Purification of the human blood platelet thromboxane A2/prostaglandin H2 receptor protein.

The human platelet thromboxane A2/prostaglandin H2 receptor has been purified 6100-fold to apparent homogeneity by a three-step chromatographic procedure with an overall yield of 6%. A 6-fold purification of the receptor was first achieved by chromatography of 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS)-solubilized membrane proteins from human platelets on a diethylaminoethyl (DEAE)-Sepharose column. The DEAE eluate fractions containing receptor activity were then applied to a newly developed affinity column using the cyclohexyl derivative of SQ30,741 (SQ31,491) as the immobilized ligand. Elution of the receptor from the affinity column with BM13.177 yielded a further purification of 1700-fold. An additional 4-fold receptor purification from the affinity column eluate was achieved by HPLC using GPC 500 and GPC 100 columns connected in tandem. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the HPLC eluate containing purified receptor revealed a single, distinct band with a molecular weight of 55,000. The receptor binding activity was detected with [3H]SQ29,548 using a newly developed binding assay which involved immobilization of the receptor on polyethyleneimine-treated glass fiber filters. The binding of [3H]SQ29,548 to the purified receptor was time dependent, saturable, reversible and highly specific. Unlabeled SQ29,548, BM13.505, and U46619 (but not thromboxane B2 or 6-keto prostaglandin F1 alpha) competed for [3H]SQ29,548 binding to the purified receptor in a concentration-dependent manner. Scatchard analysis of [3H]SQ29,548 binding to the purified receptor revealed the presence of a single class of high-affinity binding sites, with a Kd of 4 nM and a Bmax of 17 nmol/mg protein.     

Pharmacological and biochemical characterization of purified A2a adenosine receptors in human platelet membranes by [3H]-CGS 21680 binding.

1. The binding properties of human platelet A2a adenosine receptors, assayed with the A2a-selective agonist, [3H]-2-[p-(2-carboxyethyl)-phenethylamino]-5''-N-ethylcarboxamidoad enosine ([3H]-CGS 21680), are masked by a non-receptorial component, the adenotin site. In order to separate A2a receptors from adenotin sites, human platelet membranes were solubilized with 1% 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulphonate (CHAPS). The soluble platelet extract was precipitated with polyethylene glycol (PEG) and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. 2. The present paper describes the binding characteristics of the selective A2a agonist, [3H]-CGS 21680, to this purified platelet membrane preparation. In addition, receptor affinity and potency of several adenosine agonists and antagonists were determined in binding and adenylyl cyclase studies. 3. Saturation experiments revealed a single class of binding site with Kd and Bmax values of 285 nM and 2.07 pmol mg-1 of protein respectively. Adenosine receptor ligands competed for the binding of 50 nM [3H]-CGS 21680 to purified protein, showing a rank order of potency consistent with that typically found for interactions with the A2a adenosine receptors. In the adenylyl cyclase assay the compounds examined exhibited a rank order of potency very close to that observed in binding experiments. 4. Thermodynamic data indicated that [3H]-CGS 21680 binding to the purified receptor is totally entropy-driven in agreement with results obtained in rat striatal A2a adenosine receptors. 5. It is concluded that in the purified platelet membranes there is a CGS 21680 binding site showing the characteristic properties of the A2a receptor. This makes it possible to use this compound for reliable radioligand binding studies on the A2a adenosine receptor of human platelets.     

Covalent labeling of vasopressin receptors from LLC-PK1 cells by the use of a bifunctional reagent

The possibility of covalently attaching vasopressin to its receptors by the use of a bifunctional reagent was explored. Plasma membranes from the LLC-PK1 pig kidney cell line were purified by Percoll density gradient centrifugation. These membranes contained a single population of high affinity (Kd = 5.2 nM) and high capacity (Bmax 3.8 pmol/mg of protein) [3H]lysine vasopressin ([3H]LVP)-binding sites. [3H]LVP-labeled receptors could be solubilized with a high yield (83%) and minimal dissociation (9%) by treatment with the non-ionic detergent, octaethylene glycol mono-n-dodecyl ether (C12E8) (0.5%, v/v) in the presence of glycerol (20%). The solubilized [3H]LVP-labeled receptors were stable upon storage at 4 degrees (5% dissociation after 24 hr). They were partially purified to a specific activity of 17 pmol/mg of protein by chromatography on a Cibacron blue-Sepharose column with a yield of 90%. The [3H]LVP-receptor complexes in both intact membranes and the partially purified preparation were almost completely dissociated by incubation at 30 degrees for 30 min in the presence of 20 mM ethylenediaminetetraacetate (EDTA). This property was used to test the effect of ethylene glycol bis (succinimidyl-succinate) (EGS) as cross-linking reagent for the covalent attachment of [3H]LVP to its receptors. After treatment of [3H]LVP-labeled membranes for 30 min with 1 mM EGS at 4 degrees, about 30% of specifically bound [3H]LVP was resistant to EDTA dissociation. The amount of EDTA-resistant binding varied as a linear function of the fractional receptor occupancy and maximal binding capacity of the different batches of membranes used. Similar results were obtained with solubilized and partially purified vasopressin receptors. Upon steric exclusion high performance liquid chromatography, the EDTA-resistant [3H]LVP-labeled material, like the native [3H]LVP-labeled receptor, was eluted as a single and apparently homogeneous peak. The covalent character of the EGS-induced [3H]LVP binding to solubilized or partially purified receptors was assessed by its resistance to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The yield of EGS-induced labeling deduced from these experiments (27%) was close to that determined by the EDTA method. SDS-PAGE analysis of the [3H]LVP-labeled cross-linked material revealed the specific labeling of a major 50-kDa component and a minor component of 30 kDa. The size of these two components was not affected by dithiothreitol.     

"Gastrin" and "CCK" receptors on histamine- and somatostatin-containing cells from rabbit fundic mucosa-II. Characterization by means of selective antagonists (L-364,718 and L-365,260).

In the preceding paper, by means of selective agonists to gastrin (HG-17) and cholecystokinin (CCK-39), we evidenced the existence of "gastrin-type" receptors that could regulate histamine release and "CCK-type" receptors that could stimulate somatostatin release in isolated rabbit fundic non-parietal cells (F1 cells). Furthermore, these receptors could induce phosphoinositide breakdown. To confirm the involvement of these receptor types in these biological and biochemical processes, we used selective antagonists, L-364,718 (3-(benzoylamino)-benzodiazepine) specific to "CCK-A-type" receptor and L-365,260 (3-(acylamino)-benzodiazepine) specific to "gastrin/CCK-B-type" receptor. Neither L-364,718 nor L-365,260 alone caused any significant stimulation of [3H]inositol phosphate ([3H]InsP) production and release of histamine or somatostatin-like immunoreactivity (SLI). Each analogue inhibited in a dose-dependent manner [125I]HG-17 or [125I]CCK-39 binding to F1 cells, [3H]InsP accumulation and histamine and SLI release stimulated by HG-17 or CCK-39. L-365,260 appeared to be 30-70 times more potent than L-364,718 in inhibiting [125I]HG-17 binding to F1 cells, as well as HG-17-induced [3H]InsP accumulation and HG-17-or CCK-39-enhanced histamine release (IC50 values: approximately 5-20 nM for L-365,260 and approximately 200-1500 nM for L-364,718). In contrast, L-364,718 was 200 to 400 times more potent than L-365,260 in inhibiting [125I]CCK-39 binding to F1 cells, CCK-39-induced [3H]-InsP accumulation and SLI release stimulated by CCK-39 or HG-17 (IC50 values: approximately 0.3-1 nM for L-364,718 and 100-200 nM for L-365,260). These results led to conclude: (i) the existence of a "gastrin-type" receptor related to histamine release: (ii) the existence of a "CCK-A-type" receptor related to somatostatin release; (iii) the existence of "gastrin type" and "CCK-A-type" receptors linked to the phosphoinositide breakdown pathway.     

Evaluation of 11-[3H]-tetrodotoxin use in a heterologous receptor binding assay for PSP toxins.

This report describes the preparative scale production of 11-[3H]-tetrodotoxin (TTX) and its evaluation as a substitute for [3H]-saxitoxin (STX) as the radioligand in a receptor binding assay for paralytic shellfish poisoning (PSP) toxins. Restrictions on the world-wide distribution of [3H]-STX imposed by the international Chemical Weapons Convention served as the primary impetus for this study. We have incorporated on a preparative scale, a nonexchangeable tritium label into the TTX molecule at a specific activity of 12.90 Ci/mmol and recovered material of high radiochemical purity (98%). The resulting 11-[3H]-TTX was found to exhibit site-specific binding characteristics in the receptor assay (dissociation constant(K(d))=4.77+/-1.54nM; maximum binding(B(max))=1. 62+/-0.24pmol/mg of synaptosomal protein). The inhibition constant (K(i)) for the assay was 1.46+/-0.28 nM STX equiv. (n=6), with an estimated detection limit of ca. 2-4 ng STX equiv./ml in a sample extract. Moreover, quantitative comparisons indicated that 11-[3H]-TTX could be used interchangeably with [3H]-STX in the receptor assay for determination of PSP toxicity in shellfish and algal extracts without compromising assay performance. We conclude that the 11-[3H]-TTX produced and evaluated herein exhibits physical, chemical and biological characteristics suitable not only for use in the PSP receptor binding assay, but likely for other applications employing [3H]-STX as the radioligand.     

Improvement of 5-HT3 receptor binding assay: enhancement of specific [3H]quipazine binding with Triton X-100-treated membranes from rat cortex.

The 5-hydroxytryptamine (5-HT)3 receptor binding assay using [3H]quipazine was examined. It was impossible to obtain specific [3H]quipazine binding with the membrane fractions from rat cortex prepared by the usual procedure. When the membranes were pretreated with detergent Triton X-100, the ratio of specific [3H]quipazine binding markedly increased, depending upon the concentration of Triton X-100 in the range of 0.01-0.1% (w/v). At a concentration of more than 0.05%, the specific binding reached a maximum of 55 to 60% of the total binding. The specific [3H]quipazine binding to the Triton X-100-treated membranes was reversible and was potently inhibited by several 5-HT3 antagonists, while 5-HT1, 5-HT2 receptor antagonists and other receptor-specific ligands had no effect on the binding. Scatchard analysis indicated a single class of binding sites with a Kd of 0.62 nM and Bmax of 97 fmol/mg protein. Thus, the Triton X-100-treated membranes retained the characteristics of 5-HT3 binding sites, making it possible to use [3H]quipazine for a 5-HT3 receptor binding assay with a high ratio of specific binding.     

Structural requirements for prostaglandin analog interaction with the ovine corpus luteum prostaglandin F2 alpha receptor. Implications for development of a photoaffinity probe

The capacity of structurally modified analogs of prostaglandin F2 alpha (PGF2 alpha) to inhibit binding of [3H]PGF2 alpha to receptors on ovine luteal cells was evaluated by radioreceptor assay using dispersed, viable, ovine luteal cells. Binding assays were conducted at pH 5.75, since binding to both high (Kd 17.4 +/- 2.3 nM) and low (Kd 409 +/- 166 nM) affinity sites was enhanced markedly at reduced pH. The capability to compete with [3H]PGF2 alpha for binding was evaluated for different prostaglandin analogs having modifications in the C-8 "upper" side-chain, in the cyclopentane ring, or in the C-12 "lower" side-chain. Prostaglandin J2 was a surprisingly potent competitor for binding to the PGF2 alpha receptor. Several phenyl-substituted analogs exhibited receptor-binding potency greater than or equal to native PGF2 alpha, while most other analogs had reduced capacity to compete with native PGF2 alpha for binding. Several 17-azidophenol PGF2 alpha analogs were synthesized and tested, but analogs having hydroxyl groups on the aryl ring had low affinity for receptors. However, 17-(4-azidophenyl)-18,19,20-trinor-PGF2 alpha as well as 17-(3-iodo-4-azidophenyl)-18,19,20-trinor-PGF2 alpha exhibited binding affinities that were approximately 10% of native PGF2 alpha, and the radioiodinated analogs of PGF2 alpha may be useful as probes of the PGF2 alpha receptor.     

3H]Pirenzepine and [3H]quinuclidinyl benzilate binding to brain muscarinic cholinergic receptors. Differences in measured receptor density are not explained by differences in receptor isomerization

Muscarinic receptor densities were measured in membranes prepared from rat cerebral cortex using [3H]pirenzepine and [3H]quinuclidinyl benzilate. Isotherms of equilibrium binding data modeled to a single apparent binding site for both ligands. However, as has been reported previously, [3H]pirenzepine labeled only a small fraction of the binding sites that were labeled by [3H]quinuclidinyl benzilate. This observation has been used to support the hypothesis that subtypes of muscarinic receptors exist. Several investigators have previously suggested that antagonist binding to muscarinic receptors involves an isomerization of the receptor-antagonist complex, and it is only the isomerized form of the receptor that is identified by radioligand binding studies. To examine the possibility that the difference in the density of binding sites identified by [3H]pirenzepine and [3H]quinuclidinyl benzilate is due to differences in the degree of isomerization of the receptor associated with the binding of each ligand, the kinetics of the binding of [3H]pirenzepine and [3H]quinuclidinyl benzilate to membranes prepared from rat cerebral cortex were examined. The pseudo-first-order rate constant of association for both ligands showed a nonlinear (hyperbolic) dependence on ligand concentration. These results suggested that a rapidly equilibrating initial binding step was followed by a more slowly equilibrating isomerization of the initially formed ligand-receptor complex. The kinetic data were computer-modeled to obtain estimates of the equilibrium constants for both reaction steps. The equilibrium constants for the isomerization step were 0.1 and 0.004 for [3H]pirenzepine and [3H]quinuclidinyl benzilate, respectively. Our measurements, in agreement with others, suggested that only the fraction of receptors which isomerized were measurable using filtration binding assays. Although essentially all (99.6%) of the [3H]quinuclidinyl benzilate binding sites appeared to isomerize, only 90% of the [3H]pirenzepine binding sites isomerized, and thus only 90% were measured in our assay. It therefore appears that differences in receptor isomerization can partially, but not wholly, account for the differences between [3H]pirenzepine and [3H]quinuclidinyl benzilate binding in rat cerebral cortex.     

Comparison of two putatively selective radioligands for labeling central nervous system beta-adrenergic receptors: inadequacy of [3H]dihydroalprenolol

[3H]Dihydroalprenolol ([3H]DHA) has been used extensively in receptor binding studies to measure beta-adrenergic receptors in the central nervous system. Usually, nonspecific binding has been defined by high concentrations of the beta-adrenergic receptor agonist isoproterenol or antagonists such as alprenolol or propranolol. Scatchard plots of such "specific" [3H]DHA saturation data in rat cerebral cortex membranes are linear. However, computer analysis demonstrated that the competition curves of these drugs for 2.0 nM [3H]DHA binding are biphasic, with a continuous inhibition of [3H]DHA binding in the concentration range usually used to determine nonspecific binding. These data indicate that another saturable high affinity site was being labeled by the radioligand and that the definition of nonspecific binding with any of these unlabeled drugs is not satisfactory. We used the nonlinear, least squares, curve-fitting program LIGAND to analyze total [3H]DHA binding, allowing the program to mathematically define nonspecific binding as a function of 3H-ligand concentration. Significantly lower Bmax (-44%) and Kd (-58%) values for beta-adrenergic receptors were found, indicating that under normal experimental procedures (defining [3H]DHA non-specific binding with these nonradioactive drugs) a second binding site was being labeled. We found that [3H]DHA binding to this site could be inhibited by drugs such as RU24969, a 5-hydroxytryptamine1A (5HT1A) and 5HT1B receptor subtype-selective agonist, and CGS12066B, a 5HT1B receptor subtype-selective agonist, which were able to compete for 15-20% of [3H]DHA binding in the nanomolar concentration range, whereas drugs that are selective for other serotonin receptor subtypes inhibited [3H]DHA binding only at much higher concentrations. Another beta-adrenergic receptor antagonist radioligand, [3H]CGP-12177, was found to be more selective for beta-adrenergic receptors. Alprenolol competition curves for [3H]CGP-12177 binding were monophasic and saturation curves, with nonspecific binding defined either by 10 microM alprenolol or by LIGAND, yielded Bmax values close to those obtained with [3H]DHA when its nonspecific binding was defined by LIGAND. [3H]DHA cannot be considered a suitable radioligand to quantify central nervous system beta-adrenergic receptors in the manner in which it has been typically used.     

Functional coupling of gamma-aminobutyric acid (GABA)A and benzodiazepine type II receptors: analysis using purified GABA/benzodiazepine receptor complex from bovine cerebral cortex

Possible functional coupling between gamma-aminobutyric acid (GABA) and benzodiazepine receptors was examined using a purified GABA/benzodiazepine receptor complex. The purified receptor complex was obtained by 1012-S-acetamide adipic hydrazide Sepharose 4B affinity column chromatography, following the solubilization of synaptic membrane from the bovine cerebral cortex with Nonidet P-40. The binding of [3H]GABA to the purified GABA receptor was displaced significantly by muscimol and bicuculline, GABAA receptor agonists and antagonists, respectively, but not by baclofen, a GABAB receptor agonist. On the other hand, the binding of [3H]flunitrazepam to the purified benzodiazepine receptor was found to be displaced by microM ranges of CL 218,872, which is known to be sensitive to the benzodiazepine type II receptor. Furthermore, it was found that the binding of [3H]muscimol to these purified GABAA receptors was enhanced by benzodiazepines, while the binding of [3H]flunitrazepam to these benzodiazepine type II receptors was increased by GABA receptor agonists. These enhancements by GABA agonists and benzodiazepine agonists were found to be blocked by bicuculline and a benzodiazepine receptor antagonist, Ro15-1788, respectively. These results strongly suggest that the purified receptor may consist of GABAA and benzodiazepine type II receptors and possess a functional coupling of these sites, as shown in cerebral synaptic membranes.     

Chronic neuroleptic treatment increases D-2 but not D-1 receptors in rat striatum

In vitro binding of [3H]cis(Z)-flupentixol (FPT) to rat striatal membranes was evaluated as an assay for D-1 receptors. It was found that under the appropriate assay conditions [3H]FPT bound to a single saturable site which was most abundant in striatum and bound dopaminergic agonists in the potency order that these drugs demonstrate for adenylate cyclase. These data support previous work suggesting that [3H]FPT labels the D-1 receptor. Next, rats received haloperidol or fluphenazine for 54 days and striatal dopamine receptors were assayed 72 h later. The drug treatments increased the density of D-2 receptors as measured by [3H]spiperone binding by 40% and 25% respectively. However, no change was observed in D-1 receptor density. We conclude that effects of chronic neuroleptic treatment that depend upon increased dopamine receptor density are mediated via the D-2 receptor subtype.     

Solubilization and preliminary characterization of mu and kappa opiate receptor subtypes from rat brain

The opiate agonists [3H]dihydromorphine (DHM, mu-selective ligand), [3H]bremazocine (potent kappa ligand), and [3H]etorphine bound stereospecifically, with high affinity, and reversibly to partially purified 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS)-solubilized extract from rat brain membranes. Recoveries of the three binding activities were as follows: [3H]DHM, 47%; [3H]bremazocine, 55%; and [3H]etorphine, 17%. Each ligand exhibited (by Scatchard analysis) binding to a class of high-affinity sites (Kd = 0.8-2 nM). Hill analyses revealed Hill coefficients of n = 1.1-1.3. Many of the properties of solubilized brain opiate receptors are similar to those of membrane-associated opiate receptors. Opiate binding in soluble fractions was inhibited by a variety of protein-modifying agents, including trypsin, proteinase K, and N-ethylmaleimide as well as by heat treatment (60 degrees, 15 min). The relative potencies of a series of opiate narcotic agonists and antagonists in displacing 2 nM [3H]etorphine binding to the CHAPS-solubilized extract was similar to that determined for rat brain homogenates. In contrast, D-Ala2, D-Leu5-enkephalin (DADLE, putative delta-selective ligand) exhibited a much lower affinity for solubilized brain opiate receptors than for the membrane-bound receptors unless assayed in the presence of manganese chloride, sodium chloride, and GTP. Mu agonist binding to solubilized receptors was inhibited relatively selectively by sodium and guanyl nucleotides. These findings lend support to the pharmacological relevance of the solubilized opiate-binding component(s). The pI of the solubilized brain opiate receptor(s) was estimated by liquid isoelectrofocusing to be pH 4. The sizes of the solubilized, prelabeled [3H]etorphine-receptor complex of the solubilized mu and kappa receptor subtypes, as assayed by stereospecific binding of [3H]DHM and [3H]bremazocine binding, respectively, were estimated by molecular exclusion chromatography. The [3H]etorphine-receptor complex migrated as a broad radioactive peak at a position corresponding to a protein of Stoke radius 63 A. A secondary peak of radioactivity was observed at the salt peak. Mu receptor activity chromatographed as two major peaks. The first of these eluted just behind, but significantly separated from, the protein void peak and corresponded to a Stokes radius of 70 A; the second eluted just ahead of the salt peak and corresponded to a radius of less than 20 A. Kappa receptor activity eluted at positions corresponding to macromolecules of 50 A and less than or equal to 20 A. Together, these findings indicate that selective mu and kappa ligands interact with high molecular weight species of somewhat different sizes as well as a lower molecular weight species, which may represent a common subunit that can bind both ligands.     

Competitive inhibition of platelet thromboxane A2 receptor binding by picotamide

On the basis of indirect pharmacological evidence, picotamide, a methoxy derivative of 4-hydroxy-isophthalic acid (N,N'bis(3-picolyl)-4-methoxy-isophthalamide) has been postulated to inhibit platelet aggregation by competitively interfering with the thromboxane A2 (TxA2) platelet receptor. In the present study the interaction between picotamide and TxA2 receptors on human platelets was investigated by a direct radioligand assay method with [125I]PTA-OH and [3H]U46619 as labelled radioligands. The ONO11120 and U46619 inhibitory constants (Ki) for [125I]PTA-OH binding were 19 +/- 4 and 17 +/- 3 nM, respectively. Picotamide displaced [125I]PTA-OH binding with a Ki of 1472 +/- 321 nM. The Ki for ONO 11120 and U46619 on [3H]U46619 binding were 42 +/- 12 and 16 +/- 5 nM, respectively, whereas the Ki for picotamide was 1648 +/- 431 nM. These data provide evidence that picotamide can directly inhibit the TxA2 platelet receptor.     

Effect of the umami peptides on the ligand binding and function of rat mGlu4a receptor might implicate this receptor in the monosodium glutamate taste transduction

1. The effect of several metabotropic ligands and di- or tripeptides were tested on the binding of [3H]-L(+)-2-amino-4-phosphonobutyric acid ([3H]-L-AP4) on rat mGlu4 receptor. For selected compounds, the functional activity was determined on this receptor using the guanosine-5'[gamma-35S]-thiotriphosphate [gamma-35S]-GTP binding assay. 2. Using the scintillation proximity assay, [3H]-L-AP4 saturation analysis gave binding parameters K(D) and Bmax values of 150 nM and 9.3 pmoles mg-1 protein, respectively. The specific binding was inhibited concentration-dependently by several mGlu receptor ligands, and their rank order of affinity was established. 3. Several peptides inhibited the [3H]-L-AP4 binding with the following rank order of potency: glutamate-glutamate>glutamate-glutamate-leucine=aspartate - glutamate>glutamate - glutamate-aspartate>lactoyl-glutamate>aspartate-aspartate. Aspartate-phenylalanine-methyl ester (aspartame) was inactive up to 1 mM and guanosine-5'-monophosphate and inosine-5'-monophosphate were inactive up to 100 micronM. 4. The [gamma-35S]-GTP binding functional assay was used to determine the agonist activities of the different compounds. For the rat mGlu4 agonists, L-AP4 and L-glutamate, the correlation between their occupancy and activation of the receptor was close to one. The peptides, Glu-Glu, Asp-Glu and Glu-Glu-Asp failed to stimulate the [gamma-35S]-GTP binding at receptor occupancy greater than 80% and Glu-Glu-Leu appeared to be a weak partial agonist. These peptides did not elicit a clear dose-dependent umami perception. However, Glu-lac showed a good correlation between its potency to stimulate the [gamma-35S]-GTP binding and its affinity for displacement of [3H]-L-AP4 binding. These data are in agreement with the peptide taste assessment in human subjects, which showed that the acid derivatives of glutamate had characteristics similar to umami.     

The levamisole receptor, a cholinergic receptor of the nematode Caenorhabditis elegans

We describe a glass fiber filter binding assay for the levamisole receptor, a putative acetylcholine receptor of the nematode Caenorhabditis elegans, and we show that receptor detected in vitro binds both levamisole derivatives and cholinergic agonists with the pharmacological specificity expected of the physiologically functional nematode receptor. The receptor is detected by the binding of tritiated meta-aminolevamisole ([3H]MAL, 27 Ci/mmol). In extracts of the wild-type nematode, there is a saturable, high affinity binding activity for [3H]MAL (Kd approximately 5-10 nM). Well fed wild-type worms contain as much as 3 fmol of high affinity binding activity per mg of extract protein (0.14 pmol/g of wet weight of worms) and dauer larvae, a special juvenile stage, contain as much as 15 fmol of activity per mg of protein. Specific binding activity per mg of protein is highest in larval stages and decreases severalfold in the adult worm. The rates of formation and dissociation of the [3H]MAL-receptor complex are relatively slow (dissociation half-life, 17 min), in agreement with physiological studies of levamisole on Ascaris muscle strips. Levamisole derivatives and cholinergic agonists have the same relative potencies in inhibiting [3H]MAL binding as they do in causing nematode muscle contraction. Vertebrate cholinergic antagonists do not inhibit [3H]MAL binding, but several antagonists (mecamylamine, alpha-bungarotoxin, and cobra venom) potentiate the binding of [3H]MAL and can be used to demonstrate more clearly the presence of a second, lower affinity binding activity whose ligand-binding affinity is also potentiated by these agents. Both high and low affinity wild-type binding components are missing in the extremely levamisole-resistant mutant unc-74(x19).     

Binding of [3H]apomorphine to an aporphine binding site as well as to dopamine sites in tissue from bovine caudate nucleus

Binding of the tritiated dopamine (DA) agonists, apomorphine (APO) and a dihydroxyaminotetralin (ADTN) to a membrane preparation from the caudate nucleus of calf brain was compared. Binding of [3H]dihydroxyaminotetralin at small (nM) concentrations followed simple, monophasic inhibition (over 80% at less than 500 nM) by concentrations of apomorphine between 50 pM and 1 mM. Inhibition of the binding of [3H]apomorphine by dihydroxyaminotetralin was more complex, and included in component with a low (microM) affinity for dihydroxyaminotetralin accounting for approx. 20% of total binding. The kinetics of binding of the ligands to high-affinity sites were virtually identical (apparent Kd = 0.81 nM; Bmax = 211 fmol/mg protein) and could not be distinguished by curve-fitting techniques adapted to analysis by microcomputer. In contrast, the binding of [3H]apomorphine with a "blank" defined by excess (10 microM) dihydroxyaminotetralin could be resolved into the same high-affinity component and a lower-affinity site (Kd = 124 nM; Bmax = 5740 fmol/mg). The pharmacology of the lower-affinity binding of [3H]apomorphine was evaluated by coincubating with 0.5 microM dihydroxyaminotetralin to "mask" high-affinity sites, and was compared to high-affinity binding of [3H]apomorphine and [3H]dihydroxyaminotetralin. The high-affinity binding was stereoselective for DA receptor agonists and antagonists. The pharmacology of the lower-affinity site resembled no known DA receptor type and showed highest affinities for aporphines but was not stereoselective and reacted weakly and nonspecifically with dihydroxyaminotetralin, DA, other catecholamines and neuroleptics. Thus, [3H]apomorphine, under certain conditions, may detect an aporphine binding site of uncertain pharmacological significance, as well as high-affinity DA agonist (D-3) sites.     

Effect of proteases and phospholipases on [3H]yohimbine binding to human platelet membranes

Trypsin and chymotrypsin inactivated specific [3H]yohimbine binding sites in the partially purified human platelet membranes in a concentration- and time-dependent fashion. The maximal inactivation (70-80% of control) was incomplete regardless of the concentrations of the proteases used or the incubation time. Scatchard analysis of the binding data showed that the total number of binding sites was reduced, but the affinity of the receptor to the ligand remained unaffected. Pretreatment of the membranes with unlabeled yohimbine or epinephrine produced a 20-30% increase in the specific [3H]yohimbine binding; however, this treatment offered only a slight protection (10-15%) against trypsin-induced inactivation of [3H]yohimbine binding. Pretreatment with phospholipase A2 produced a complete inhibition, while pretreatment with phospholipase C resulted in only a partial (70-80% of control) reduction in [3H]yohimbine binding. The inhibitory effects were not reversed when the specific binding of [3H]yohimbine was carried out with membranes treated with phospholipases and subsequently washed with defatted bovine serum albumin, suggesting that products released from phospholipolysis were not involved in the inhibition of [3H]yohimbine binding. These results suggest that the integrity of the receptor proteins and phospholipids is necessary for the specific binding of the ligand to the alpha 2-adrenoreceptor proteins of the human platelet membranes.     

Solubilization and characterization of pituitary thyrotropin-releasing hormone receptors

Thyrotropin-releasing hormone (TRH) receptors were solubilized from a rat pituitary tumor cell line, GH4C1, with digitonin. Convenient assays were developed based on the ability of hydroxylapatite and polyethyleneimine-soaked glass fiber filters to adsorb the solubilized [3H]methyl-TRH-receptor complex but not free [3H]methyl-TRH. The kinetics of [3H]methyl-TRH binding to solubilized receptors were extremely temperature dependent. Binding reached equilibrium at 10-20 nM [3H]methyl-TRH in 30 min at 23 degrees and 6 hr at 0 degree. The half-times for dissociation were less than 5 min at 23 degrees and 7.6 hr at 0 degree. Equilibrium binding experiments yielded linear Scatchard plots at 0 degree with Kd = 3 nM, whereas the Kd was greater than 20 nM at 23 degrees. A series of TRH congeners displaced [3H]methyl-TRH with the rank order reported for membrane receptors, N3-methyl-HisTRH greater than or equal to TRH greater than pGlu-N3-methyl-HisProNH(CH2)6NH2 greater than or equal to pGluHisProNH(CH2)6NH2 greater than pGluHisTyrNH2 much greater than TRH free acid. The antagonist chlordiazepoxide exhibited an IC50 of 10 microM. [3H]methyl-TRH binding to solubilized receptors displayed a broad pH optimum, from 6.5 to 7.5. The solubilized receptor could be obtained from cultured GH4C1 cells and in much larger quantities from GH4C1 tumors. Tumors from 12 rats yielded greater than 700 pmol of specific soluble TRH binding activity (1 g of protein). The solubilized receptor could be purified 10-20-fold by chromatography on wheat germ agglutinin columns and could be concentrated by adsorption on either DEAE-Sephadex or hydroxylapatite. The procedures outlined allow the solubilization of pituitary TRH receptors from a rich and abundant source, the rapid and reproducible assay of [3H]methyl-TRH binding, and substantial enrichment of receptor activity. These findings should be valuable for the purification and identification of the TRH receptor protein.     

Solubilization, characterization and partial purification of [3H]mepyramine-binding protein, a possible histamine H1 receptor, from rat liver membrane

[3H]Mepyramine binding protein, a possible subtype of histamine H1 receptors, was solubilized from rat liver membrane with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and Tween 60 as detergents and glycerol as an enhancer of solubilization. The optimal concentration of CHAPS was 10 mM and that of glycerol was 20% or more (v/v). The molecular weight of the [3H]mepyramine binding protein-detergent complex was determined to be 670K by Sepharose CL-4B gel filtration and 800K by sucrose density gradient sedimentation. By target size analysis, the molecular weights of both the membrane-bound and solubilized [3H]mepyramine binding protein were determined to be 162K. These values are similar to those of other well-characterized H1-receptor proteins, though slightly different. Simultaneous computerized analysis of the data obtained by [3H]mepyramine binding to the solubilized [3H]mepyramine binding protein indicated the presence of a single binding site with a KD value of 19.0 +/- 5.6 nM and a binding capacity (Bmax) of 6.6 +/- 2.1 pmole/mg protein. The Ki value of cold mepyramine for [3H]mepyramine binding to the solubilized receptor was 20 +/- 4 nM, whereas those of diphenhydramine, d-chlorpheniramine and triprolidine were all 2.9 +/- 0.8 microM, or about 150 times that of mepyramine. These data on the molecular and binding characteristics of the solubilized protein reported here suggest that there is a subtype of histamine H1 receptor in rat liver membrane. The solubilized preparation retained 90% and 75% of its [3H]mepyramine binding activity after storage at -80 degrees C and 4 degrees C, respectively, for 20 days. The solubilized [3H]mepyramine binding protein was purified 30-fold by Sepharose CL-4B gel filtration, Bio Gel HTP hydroxylapatite, Octyl Sepharose 4B and hydroxylapatite HPLC column chromatographies.     

3H]diazepam specific binding to rat cortex in vitro is enhanced by oleic, arachidonic and docosahexenoic acid isolated from pig brain

Substances were found in purified fractions from pig brain that enhanced the specific binding of [3H]diazepam to membranes from rat brain in vitro. These substances were identified as oleic acid, arachidonic acid and docosahexenoic acid. Oleic acid (10(-5) - 10(-4)M) increased the affinity for agonists binding to the benzodiazepine receptor, whereas the binding of antagonists to this receptor was only slightly enhanced. The number of [3H]muscimol binding sites was increased, whereas binding of [3H]SR 95331, a GABA receptor antagonist, was unchanged. The effect of oleic acid was additional to the GABA-induced enhancement of [3H]diazepam binding.