Glycoprotein nature of alpha 2-adrenergic receptors labeled with p-azido[3H] clonidine in calf retina membranes.

alpha 2-Adrenergic receptors in calf retina membranes can be specifically labeled with the tritiated agonist p-azido[3H]clonidine. Saturation binding in the dark occurs with high affinity (1.3 +/- 0.3 nM) to a single class of sites (1122 +/- 67 fmol/mg protein). Irradiation of the membrane-bound radioligand results in the labeling of a peptide band with an apparent size of 65 kDa and a characteristic pharmacological profile for an alpha 2-adrenergic receptor. The carbohydrate moieties of the alpha 2-receptor are characterized by lectin affinity chromatography and glycosidase treatment. The Nonidet P-40-solubilized, p-azido[3H]clonidine-labeled receptors are completely retained by Con A- as well as WGA-Sepharose columns. Neuraminidase, alpha-mannosidase and TFMS do not affect the electrophoretic mobility of the receptor on SDS-PAGE whereas endoglycosidase F reduces the apparent size to 45 kDa.

Characterization of alpha 2-adrenergic receptors of calf retina membranes by [3H]-rauwolscine and [3H]-RX 781094 binding.

Alpha 2-adrenergic receptors were identified in calf retina membranes by binding of the radiolabelled antagonists [3H]-RX 781094 and [3H]-rauwolscine. When 10 microM phentolamine was used to determine the non-specific binding, both radioligands labelled a single class of non-cooperative sites: Bmax = 1051 +/- 252 fmol/mg protein, Kd = 5.1 +/- 1.5 nM for [3H]-RX 78104 and Bmax = 1167 +/- 449 fmol/mg protein, Kd = 21.0 +/- 4.1 nM for [3H]-rauwolscine. Competition binding experiments showed the typical pharmacological potency order of alpha 2-adrenergic receptors, i.e. phentolamine greater than yohimbine greater than prazosin. Agonist competition binding curves revealed the presence of two receptor populations, having respectively high affinity (70% of the total receptor population) and low affinity for agonists, but with the same affinity for the antagonists. The high affinity sites could be converted into low affinity sites by guanine nucleotides. The non-specific binding of [3H]-RX 781094 was the same if 0.1 mM (-)-epinephrine was used instead of phentolamine. In contrast, the non-specific binding of [3H]-rauwolscine was markedly lower with (-)-epinephrine than with phentolamine. Under this condition, the Scatchard plot of [3H]-rauwolscine saturation binding was curvilinear, indicating the presence of low affinity sites for the radioligand in addition to alpha 2-adrenergic receptors. Competition binding experiments revealed that these low affinity sites were distinct from adrenergic receptors: the catecholamine agonists (-)- and (-)-epinephrine, (-)-norepinephrine, (-)-isoproterenol and dopamine competed with similar Ki values (microM range) whereas clonidine did not interact. Furthermore, these sites bound reserpine and the alpha 2-adrenergic antagonists yohimbine and rauwolscine but not phentolamine.

High affinity binding of 3H rauwolscine and 3H RX781094 to alpha 2 adrenergic receptors and non-stereoselective sites in human and rabbit brain cortex membranes.

The radiolabeled antagonists 3H RX 781094 and 3H rauwolscine bind with high affinity to alpha 2 adrenergic receptors as well as to non-receptor sites in human and rabbit brain cortex membranes. These non-receptor sites form an important contaminant of the specific binding when non-specific binding is determined in the presence of 10 microM phentolamine or more. While phentolamine is no suitable ligand to discriminate both sites, (-)-epinephrine displays a sufficient affinity ratio to separate radioligand binding to these sites. When 1 microM (-)-epinephrine is used for the determination of the non-specific binding, both radioligands bind specifically to alpha 2 receptors. Under these conditions, 3H rauwolscine and 3H RX 781094 bind to the same amount of non-cooperative sites; binding isotherms for human brain are Bmax = 113 +/- 15 fmol/mg protein and Kd = 22.8 +/- 4.2 nM for 3H RX781094 and Bmax = 110 +/- 17 fmol/mg protein and Kd = 4.7 +/- 2.5 nM for 3H rauwolscine. Competition binding experiments show, for both radioligands and in both species, the typical pharmacological potency order of alpha 2 adrenergic receptors, i.e. phentolamine greater than yohimbine greater than prazosin for the antagonists and UK 14304 greater than p-aminoclonidine greater than or equal to (-)-epinephrine greater than (+)-epinephrine greater than isoproterenol for the agonists. Whereas the alpha 2 receptor sites display high affinity and stereoselectivity towards (-)-epinephrine and (+)-epinephrine, the non-receptor sites bind both epinephrine isomers with equal low affinity. Specific binding of both radioligands to these sites can be determined when total binding is performed in the presence of 1 microM (-)-epinephrine and non-specific binding the presence of 1 mM phentolamine. 3H rauwolscine binding to the non-stereoselective sites can be displaced with high affinity by 5-HT, suggesting binding to a 5-HT1-receptor. The 3H RX 781094 binding displays low affinity for most alpha adrenergic ligands and do not correspond to beta adrenergic, dopaminergic or serotonergic receptors.

Conus venom interaction with ?(2)-adrenergic receptors in calf retina membranes.

?(2)-Adrenergic receptors were identified in calf retina membranes by the specific binding of the radiolabelled antagonist [(3)H]RX 781094. Crude venoms from various Conus species did not interact with the radioligand but were able to inhibit radioligand binding to the ?(2)-receptors with the following order of potency: C. planorbis (IC(50) = 2.1 ?g protein/ml) ? C. tessulatus (IC(50) = 2.7) >C. eburneus (IC(50) = 19) >C. textile (IC(50) = 54) >C. geographus (IC(50) = 130). Venom from 17 other species was less or not active at all. Venom competition binding curves were steep and not affected by GTP. In contrast, the ( ? )-epinephrine competition binding curve was shallow and underwent a rightward shift and steepening in the presence of GTP. The venom-?(2)-receptor interaction was completely inhibited by the calcium chelating reagent EGTA. These data suggest that the venom of certain Conus species contains peptide toxins which are capable of shielding the binding site of ?(2)-receptors in an antagonistic manner.

Autoradiographic distribution of alpha 2 adrenoceptors, NAIBS, and 5-HT1A receptors in human brain using [3H]idazoxan and [3H]rauwolscine.

The regional distribution of [3H]idazoxan and [3H]rauwolscine was studied autoradiographically in human brain. [3H]Idazoxan binds with high affinity to alpha 2 adrenoceptors as well as to non-adrenergic sites (NAIBS). [3H]Rauwolscine, besides binding to alpha 2 adrenoceptors, also binds to 5-HT1A receptors. Both radioligands labelled the same population of alpha 2 adrenoceptors, defined as the epinephrine-displaceable binding component. The highest densities of alpha 2 adrenoceptors occur in the leptomeninges, cerebral cortex and claustrum; lower densities were visualised in the basal ganglia, thalamus, pons, substantia nigra, cerebellum and medulla oblongata; no alpha 2 adrenoceptors were detected in amygdala and nucleus ruber. NAIBS were present in all the examined brain areas, with the highest densities found in the basal ganglia and substantia nigra. The finding that certain brain regions, such as the amygdala, contained NAIBS but no detectable alpha 2 adrenoceptors, suggests that the binding sites are independent from each other. The regional distribution of 5-HT1A receptors labelled by [3H]rauwolscine is in agreement with previous studies using [3H]8-OH-DPAT.

[3H]rauwolscine labels alpha 2-adrenoceptors and 5-HT1A receptors in human cerebral cortex.

[3H]Rauwolscine binds with high affinity to alpha 2-adrenoceptors (Kd = 4.8 +/- 1.3 nM, Bmax = 79 +/- 26 fmol/mg protein, micromolar affinity for 5-HT) as well as to 5-HT1-like receptors (Kd = 13 +/- 2.7 nM, Bmax = 147 +/- 11.4 fmol/mg protein, nanomolar affinity for 5-HT) in human brain cortex membranes. The Ki values of 11 serotonergic compounds for the latter receptors agreed closely with those previously reported for 5-HT1A sites but not with those for 5-HT1B, 5-HT1C and 5-HT1D sites.

[3H]SCH 23390 labels a novel 5-hydroxytryptamine binding site in human blood platelet membranes.

In human blood platelet membranes, 5-HT displaced the binding of the putative selective D-1 dopamine receptor antagonist [3H]SCH 23390 in a competitive manner with a Ki value of 5.7 +/- 0.8 nM, which was about 1000-fold lower than the Ki value for dopamine (Ki = 4400 +/- 150 nM). Thus the 'D-1 dopamine-like' site in human blood platelet membranes described previously corresponds to a 5-HT1-type site. [3H]SCH 23390 competition experiments with a number of serotonergic drugs disclosed a pharmacological profile that was distinct from the four 5-HT1 site subtypes reported previously. We therefore propose that this novel 5-HT site be designated the 5-HT1E site. Binding of [3H]SCH 23390 to 5-HT1-type sites could not be detected in several regions of the human brain. In some regions, however, 5-HT displaced part of the [3H]SCH 23390 binding with a K1 value of 320-380 nM. These sites correspond to 5-HT2 receptors.

Subtypes of adrenergic and dopaminergic receptors in bovine cerebral blood vessels.

Binding of the radiolabelled antagonists [3H]rauwolscine, [3H]SCH 23390 and (-)-[3H]dihydroalprenolol revealed the presence of alpha 2-adrenergic greater than DA1 dopaminergic greater than beta-adrenergic receptors in membrane preparations of calf basal cerebral arteries (basilar artery and circle of Willis) and pial vessels of the cerebral convexity. Computer-assisted analysis of ICI 118 551/(-)-[3H]dihydroalprenolol competition binding curves indicated the existence of beta 1- and beta 2-adrenergic receptor subtypes (beta 2/beta 1 ratio 7:3). No specific binding of [3H]prazosin (to alpha 1-adrenergic receptors) and [3H]spiroperidol (to DA2 dopaminergic receptors) was detected. Whereas DA1, beta 1- and beta 2-receptor densities were very similar in both blood vessel types, the alpha 2-receptor density was 3-fold higher in the pial vessels of the convexity. This suggests a functionally more important vasoconstrictor adrenergic control of the cerebral circulation in pial vessels of the convexity than in the arteries at the base of the brain.

Cyclic AMP content and invasive capacity of metastatic variants of the BW-5147 murine T-cell lymphoma.

The invasive behaviour of 8 lymphoma cell lines were tested by an in vitro monolayer invasion assay. The metastatic cell lines (TAM 4D1.2, DCH10Sp, TAM 4D6.2, E4 and BWLi) were more invasive than their non-metastatic counterparts (TAS 5C4, BWO and DCH 10). There was a positive correlation between their invasiveness and the PGE1- and forskolin stimulated cellular cAMP levels. Invasiveness and basal cAMP levels could not be correlated. Pretreatment with pertussis toxin (50 ng/ml) for 24 hours provoked did not significantly affect the basal and PGE1-stimulated cAMP levels in all cells. Yet, the toxin catalysed the ADP-ribosylation of 40 kDa components in all cells and provoked a significant increase in the invasiveness of non-metastatic cell lines and a decrease in the invasiveness of metastatic cell lines. These data suggest that the invasiveness of T-lymphoma cell lines might be controlled by a complex interplay between different signal transducing pathways in the membrane, rather than by the intracellular level of cAMP.

Identification of D1-like dopamine receptors on human blood platelets.

Dopamine is able to inhibit the epinephrine-induced aggregation of human blood platelets, but the mechanism of action has not been elucidated. In this study we report that membranes from human blood platelets possess high affinity, saturable and stereoselective binding sites for the D1 dopamine receptor antagonist (3H) SCH 23390. (3H) SCH 23390 appeared to label a single class of binding sites with a Bmax of 18.6 +/- 1.6 fmol/mg protein and a KD of 0.8 nM. The potencies of different dopaminergic antagonists and agonists in displacing (3H) SCH 23390 from blood platelet membranes were similar to those obtained for striatal membranes. Unlike the classically defined D1 receptors, e.g. those in striatum, the D1 receptor sites on platelets appeared not to be coupled to the adenylate cyclase system, hence the term "D1-like". The D1 agonist SKF 38393 was more potent than dopamine in inhibiting platelet aggregation induced by epinephrine, and the effects of dopamine and SKF 38393 were prevented by SCH 23390. These results suggest that the inhibitory action of dopamine on the epinephrine-induced platelet aggregation is mediated through these D1-like receptors.

Desensitization of alpha 2-adrenergic receptors in NG 108 15 cells by (-)-adrenaline and phorbol 12-myristate 13-acetate.

alpha 2-Adrenergic receptors on NG 108 15 cell membranes were identified by [3H]rauwolscine binding: Bmax. = 661 +/- 81 fmol/mg of protein, Kd = 6.9 +/- 2.5 nM (mean +/- S.E.M., n = 6). On intact cells, stimulation of these receptors by (-)-adrenaline inhibited the prostaglandin-E1-stimulated adenylate cyclase activity by about 60%. The effect of (-)-adrenaline was pertussis-toxin-sensitive, indicating the involvement of an inhibitory G protein. (-)-Adrenaline/[3H]rauwolscine competition-binding experiments revealed that only 50% of the alpha 2 receptors were coupled to G proteins (i.e. displayed high agonist affinity). Pre-treatment of the cells with 20 microM-(-)-adrenaline provoked homologous desensitization of the alpha 2 receptors. The alpha 2-adrenergic response decreased after a time lag of about 2 h, to reach a minimum after 12 h. The bradykinin and muscarinic responses were not affected. The alpha 2-receptor concentration decreased without time lag. The high-agonist-affinity sites disappeared more rapidly (t1/2 = 42 min) than did the low-affinity uncoupled sites (t1/2 approx. 20 h). In contrast, pertussis-toxin-mediated [32P]ADP-ribosylation of inhibitory G proteins was unaffected by the pre-treatment. Pretreatment of intact NG 108 15 cells with 1 microM-phorbol 12-myristate 13-acetate (PMA) provoked a rapid decrease of the alpha 2-adrenergic response. The effect was nearly complete after 40 min. PMA also decreased the bradykinin response, suggesting a heterologous desensitization process. The alpha 2-receptor concentration, the (-)-adrenaline competition-binding curves and the pertussis- and cholera-toxin-mediated [32P]ADP-ribosylation of their respective G proteins were not affected.

Tight agonist binding may prevent the correct interpretation of agonist competition binding curves for alpha 2-adrenergic receptors.

alpha 2-Adrenergic receptors in calf retina membranes can be specifically labeled with the tritiated antagonist 3H-RX 781094. Saturation binding occurs to a single class of noncooperative sites. The number of sites amounts to 1070 +/- 243 and 935 +/- 178 fmol/mg of protein, and the equilibrium dissociation constants equal 1.8 +/- 0.4 and 3.8 +/- 0.3 nM at 25 degrees and 37 degrees, respectively. Binding is rapid, equilibrium being reached within 5 min, and is reversible. At both temperatures, (-)-epinephrine competition binding curves are shallow in the presence of magnesium ions. The curves, obtained for incubation periods varying between 5 and 60 min, are superimposable at 37 degrees. Computer-assisted analysis indicates that approximately 75% of the receptors (RH sites) display high agonist affinity for (-)-epinephrine as well as for the other agonists tested: (-)-norepinephrine, clonidine, and UK 14304. However, the (-)-epinephrine competition curves display a time-dependent leftward shift at 25 degrees. This can be attributed to an increase in agonist affinity for the RH sites. Addition of 0.1 mM Gpp(NH)p causes a marked steepening and rightward shift of the curves, at both 25 and 37 degrees. These curves are superimposable for all of the incubation times tested. The nonequilibrium of agonist competition binding at 25 degrees can be attributed to slow dissociation of the agonist (i.e., tight binding) when the receptor is coupled to the regulatory component Ni. This dissociation rate can be measured by preincubation of the membranes with 10 microM (-)-epinephrine, followed by extensive washing and incubation with 3H-RX 781094 for increasing lengths of time. The first order rate of agonist dissociation (i.e., receptor recovery) is appreciably faster at 37 degrees than at 25 degrees: i.e., 0.029 min-1 and 0.0044 min-1, respectively. These findings are confirmed by kinetic experiments using the radiolabeled agonist 3H-UK 14304. Slow agonist dissociating kinetics may prevent the correct evaluation of the agonist binding parameters by computerized analysis of competition binding curves when the incubation time is too short, especially at low temperature.

D2 dopamine receptors in calf globus pallidus: agonist high- and low-affinity sites not regulated by guanine nucleotide.

By use of the radioligand [3H]spiroperidol, D2 3,4-dihydroxyphenylethylamine (dopamine) receptor binding characteristics were studied in calf globus pallidus and compared with those of neostriatum. Antagonist competition curves were monophasic and revealed similar affinities for neostriatum and globus pallidus, suggesting a uniform receptor population with one affinity state for antagonists. In both regions, competition curves with the agonist dopamine were biphasic, distinguishing a high- and low-agonist-affinity state. In neostriatum and globus pallidus, respectively, 45% and 19% of [3H]spiroperidol binding was displaced with high affinity and the remainder with low affinity. In neostriatum, the addition of 0.4 mM GTP resulted in a partial conversion from high- to low-affinity state with a remaining high-affinity component of 15%. In globus pallidus, dopamine binding was not altered by GTP. The capability of GTP to modulate agonist binding to D2 receptors appears to be dependent on their neuroanatomical localization.

Immunochemical studies of the muscarinic acetylcholine receptor.

Muscarinic receptors have been purified from calf forebrain plasma cell membranes by affinity chromatography on a dexetimide-agarose gel. SDS-PAGE analysis showed a single 70 kDa band. Monoclonal antibodies have been prepared against these affinity purified 70 kDa protein(s). One antibody, M-35, immunoprecipitated up to 80% of digitonin-solubilized muscarinic receptors. M-35 had agonist-like effects on guinea-pig myometrium: it increased the intracellular cyclic GMP content, decreased prostaglandin-induced cyclic AMP accumulation and caused muscle contractions. The two first effects were inhibited by atropine. M-35 was used to visualize muscarinic receptors at the surface of human fibroblastic cells. In the particular cell line used, the receptors have a low affinity for pirenzepine, were negatively coupled to adenylate cyclase and mediated increase in the phosphatidyl-inositol breakdown.

A human embryonic lung fibroblast with a high density of muscarinic acetylcholine receptors.

Binding studies with the radiolabeled muscarinic antagonists dexetimide, quinuclidinyl benzilate and N-methylscopolamine showed that the human embryonic lung fibroblast CCL137 possesses approximately 2 X 10(5) muscarinic receptors/cell, i.e. 2.1 pmol/mg membrane protein. These receptors showed a marked stereoselectivity towards dexetimide and levetimide and only low affinity for another antagonist, pirenzepine. The muscarinic agonist carbamylcholine inhibited forskolin-stimulated adenylate cyclase and induced phosphatidylinositide turnover in the intact cells. Both effects were inhibited by the muscarinic antagonist atropine. Affinity labeling with tritiated propylbenzylcholine mustard revealed a protein of 72 kDa. Finally, down-regulation of the membrane receptors following prolonged treatment with the agonist carbamylcholine was assessed by means of the hydrophilic antagonist N-methylscopolamine.