamma globulin (BGG) was ineffective. The presence of 0.25% BSA, however, did not protect [3H]lyso-PAF C18:0 from extensive catabolism: the major product formed was [3H]alkylacyl-GPC. Insignificant amounts of [3H]PAF were formed. Under the same conditions (25 degrees C, 150 min) in the presence of 0.25% BSA, saturation analysis revealed the presence of two types of PAF C18:0 receptors in the endometrial membranes. Type 1 sites had a Kd of 0.42 +/- 0.03 nM (mean +/- SD; n = 3) and binding capacity of 0.11 +/- 0.01 pmol/mg protein. Type 2 receptor sites had a Kd of 5.96 +/- 0.35 nM and a binding capacity of 1.59 +/- 0.22 pmol/mg protein. Thus, in the presence of BSA, the binding capacities of the two classes of receptors were markedly reduced compared to values generated previously in its absence. The Kd of the Type 1 sites was not significantly changed by the presence of BSA. A single class of saturable high-affinity binding sites was demonstrable for lyso-PAF C18:0: Kds ranged from 0.76 +/- 0.58 to 11.1 +/- 0.62 nM, depending on which method of analysis was used (Eadie-Hofstee, Scatchard-Rosenthal, or the Lundon nonlinear method). The binding capacities were equally varied, ranging from 0.15 +/- 0.08 to 15.17 +/- 4.95 pmol/mg protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of histamine-H1 receptors in bovine intracerebral microvessels with cerebral grey matter by [H]mepyramine binding

Small intracerebral blood vessels (microvessels) of bovine brain are known to contain the vasoactive amine histamine, and the presence of histamine-H₁ receptors in microvessels was examined using the radioligand, [³H]mepyramine. Microvessels were isolated from cerebral cortex grey matter, striatum and hippocampus by a sieving technique and membranes prepared for binding studies. [³H]Mepyramine bound to a single, high affinity site, which displayed stereoselectivity for (+) chlorpheniramine relative to its (−) isomer and was consistent with binding to H₁-receptors. The density of binding sites (B_max), in microvessel membranes from cortical grey matter, was approximately one-third of that seen in membranes prepared from cortical grey matter. Microvessels isolated from striata and hippocampi had a similar density of H₁-receptor sites to that seen in cortical microvessels.

These results demonstrate that bovine intracerebral microvessels contain significant numbers of histamine-H₁ receptors and strengthen the hypothesis that histamine could regulate the calibre of intracerebral blood vessels.     

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.     

Effect of mequitazine a non sedative antihistamine on brain H1 receptors

Marked species variations occured in the relative activity of mequitazine (10-[3-quinuclidinylmethyl]-phenothiazine) on the binding of [³H]mepyramine to brain cortical membranes. Mequitazine was as potent as promethazine in the mouse but about 6 times less effective than promethazine in the guinea pig and human. On the other hand in the guinea pig mequitazine was as potent as promethazine on [³H]mepyramine binding in a peripheral organ (lung). Although mequitazine did not displace [³H]mepyramine in vivo in the mouse and guinea pig, its brain concentration (measured by [³H]mequitazine) was largely sufficient and corresponds to 90% of inhibition in vitro. Moreover in the mouse the brain regional distribution of [³H]mequitazine was very different from that of [³H]mepyramine, highest level was obtained in the cerebellum and hypothalamus was the poorest region with mequitazine whereas the reverse was true with mepyramine. All these results could suggest that mequitazine possesses a greater affinity for peripheral H₁ receptors which could explain the absence of sedative side-effects of this potent H₁ antagonist.     

A reliable assay for beta-adrenoceptors in intact isolated human fat cells with a hydrophilic radioligand, [3H]CGP-12177

The beta-adrenergic receptors of isolated human fat cells were identified using a new hydrophilic beta-adrenergic radioligand (+/-)[3H]CGP-12177. The results were compared with those from [3H]dihydroalprenolol binding to fat cells and membranes. [3H]CGP-12177 binding to isolated fat cells showed lower nonspecific binding (less than 15% of total binding) than the lipophilic [3H]dihydroalprenolol (40-60%) at 3 times the KD. At 37 degrees C, [3H]CGP-12177 binding was rapid, reversible, of high affinity (1.2 +/- 0.3 nM) and saturable. The total number of binding sites per cell in subcutaneous adipocytes was 25,000 +/- 6,000 and was equivalent to that found using membrane fractions. Displacement of [3H]CGP-12177 bound to adipocytes by propranolol was stereoselective, consistent with competition at a single site, and had the same characteristics as in membranes. The displacement curves of the beta 1-selective antagonists (atenolol and betaxolol) were biphasic, the high affinity displacement accounting for 70% of the total binding sites. Beta-adrenergic agonists also competed with [3H]CGP-12177 binding in the order of potency: (-) isoproterenol greater than (-) norepinephrine greater than (-) epinephrine, similar to that found in membranes and in in vitro studies on the lipolytic activity of isolated fat cells. This study demonstrates that the sites specifically labeled by [3H]CGP-12177 are the physiological beta-adrenoceptors and also shows that the ligand is better than [3H]dihydroalprenolol for the accurate identification of these receptors in intact human adipocytes. The methodology, which requires biopsies of less than 1 gram of adipose tissue, can be of potential interest for clinical studies investigating the status of fat cell beta-adrenoceptors in various pathophysiological situations.     

Characterization of alpha 2-adrenergic receptors in human platelets by binding of a radioactive ligand [3H]yohimbine

[3H]Yohimbine, a potent alpha 2-adrenergic antagonist, was used to label the alpha-adrenergic receptors in membranes isolated from human platelets. Binding of [3H]yohimbine to platelet membranes appears to have all the characteristics of binding to alpha-adrenergic receptors. Binding reached a steady state in 2-3 min at 37 degrees C and was completely reversible upon the addition of excess phentolamine or yohimbine (both at 10(-5) M; t1/2 = 2.37 min). [3H]Yohimbine bound to a single class of noncooperative sites with a dissociation constant of 1.74 nM. At saturation, the total number of binding sites was calculated to be 191 fmol/mg protein. [3H]Yohimbine binding was stereo-specifically inhibited by epinephrine: the (-) isomer was 11-times more potent that the (+) isomer. Catecholamine agonists competed for the occupancy of the [3H]yohimbine-binding sites with an order of potency: clonidine greater than (-)-epinephrine greater than (-)-norepinephrine much greater than (-)-isoproterenol. The potent alpha-adrenergic antagonist, phentolamine, competed for the sites whereas the beta-antagonist, (+/-)-propranolol, was very weak inhibitor. 0.1 mM GTP reduced the binding affinity of the agonists, while producing no change in antagonist-binding affinity. Dopamine and serotonin competed only at very high concentrations. Similarly, muscarinic cholinergic ligands were also poor inhibitors of [3H]yohimbine binding. These results suggest that [3H]yohimbine binding to hunan platelet membranes is specific, rapid, saturable, reversible and, therefore, can be successfully used to label alpha 2-adrenergic receptors.     

Modulation of A2A adenosine receptor(s) by K+(ATP) channels in bovine brain striatal membranes

The modulation of adenosine receptor with K+(ATP) channel blocker, glibenclamide, was investigated using the radiolabeled A2A-receptor selective agonist [3H]CGS 21680. Radioligand binding studies in bovine brain striatal membranes (BBM) indicated that unlabeled CGS 21680 displaced the bound [3H]CGS 21680 in a concentration-dependent manner with a maximum displacement being approximately 65% at 10(-4) M. In the presence of 10(-5) M glibenclamide, unlabeled CGS 21680 increased the displacement of bound [3H]CGS 21860 by approximately 28% at 10(-4) M. [3H]CGS 21680 bound to BBM in a saturable manner to a single binding site (Kd = 10.6+/-1.71 nM; Bmax = 221.4+/-6.43 fmol/mg of protein). In contrast, [3H]CGS 21680 showed saturable binding to two sites in the presence of 10(-5) M glibenclamide; (Kd = 1.3+/-0.22 nM; Bmax = 74.3+/-2.14 fmol/mg protein; and Kd = 8.9+/-0.64 nM; Bmax = 243.2+/-5.71 fmol/mg protein), indicating modulation of adenosine A2A receptors by glibenclamide. These studies suggest that the K+(ATP) channel blocker, glibenclamide, modulated the adenosine A2A receptor in such a manner that [3H]CGS 21680 alone recognizes a single affinity adenosine receptor, but that the interactions between K+(ATP) channels and adenosine receptors.     

Autoradiographic Characterization and Localization of Quisqualate Binding Sites in Rat Brain Using the Antagonist [3H]6-Cyano-7-Nitroquinoxaline-2,3-Dione: Comparison with (R,S)-[3H]α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites

Using quantitative autoradiography, we have investigated the binding sites for the potent competitive non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist [3H]6-cyano-7-nitro-quinoxaline-2,3-dione ([3H]-CNQX) in rat brain sections. [3H]CNQX binding was regionally distributed, with the highest levels of binding present in hippocampus in the stratum radiatum of CA1, stratum lucidum of CA3, and molecular layer of dentate gyrus. Scatchard analysis of [3H]CNQX binding in the cerebellar molecular layer revealed an apparent single binding site with a KD = 67 +/- 9.0 nM and Bmax = 3.56 +/- 0.34 pmol/mg protein. In displacement studies, quisqualate, L-glutamate, and kainate also appeared to bind to a single class of sites. However, (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) displacement of [3H]CNQX binding revealed two binding sites in the cerebellar molecular layer. Binding of [3H]AMPA to quisqualate receptors in the presence of potassium thiocyanate produced curvilinear Scatchard plots. The curves could be resolved into two binding sites with KD1 = 9.0 +/- 3.5 nM, Bmax = 0.15 +/- 0.05 pmol/mg protein, KD2 = 278 +/- 50 nM, and Bmax = 1.54 +/- 0.20 pmol/mg protein. The heterogeneous anatomical distribution of [3H]CNQX binding sites correlated to the binding of L-[3H]glutamate to quisqualate receptors and to sites labeled with [3H]AMPA. These results suggest that the non-NMDA glutamate receptor antagonist [3H]CNQX binds with equal affinity to two states of quisqualate receptors which have different affinities for the agonist [3H]AMPA.     

Purification of a histamine H3 receptor negatively coupled to phosphoinositide turnover in the human gastric cell line HGT1.

The histamine H3 receptor agonist (R)alpha-methylhistamine (MeHA) inhibited, in a nanomolar range, basal and carbachol-stimulated inositol phosphate formation in the human gastric tumoral cell line HGT1-clone 6. The inhibition was reversed by micromolar concentrations of the histamine H3 receptor antagonist thioperamide and was sensitive to cholera or pertussis toxin treatment. Using [3H]N alpha-MeHA as specific tracer, high affinity binding sites were demonstrated with a Bmax of 54 +/- 3 fmol/mg of protein and a KD of either 0.61 +/- 0.04 or 2.2 +/- 0.4 nM, in the absence or presence of 50 microM GTP[gamma]S, respectively. The binding sites were solubilized by Triton X-100 and prepurified by gel chromatography. They were separated from the histamine H2 receptor sites by filtration through Sepharose-famotidine and finally retained on Sepharose-thioperamide. The purified sites concentrated in one single silver-stained protein band of 70 kDa in SDS-polyacrylamide gel electrophoresis. They specifically bound [3H]N alpha-MeHA with a KD of 1.6 +/- 0.1 nM and a Bmax of 12,000 +/- 750 pmol/mg of protein. This corresponds to a 90,225-fold purification over cell lysate and a purity degree of 84%. Binding was competitively displaced by N alpha-MeHA (IC50 = 5.8 +/- 0.7 nM), (R) alpha-MeHA (IC50 = 9 +/- 1 nM), and thioperamide (IC50 = 85 +/- 10 nM), but not by famotidine (H2 antagonist) or by mepyramine (H1 antagonist). These findings provide the first evidence for solubilization, purification, and molecular mass characterization of the histamine H3 receptor protein and for the negative coupling of this receptor phosphatidylinositol turnover through a so far unidentified G protein.     

Histamine H(2) -like receptors in chick cerebral cortex: effects on cyclic AMP synthesis and characterization by [(3) H]tiotidine binding

In this study, histamine (HA) receptors in chick cerebral cortex were characterized using two approaches: (1) analysis of the effects of HA-ergic drugs on the cAMP-generating system, and (2) radioreceptor binding of [(3) H]tiotidine, a selective H(2) antagonist. HA was a weak activator of adenylyl cyclase in a crude membrane preparation of chick cerebrum. On the other hand, HA (0.1-1000 microm) potently and concentration dependently stimulated cAMP production in [(3) H]adenine pre-labelled slices of chick cerebral cortex, displaying an EC(50) value (concentration that produces 50% of maximum response) of 2.65 microm. The effect of HA was mimicked by agonists of HA receptors with the following rank order of potency: HA >or= 4-methylHA (H(2)) >or= N alpha,N alpha-dimethylHA (H(3) > H(2) = H(1)) > 2-methylHA (H(1)) > 2-thiazolylethylamine (H(1)) >or= R alpha-methylHA (H(3)) > amthamine, dimaprit (H(2)), immepip (H(3), H(4)). The HA-evoked increase in cAMP production in chick cerebral cortex was antagonized by selective H(2) receptor blockers (aminopotentidine >or= tiotidine > ranitidine > zolantidine), and not significantly affected by mepyramine and thioperamide, selective H(1) and H(3) /H(4) receptor blockers, respectively. A detailed analysis of the antagonistic action of aminopotentidine (vs. HA) revealed a non-competitive mode of action. The binding of [(3) H]tiotidine to chick cortical membranes was rapid, stable and reversible. Saturation analysis resulted in a linear Scatchard plot, suggesting binding to a single class of receptor binding site with high affinity [equilibrium dissociation constant (K (d)) = 4.42 nm] and high capacity [maximum number of binding sites (B (max) ) = 362 fmol/mg protein]. The relative rank order of HA-ergic drugs to inhibit [(3) H]tiotidine binding to chick cerebrum was: antagonists - tiotidine > aminopotentidine = ranitidine >or= zolantadine > thioperamide - triprolidine; agonists - HA >or= 4-methylHA > 2-methylHA >or=R alpha-methylHA - dimaprit. In conclusion, chick cerebral cortex contains H(2) -like HA receptors that are linked to the cAMP-generating system and are labelled with [(3) H]tiotidine. The pharmacological profile of these receptors is different from that described for their mammalian counterpart. It is suggested that the studied receptors represent either an avian-specific H(2) -like HA receptors or a novel subtype of HA receptors.     

Interactions of radiolabelled ligands with specific receptors: an analysis

The binding and displacement of beta-adrenoceptor blockers, [3H]propranolol ([3H]PRP) and [3H]dihydroalprenolol ([3H]DHA), were studied on isolated rat erythrocytes, their membranes and ghosts; the binding of [3H]DHA and a M-cholinoceptor blocker, [3H]quinuclidinylbenzylate ([3H]QNB), on cerebral cortex membranes. In all experiments, ligand-receptor interactions conformed to a model of two pools of receptors in the same effector system and the binding of two ligand molecules to the receptor. The results were similar for the displacement of [3H]PRP, [3H]DHA and [3H]QNB with propranolol, dihydroalprenolol and quinuclidinyl-benzylate, respectively. The parameters of [3H]PRP to beta-adrenoceptor binding for intact erythrocytes were: Kd1 = 0.74+/-0.07 nM, Kd2 = 14.40+/-0.41 nM, B1 = 24+/-2 unit/cell, B2 = 263+/-5 unit/cell; for ghosts, Kd1 = 0.70+/-0.17 nM, Kd2 = 19.59+/-2.59 nM, B1 = 9+/-1 fmol/mg protein, B2 = 39+/-4 fmol/mg protein. Receptor affinities were similar in erythrocytes and ghosts; on the ghost membrane, the number of receptors was considerably lower (B1 = 2 unit/cell, B2 = 6 unit/cell). The parameters of [3H]QNB to M-cholinoceptor binding of the cerebral cortex membrane were the following: Kd1 = 0.43 nM, Kd2 = 2.83 nM, B1 = 712 fmol/mg, B2 = 677 fmol/mg.