Agonist binding to rat brain somatostatin receptors alters the interaction of the receptors with guanine nucleotide-binding regulatory proteins.

To investigate the interaction of guanine nucleotide-binding regulatory proteins (G proteins) with the agonist-bound brain somatostatin (SRIF) receptor, rat brain SRIF receptor/G protein complexes were solubilized and immunoprecipitated with peptide-directed antisera selective for the different subtypes of G protein alpha subunits (G alpha). In the absence of agonist, solubilized SRIF receptor/G proteins complexes could be immunoprecipitated by antiserum 8730, which is directed against the carboxyl-terminal region of Gi alpha and recognizes all Gi alpha subtypes, and by antiserum 3646, which selectively interacts with internal regions of Gi alpha 1. In contrast, antiserum 1521, which is directed against an internal region of Gi alpha 2, and antiserum 9072, which is directed against the carboxyl-terminal region of Go alpha, did not immunoprecipitate the SRIF receptor. After the binding of agonist to solubilized SRIF receptors, antisera 9072 and 1521, as well as antisera 8730 and 3646, were able to immunoprecipitate the agonist-bound SRIF receptor/G protein complexes, indicating that agonist interaction with SRIF receptors maintained receptor association with Gi alpha 1 and promoted receptor association with Go alpha and, to a lesser extent, Gi alpha 2. Antiserum 1518, which is directed against Gi alpha 3, uncoupled SRIF receptors from Gi alpha and did not immunoprecipitate the agonist-bound or agonist-free brain SRIF receptor. These findings indicate that differences exist in the interaction of the agonist-free and agonist-bound SRIF receptors with G proteins. The binding of agonists to SRIF receptors promotes the association of the receptor with Go alpha and, to a lesser extent, Gi alpha 2, indicating that these G proteins, along with Gi alpha 1 and Gi alpha 3, may be involved in coupling SRIF receptors to cellular effector systems.     

Tissue-dependent association of muscarinic acetylcholine receptors with guanine nucleotide-binding regulatory proteins.

The muscarinic acetylcholine receptors in heart and cerebellum form a stable association with guanine nucleotide-binding regulatory proteins (G proteins) in the presence of receptor agonists. This has been confirmed by purification of the muscarinic receptor-G protein complexes using an immunoprecipitation protocol. The isolated complexes were subjected to Western blotting to identify the G protein subunits present in the complexes. At saturating concentrations of carbachol, the muscarinic receptors in atrial membranes co-purified exclusively with Go, whereas in cerebellar and ventricular membranes an association with both Gi and Go was demonstrated. Further characterization of the G protein subunits allowed identification of the species of Gi alpha subunits present in the complexes of muscarinic receptor and G protein; in ventricle Gi alpha 2 was the only subtype present, whereas in cerebellum both Gi alpha 1 and Gi alpha 2 were present. These results demonstrate that a single muscarinic receptor subtype, depending on the tissue studied, is capable of interacting with more than one G protein subtype. The concentrations of agonist required to promote receptor-G protein association in atrial and ventricular membranes correlated with the high affinity component of receptor occupancy by agonist, as measured in equilibrium binding assays. Furthermore, incubation of cardiac membranes with saturating concentrations of pilocarpine or McN A343 resulted in reduced amounts of receptor-G protein complexes, compared with carbachol. Overall, our results suggest that the specificity of cellular effects of muscarinic agonists may relate, in part, to the selective interaction of receptor with G proteins.     

Interaction of beta-adrenergic receptors with the inhibitory guanine nucleotide-binding protein of adenylate cyclase in membranes prepared from cyc- S49 lymphoma cells

beta-Adrenergic receptors on membranes prepared from L6 myoblasts, wild-type S49 lymphoma cells, and an adenylate cyclase-deficient variant (cyc-) of S49 lymphoma cells bind the agonist [3H]hydroxybenzylisoproterenol ([3H]HBI) with high affinity. In each case the agonist [3H]HBI is associated with a larger complex than is the antagonist [125I]iodopindolol, and the binding of [3H]HBI can be inhibited by GTP. These observations suggest that there is an agonist-dependent association of the receptor with a guanine nucleotide-binding protein. The goal of the present experiments was to investigate the possibility that an interaction of beta-adrenergic receptors with the inhibitory guanine nucleotide-binding protein of adenylate cyclase was responsible for these observations. Treatment of S49 cells with pertussis toxin decreased the extent of pertussis toxin-catalyzed [32P]ADP-ribosylation of a 41,000-dalton protein, measured in vitro, and decreased the inhibition of adenylate cyclase activity observed in the presence of somatostatin or analogues of GTP. Isoproterenol-stimulated adenylate cyclase activity was potentiated following treatment of wild-type S49 cells and L6 myoblasts with pertussis toxin. Although the ability of receptors on membranes prepared from L6 myoblasts to bind the agonist [3H]HBI was not affected by treatment of cells with pertussis toxin, treatment of cyc- S49 cells with pertussis toxin markedly decreased the ability of receptors to bind [3H]HBI. The observed inhibition of the binding of the agonist [3H]HBI to beta-adrenergic receptors on membranes prepared from cyc- S49 cells after treatment with pertussis toxin could be explained by an interaction between beta-adrenergic receptors and the inhibitory guanine nucleotide-binding protein. Such an interaction may represent a mechanism through which stimulation of the activity of adenylate cyclase by beta-adrenergic receptors can be regulated or through which beta-adrenergic receptors can affect the activity of cyclic AMP-independent cellular processes.     

Differential coupling of mu-competitive and mu-noncompetitive delta opiate receptors to guanine nucleotide binding proteins in rat brain membranes

The effects of fentanyl isothiocyanate (FIT) and pertussis toxin on the binding of [3H]D-Ala2, D-Leu5-enkephalin ([3H]DADLE) to rat brain membranes were compared. The site of action of pertussis toxin was confirmed by the labeling of a 41,000 dalton protein in the presence of [alpha-32P]NAD. Both reagents produced inhibition of [3H]DADLE binding when binding was assayed in 10 mM Tris-HCl buffer alone. FIT inhibited binding 91% whereas pertussis toxin treatment resulted in 27% inhibition. However, when binding was assayed in 10 mM Tris-HCl containing SMG (100 mM NaCl, 3 mM manganese acetate, and 2 microM guanosine triphosphate), inhibition due to both reagents was attenuated markedly: 66% for FIT and 5% for toxin. In addition, both reagents markedly potentiated enhancement of binding by SMG. Thus, the effects of FIT and pertussis toxin on [3H]DADLE binding were qualitatively similar. These results suggest that FIT and pertussis toxin affect binding of [3H]DADLE to the same population of delta receptors. This was further supported by the observation that treatment of membranes with FIT prior to pertussis toxin treatment blocked the effect of toxin on [3H]DADLE binding. FIT selectively eliminates the SMG-insensitive, mu-competitive [3H]DADLE binding site [Rothman et al., Neuropeptides 4, 201 (1984); Rothman et al., Molec. Pharmac. 27, 399 (1985)]. These results indicate that this site is coupled to G protein substrates for pertussis toxin and that it mediates the inhibitory effects of delta ligands on adenylate cyclase. The FIT-insensitive, SMG-sensitive mu-noncompetitive [3H]DADLE site appears not to be coupled to G protein substrates for pertussis toxin and may mediate some other biochemical effects of delta ligands.     

Selective interaction of beta 2- and alpha 2-adrenergic receptors with stimulatory and inhibitory guanine nucleotide-binding proteins.

In Chinese hamster ovary cells expressing recombinant beta 2-adrenergic receptors, isoproterenol enhanced cholera toxin-catalyzed ADP-ribosylation of the large form of G5 alpha. The effect was stereoselectively blocked by the enantiomers of propranolol, indicating receptor mediation. The ADP-ribosylated form of Gs alpha-subunit was resolved into a triplet in gradient gels. beta 2-Adrenergic receptors increased both the labelling and the apparent mass of the slower migrating forms of large Gs alpha, as determined by autoradiography and immunoblotting, suggesting that Gs alpha, can incorporate more than one ADP-ribose per molecule. In cells coexpressing similar amounts of beta 2-adrenergic, alpha 2-adrenergic, and m1 muscarinic receptors, beta 2 receptors stimulated the ADP-ribosylation of only large Gs and alpha 2 receptors that of only Gi; muscarinic receptors had no apparent effect. Thus, in native membranes there appears to be a selectivity for the interaction between adrenergic receptor subtypes and Gs alpha or Gi alpha subunits.     

Agonist and antagonist binding to alpha 2-adrenergic receptors in purified membranes from human platelets. Implications of receptor-inhibitory nucleotide-binding protein stoichiometry

The agonist- and antagonist-binding properties of the alpha 2-adrenergic receptor in a purified plasma membrane preparation from human platelets were determined both by direct binding of radiolabeled ligands and by competition with the labeled alpha 2-antagonist, [3H] yohimbine. Binding of [3H]yohimbine was characterized by a single high affinity binding site (Kd = 6.2 +/- 1.4 nM, Bmax = 507 +/- 53 fmol/mg). In direct binding studies, the imidazoline full alpha 2-agonist, [3H]-5-bromo-6-N(2-4,5-dihydroimidazolyl)quinoxaline ([3H] UK 14,304), bound to only one quantifiable high affinity site (Kd = 0.88 +/- 0.17 nM), representing 65 +/- 6% of the number of [3H]yohimbine sites. Binding of the partial agonist [3H]-p-aminoclonidine (PAC) showed nonlinear Scatchard plots. Analysis according to a model of multiple independent binding sites showed the data to be consistent with two sites (Kd1 = 0.62 +/- 0.18 nM and Kd2 = 7.9 +/- 1.4 nM). The high affinity site corresponded to 15 +/- 6% and the low affinity site corresponded to 39 +/- 6% of the number of [3H]yohimbine sites. Competition for binding of the alpha 2-antagonist, [3H]yohimbine, with nonradiolabeled ligands revealed a single affinity for yohimbine. In contrast, competition for [3H]yohimbine binding by the full agonist UK 14,304 and epinephrine is best fit by a model with two independent binding sites. The partial agonist PAC was best characterized by a model with three distinct binding sites. The full agonists UK 14,304 and epinephrine inhibited adenylate cyclase approximately 30%, whereas PAC produced only 12% inhibition. The inhibitory guanine nucleotide-binding protein (Ni) with Mr 40,700 was the sole pertussis toxin substrate in the purified membranes. It was quantitated by pertussis toxin-catalyzed [32P]ADP ribosylation in cholate extracts. There is a 20- to 100-fold excess of Ni over alpha 2-adrenergic receptors. Comparisons made between the experimental data for agonist binding and theoretical predictions of the simple ternary complex model suggest that there is compartmentalization of Ni and/or that the alpha 2 receptors are heterogeneous.     

Fractionation of the beta subunit common to guanine nucleotide-binding regulatory proteins with the cytoskeleton

Gs and Gi are guanine nucleotide-binding proteins that mediate the stimulation and inhibition, respectively, of adenylate cyclase. The extent to which the beta subunit common to these proteins may be associated with the cytoskeleton of S49 mouse lymphoma cells was assessed by procedures of differential detergent extraction and immunotransfer blotting. Treatment of cells with 1% Triton X-100 results in nearly quantitative extraction of cellular protein, phospholipid, tubulin, and the catalytic component of adenylate cyclase. Approximately 65% of the beta subunit is refractory to extraction. This population of the beta subunit, along with a population of actin presumed to originate from actin filaments, is subsequently solubilized with hypotonic medium containing 0.5% sodium deoxycholate and 1% Tween-40. The pattern of distribution of the beta subunit among sequential detergent extracts is corroborated by that of generated immunoreactive tryptic fragments. These results are consistent with the interaction of guanine nucleotide-binding proteins with the cytoskeleton.     

Cerebral muscarinic acetylcholine receptors interact with three kinds of GTP-binding proteins in a reconstitution system of purified components

A new GTP-binding protein, which serves as a substrate for pertussis toxin, was prepared from porcine brain. The new G protein was separated from other GTP-binding proteins, Gi and Go, by an anion-exchange column chromatography. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the alpha subunit of the new G protein was between those of alpha subunits of Gi and Go. Evidence that the alpha subunit is not a proteolytic fragment of the alpha subunit is not a proteolytic fragment of the alpha subunit of Gi or Go was provided by experiments involving partial hydrolysis of these G proteins with thermolysin and their interaction with an antibody raised against the amino terminal peptide of the alpha subunit of Gi. In addition, the gamma subunit of the new G protein was indicated to be different from the gamma subunits of Gi and Go, because the latter were found to be phosphorylated by protein kinase C but the former was not. GTP-sensitive high affinity binding of muscarinic receptors with acetylcholine was observed when muscarinic receptors purified from porcine cerebrum were reconstituted in phospholipid vesicles with the new G protein as well as with Gi or Go. The proportion of the high affinity sites increased with the concentrations of the G proteins, the potency of the new G protein being similar to that of Gi but a little lower than that of Go. This GTP-sensitive high affinity binding was not observed when each G protein was pretreated with pertussis toxin and then reconstituted with muscarinic receptors. Acetylcholine accelerated the dissociation of [3H]GDP from the new G protein as well as from Gi and Go, which were reconstituted with muscarinic receptors. These results indicate that muscarinic receptors interact with at least the above three kinds of G proteins, in a pertussis toxin-sensitive manner.     

Influence of halothane on the interactions of serotonin1A and adenosine A1 receptors with G proteins in rat brain membranes.

The influence of halothane on the interactions of 5-HT1A and adenosine A1 receptors with G proteins was determined by monitoring the guanine nucleotide sensitivity of agonist binding to these receptors. Halothane inhibited the binding of radiolabeled agonists to 5-HT1A and adenosine A1 receptors by up to 30%, but only at concentrations considerably greater than those necessary for the maintenance of the anesthetic state. The sensitivity of high-affinity agonist binding to a guanine nucleotide (guanylyl-5'-imidodiphosphate) was not affected by halothane, indicating no disruption of receptor-G protein coupling. Thus, it appears that the ability of halothane to disrupt receptor-mediated signal transduction by interference with receptor-G protein interactions is receptor specific.     

Interactions of beta-adrenergic receptors with a membrane protein other than the stimulatory guanine nucleotide-binding protein

Beta-adrenergic receptors on membranes prepared from rat lung, wild-type S49 lymphoma cells, and the adenylate cyclase-deficient variant of S49 lymphoma cells (cyc-) bind the agonist [3H]hydroxybenzylisoproterenol ([3H]HBI) with high affinity and this binding of [3H]HBI can be inhibited by GTP. Membranes prepared from these tissues were incubated with the agonist [3H]HBI or the antagonist [125I]iodopindolol ([125I]IPIN), labeled receptors were solubilized with digitonin, and the apparent molecular sizes of the ligand-bound receptor complexes were determined by high-performance size-exclusion chromatography. Results with all three tissues demonstrated that receptors labeled with [125I]IPIN were retained by the size-exclusion columns longer than receptors labeled with [3H]HBI. Thus, the apparent molecular size of soluble beta-adrenergic receptors from rat lung, wild-type S49 cells, and cyc- S49 cells was larger when receptors were occupied with an agonist rather than an antagonist. The results suggest that receptors, including those on cyc- S49 cells, interact with a membrane protein, presumably a guanine nucleotide-binding protein. Since cyc- S49 cells do not contain a functional stimulatory guanine nucleotide-binding protein, but do contain a functional inhibitory guanine nucleotide-binding protein, an interaction between beta-adrenergic receptors and the inhibitory guanine nucleotide-binding protein may be responsible for the apparent increase in the molecular size of the receptor after occupation of the receptor with an agonist.     

Binding of bovine factor VIII-coated colloidal gold particles to receptors on platelet membranes

Bovine factor VIII/platelet aggregating factor was adsorbed into gold granules and the protein-gold complex added to either formalin-fixed or fresh washed human platelets. Following aggregation, binding of gold granules to the platelets was measured by monitoring the optical density of colloidal gold remaining in the supernatant. Scatchard analysis of binding data indicated that multiple classes of binding sites were present. The number of high affinity binding sites per formalin-fixed platelet depended on the concentration of ristocetin: 420 gold granules were calculated to bind at 1.4 mg/ml of ristocetin, 610 at 0.6 mg/ml of ristocetin and 875 when no ristocetin was added. Fresh washed platelets bound 1350 granules per cell in the absence of ristocetin. We conclude that during platelet aggregation, induced by bovine factor VIII, the binding sites on the platelet surface are only partially occupied.     

Affinity chromatography of A1 adenosine receptors of rat brain membranes

The A1 adenosine receptor of rat brain membranes has been solubilized with digitonin and purified approximately 150-fold by affinity chromatography. The digitonin-solubilized receptor, which can be labeled with 8-cyclopentyl-1,3-[3H]dipropylxanthine([3H]DPCPX), was adsorbed on xanthine amine congener (XAC)-linked agarose. The interaction of the solubilized receptor activity with the affinity gel was biospecific. Adenosine agents blocked adsorption of solubilized receptor activity to the XAC-agarose with the appropriate A1 adenosine selectivity. For agonists, 8-cyclopentyladenosine greater than (R)-phenylisopropyladenosine greater than CV-1808, whereas, for antagonists, 8-cyclopentyltheophylline (CPT) greater than XAC greater than isobutylmethylxanthine = theophylline. The same A1 adenosine receptor specificity was observed for elution of [3H]DPCPX binding activity from the gel. XAC-agarose adsorbed 65-80% of the solubilized [3H]DPCPX binding activity and, after the gel was washed, 30-40% of the adsorbed activity could be eluted with 100 microM CPT, with specific binding activity of approximately 60 pmol/mg of protein. The order of potency of adenosine agonists [8-cyclopentyladenosine greater than (R)-phenylisopropyladenosine greater than 5'-N-ethylcarboxamidoadenosine greater than (S)-phenylisopropyladenosine] and antagonists (DPCPX greater than XAC greater than CPT greater than isobutylmethylxanthine) with the affinity-purified preparation was found to be similar to that of the solubilized adenosine A1 receptor. This affinity chromatography procedure should prove to be valuable in the isolation and molecular characterization of A1 adenosine receptors.     

Interaction of purines and related compounds with photoaffinity-labelled benzodiazepine receptors in rat brain membranes

The interaction of purine-receptor agonists and antagonists with [3H]Ro15-1788 binding sites in rat brain membranes was examined before and after UV-photoaffinity labelling of a proportion of the sites with flunitrazepam. Whereas photoaffinity labelling of the receptors reduced benzodiazepine agonist affinity but not benzodiazepine antagonist affinity, the IC50S of adenosine-receptor agonists, partial agonists and antagonists were unaltered by the conformational changes in the benzodiazepine receptors which are thought to be induced by the photolabelling process. The affinity of dipyridamole, a potent adenosine uptake blocker and potent displacer of [3H]diazepam binding, was drastically reduced by photolabelling.     

Theophylline-induced upregulation of A1-adenosine receptors associated with reduced sensitivity to convulsants

Chronic administration of theophylline (50 mg/kg twice daily for 14 consecutive days) significantly increased the specific binding of [3H]CHA in membranes of the cerebral cortex and cerebellum of the rat, but not in membranes derived from the hippocampus or diencephalon. To characterize further the upregulation of A1 = adenosine receptors induced by theophylline, saturation analysis with [3H]CHA was performed in membranes of the cerebral cortex and cerebellum. In both saline- and theophylline-treated cortical membranes the binding isotherms for [3H]CHA could be resolved into receptor affinity states having respectively high (KH) and low (KL) affinity for [3H]CHA. The high and low affinity dissociation constants obtained from theophylline-exposed membranes of the cerebral cortex were 1.14 nM and 25.2 nM and did not differ significantly from the corresponding values in saline-treated animals. Chronic exposure to theophylline did, however, produce significant increases in the densities of both the high and low affinity forms of A1-adenosine receptors in the cerebral cortex. Qualitatively and quantitatively similar results were observed in cerebellar membranes. These results suggest that chronic exposure to theophylline increases the density of A1-receptor in the cerebral cortex and cerebellum with no concomitant changes in the ability of [3H]CHA to distinguish separate agonist affinity states of the receptor. The physiological significance of theophylline-induced upregulation was assessed by determining seizure thresholds for convulsants in rats treated chronically with saline or theophylline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of phomopsin A and related compounds with purified sheep brain tubulin

Phomopsins comprise a family of peptide mycotoxins containing a 13-membered ring formed by an ether bridge, produced by the fungus Phomopsis leptostromiformis, the causal agent in lupin poisoning (lupinosis). The biochemical actions of two naturally occurring phomopsins, phomopsin A and B, and the chemical derivatives, phomopsinamine A and octahydrophomopsin A, on purified sheep brain tubulin were investigated. All analogues were potent microtubule inhibitors, blocking the polymerization of tubulin at concentrations of less than 1 microM. They inhibited [3H]vinblastine binding to tubulin and, in common with vinblastine and its competitive inhibitor maytansine, enhanced the binding of [3H]colchicine to tubulin. It is postulated that phomopsin A and its analogues exert their action on tubulin by interaction at or near the vinblastine binding site. Two possible mechanisms for the interaction between vinblastine or phomopsins and colchicine binding to tubulin are proposed.     

Evidence for agonist-induced interaction of angiotensin receptor with a guanine nucleotide-binding protein in bovine adrenal zona glomerulosa

Previous studies have documented that high affinity binding of [125I]angiotensin II to adrenal cortex receptors was modulated by guanine nucleotides. Since in other receptor systems, similar properties of hormone-receptor interactions were shown to be specific for agonists, we studied the differential binding characteristics of agonists and antagonists to this receptor using a new radiolabeled antagonist [125I] [Sar1,Ile8] angiotensin II. Receptor saturation studies indicate that the antagonist is binding to a homogeneous population of sites with a Kd of 0.6-2.0 nM and with a receptor density around 1 pmol/mg of protein. Competition curves using unlabeled antagonists are characterized by a slope factor of 1 and a single Kd of 1-3 nM. Addition of guanylylimidodiphosphate to the assay is absolutely without effect on radiolabeled antagonist binding. In contrast, competition curves using the full agonists angiotensin II, [Sar1]angiotensin II, angiotensin III, and [des-Arg]angiotensin III display slope factors of 0.79, 0.87, 0.70, and 0.84, respectively. These curves can be explained by two apparent forms of the receptor having high and low affinity for the agonist. The higher affinity form associated with these four agonists is characterized by a Kd of 1.2 nM, 0.25 nM, 0.8 nM, and 3 microM, and corresponds to 60, 56, 42, and 25% of angiotensin II-binding sites, respectively. The other form displays 13- to 33-fold lower affinity. Addition of guanine nucleotide to the assay results in a 2-4-fold shift to the right and a steepening (slope factor 0.9-1.0) of agonist competition curves. Angiotensin II receptors, occupied by the full agonist [131I] [Sar1] angiotensin II or by the antagonist [125I] [Sar1, Ile8]angiotensin II, were then solubilized with the nonionic detergent octylglucoside. Dissociation of the agonist [131I] [Sar1] angiotensin II from solubilized receptors is enhanced by guanylylimidodiphosphate or sodium acetate, while dissociation of the antagonist [125I] [Sar1, Ile8]angiotensin II displays little sensitivity towards guanine nucleotides or increased ionic strength. Inclusion of bile salts in the solubilization medium preferentially destabilizes receptor-bound agonist, presumably by interfering with protein-protein interactions required for high affinity agonist binding. Separation of radiolabeled agonist and antagonist-occupied solubilized receptor complexes by steric exclusion high performance liquid chromatography reveals that the agonist-occupied receptor complex behaves as a larger protein than the antagonist-occupied receptor complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Interaction of amiloride and its analogues with adenosine A1 receptors in calf brain

Amiloride, a potassium sparing diuretic, is known to interact with a number of ion transport systems, receptors and enzymes. Here, we report on the interaction between this drug and the adenosine A1 receptor as present in calf brain membranes. Adenosine A1 receptors are characterized by a subnanomolar affinity for the antagonists [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) and the agonist [3H]N6-R-1-phenyl-2-propyladenosine ([3H]PIA). Amiloride displaces both agonist and antagonist binding with a Ki value in the low micromolar range. This inhibition is counteracted by NaCl and protons, in contrast to the binding of [3H]PIA and [3H]DPCPX. The results suggest that amiloride interacts with the adenosine A1 receptor at a site distinct from the ligand binding site. In order to elucidate the role of one of the ion transport systems known to be inhibited by amiloride, eight amiloride analogues with different sensitivities for these systems were tested. The potency and order of potency of these compounds towards adenosine A1 receptors excludes the involvement of the epithelial Na+ channel, Na+/H+ exchanger or Na+/Ca2+ exchanger.