Ligand binding properties of putative beta 3-adrenoceptors compared in brown adipose tissue and in skeletal muscle membranes.

1. The beta-adrenoceptor population was characterized in membrane preparations from rat brown adipose tissue (BAT) and from soleus muscle by use of the radioligand [125I]-iodocyanopindolol ([125I]-ICYP). In addition, atypical binding sites for [125I]-ICYP found in both tissues were examined, and the relationship between these sites and the putative rat beta 3-adrenoceptor is discussed. 2. It was established that BAT membranes host a mixed population of beta 1- and beta 2-adrenoceptors. Of these two sites, 55% showed a high affinity for the beta 1-selective ligand CGP 20712A (pK 8.5), and 45% showed a high affinity for the beta 2-selective antagonist ICI 118551 (pK 8.6). Soleus muscle membranes were found to host a population of beta 2-adrenoceptors, characterized by a high affinity for ICI 118551 (pK 9.1), but beta 1-adrenoceptors could not be detected in this preparation. 5-Hydroxytryptamine receptors were not detected in either preparation. 3. In addition to beta 1- and beta 2-adrenoceptors, atypical binding sites were identified in both tissues using high concentrations of radioligand (0.5-0.6 nM) and in the presence of 1 microM (-)-propranolol. The atypical sites were abundant, representing 80 and 81% of the total [125I]-ICYP binding sites in BAT and soleus muscle respectively. When the pK values for 11 ligands were compared, the correlation coefficient for atypical sites in BAT and soleus muscle was 0.94.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-adrenergic receptors in guinea-pig liver plasma membranes and thermal lability of [3H]dihydroalprenolol binding sites

beta-Adrenergic receptors in guinea-pig liver plasma membranes were characterized by radioligand binding, using l-[3H]dihydroalprenolol ([3H]DHA), l-3-[125I]iodocyanopindolol ([125I]CYP) and dl-[3H]4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole-2- one hydrochloride [( 3H]CGP-12177). The binding of both [125I]CYP and [3H]CGP-12177 to membranes exhibited high affinity (Kd = 3.5 +/- 0.2 pM for [125I]CYP and 0.75 +/- 0.10 nM for [3H]CGP-12177) and stereospecificity; the maximal binding sites were 130 +/- 15 and 137 +/- 8 fmoles/mg protein respectively. Catecholaminergic agonists competed for these binding sites in the order l-isoproterenol greater than l-epinephrine greater than l-norepinephrine, which is typical for beta 2-adrenergic receptors. The binding data are supported by parallel experiments on adenylate cyclase activation by catecholamines, and on antagonism of this activation by beta 1- and beta 2-selective blockers. The binding of [3H]DHA was excessive (Bmax = 21.4 pmoles/mg protein), exhibited low affinity (Kd = 34.6 nM), and lacked stereospecificity. When liver membranes were incubated at 50 degrees for 40 min in the presence of an agonist, l-isoproterenol, the binding of [3H]DHA to the heat-treated membranes exhibited high affinity (Kd = 1.07 +/- 0.17 nM) and the Bmax was reduced to 139 +/- 22 fmoles/mg protein. In such membranes, as opposed to native membranes, stereospecificity was evident and catecholaminergic agonists competed for the binding sites in the order typical for beta 2-adrenergic receptors. However, agonist competition of the binding to the heat-treated membranes could not be modulated by guanine nucleotides, indicating a loss of communication between the receptor and the guanine nucleotide regulatory protein.     

Characterization of alpha 1- and alpha 2-adrenoceptors directly associated with basolateral membranes from rat kidney proximal tubules

We have used 2-(beta-(3-125iodo-4-hydroxyphenyl)-ethylaminoethyl)-tetr alo ne ([125I]HEAT or BE2254), an alpha 1-selective antagonist, and [3H]yohimbine, an alpha 2-selective antagonist, to demonstrate and characterize binding sites in basolateral membranes from rat kidney cortex. Parathyroid hormone (PTH) stimulated the adenylate cyclase activity of the basolateral membranes, whereas thyrocalcitonin, arginine vasopressin (AVP) and isoproterenol did not. Therefore, the basolateral membranes were probably derived from the proximal tubules. The specific binding of [125I]HEAT and [3H]yohimbine to basolateral membranes was rapid, reversible, saturable and of high affinity. The maximum densities of alpha 1- and alpha 2-receptors were 364 and 1130 fmoles/mg protein, indicating that the ratio of alpha 1- to alpha 2-adrenoceptors was about 1:3. The specific binding of [125I]HEAT and [3H]yohimbine to the basolateral membranes was displaced by various adrenergic agents in a manner that suggests that the labeled sites probably represent alpha 1- and alpha 2-adrenoceptors respectively. These results suggest that the binding sites of [125I]HEAT and [3H]yohimbine, which appear to be alpha 1- and alpha 2-adrenoceptors, exist in the basolateral membranes of the proximal tubules.     

Characterization of peripheral-type benzodiazepine recognition sites in the rat spinal cord

The binding of [3H]Ro 5-4864 to membranes prepared from spinal cord of the adult rat has been studied in vitro. At 4 degrees C, the binding of [3H]Ro 5-4864 reached equilibrium by 120 min, and was rapidly reversible (dissociation t0.5 = 21 min). The [3H]Ro 5-4864 bound with a high affinity (Kd approximately equal to 3 nM) to a single, saturable population of binding sites (Bmax = 27 pmol/g tissue wet weight). Activation of receptors for gamma-aminobutyric acid with 10 microM muscimol did not alter these binding parameters. The drugs Ro 5-4864, diazepam and flunitrazepam were potent inhibitors of this binding (Kis of 10(-9)-10(-8) M) whereas clonazepam, CL 218,872 and Ro 15-1788 were weak inhibitors (Kis greater than 10(-5) M). A comparison of the binding of [3H]Ro 5-4864 in spinal cord with that in other areas of the CNS revealed that whereas the binding affinity was similar in all regions, membranes from spinal cord contained a slightly greater number of binding sites than cerebral cortex and cerebellum, and approximately one-third of the number present in the olfactory bulb. The characteristics of the binding of [3H]Ro 5-4864 obtained in this study are consistent with this ligand binding to peripheral-type benzodiazepine recognition sites in membranes from spinal cord.     

The human heart beta-adrenergic receptors. I. Heterogeneity of the binding sites: presence of 50% beta 1- and 50% beta 2-adrenergic receptors

Beta-adrenergic receptors were characterized in a particulate fraction of human auricles obtained from patients operated upon for coronary insufficiency or valvular disease. [125I] Hydroxybenzylpindolol binding was evaluated in terms of kinetics; KD and Bmax values; and inhibition of binding in the presence of 10 microM GTP and of increasing concentrations of four nonselective agonists giving a Hill coefficient of 1 (isoproterenol, salbutamol, fenoterol, and epinephrine), of two nonselective antagonists giving a Hill coefficient of 1 (pindolol and propranolol), and of a series of selective drugs giving a Hill coefficient of 0.60-0.72 that included three beta 1-selective antagonists (practolol, metoprolol, and atenolol) and two beta 2- selective agonists (procaterol and zinterol). KD values for all drugs were compatible with the coexistence in membranes from human auricles of beta 1- and beta 2-adrenergic receptors, the relative proportions of receptors of each subclass being approximately the same.     

Comparison of benzodiazepine receptor binding in membranes from human or rat brain

Specific high affinity binding of [3H]flunitrazepam to membranes from human brain was stimulated by gamma-aminobutyric acid (GABA), pentobarbital, 1-ethyl-4-(isopropylidene-hydrazino)-1H-pyrazolo[3,4b]pyridine-5-carboxy lic acid ethyl ester hydrochloride (SQ 20009) and avermectin B1a and was unaffected by 2 microM 4'-chlorodiazepam (Ro 5-4864) indicating that [3H]flunitrazepam in human brain as well as in rat brain predominantly binds to benzodiazepine receptors specific to brain, which was associated with a GABA receptor and several modulatory binding sites for drugs. The potency of several selective and non-selective ligands for benzodiazepine receptors for inhibition of the binding of [3H]flunitrazepam was compared in membranes from human or rat brain cerebellum, hippocampus and cerebral cortex. It was demonstrated that all these compounds, derived from different chemical structures, had a remarkably similar potency for inhibition of the binding of [3H]flunitrazepam in the corresponding regions of the human or rat brain. However, irreversible labelling of benzodiazepine binding sites with [3H]flunitrazepam and subsequent SDS-polyacrylamide gel electrophoresis and fluorography revealed more photolabelled protein bands in human than in rat cerebellum and hippocampus. The results seem to indicate that, although the pharmacological properties of reversible binding of [3H]flunitrazepam are remarkably similar in membranes from rat or human brain, the molecular heterogeneity of benzodiazepine binding sites is even greater in human than in rat brain.     

Comparison of the apparent irreversible beta-adrenoceptor antagonist Ro 03-7894 with propranolol in cardiac ventricular muscle by pharmacological and radioligand binding techniques

Membrane fractions were prepared from guinea-pig ventricular muscle and the specific binding of [3H]dihydroalprenolol ([3H]DHA) assessed. The dissociation constant (Ki) of (+/-)-propranolol was determined (6.9 nM) from its ability to displace [3H]DHA binding. This compared with the pA2 value of propranolol of 8.32 (dissociation constant, 4.8 nM) determined for the antagonism of isoprenaline-induced positive inotropic responses of papillary muscles from guinea-pig hearts. Scatchard analysis of the saturation curves for specific [3H]DHA binding showed that in the presence of propranolol, the displacement was characteristic of competitive antagonism. That is, there was no change in the total beta-adrenoceptor was characteristic of competitive antagonism. That is, there was no change in the total beta-adrenoceptor binding sites (Bmax) but an apparent reduction of the dissociation constant (KD) of [3H]DHA. This antagonism was fully reversed by washing membranes that had been previously incubated with propranolol. In contrast, in the presence of the beta-adrenoceptor antagonist, Ro 03-7894, the Scatchard plots were displaced in a manner characteristic of irreversible antagonism. The Bmax was significantly reduced. This antagonism was resistant to washout, with the Scatchard plots still showing a reduced Bmax and no change in the dissociation constant (KD) of [3H]DHA. This apparent irreversible antagonism by Ro 03-7894 was also demonstrated in guinea-pig isolated papillary muscles. The maximum of the dose-response curve to isoprenaline, constructed after incubation with Ro 03-7894 and a 3 hr bath-washout, was depressed by 89.5 +/- 7.5%     

Characterization of histamine H3 receptors in mouse brain using the H3 antagonist [125I]iodophenpropit

We have characterized the binding of the histamine H3 receptor antagonist [125I]iodophenpropit to mouse brain. [125I]Iodophenpropit saturably bound to mouse brain membranes with a pKd-value of 9.31+/-0.04 nM and a receptor binding density of 290+/-8 fmol per mg protein. Saturation binding analysis revealed binding of [125I]iodophenpropit to a single class of sites, showing linear Scatchard plots and Hill coefficients not different from unity (nH=0.98+/-0.02). At a concentration of 0.25 nM [125I]iodophenpropit, specific binding represented about 75% of the total binding. Competition binding curves for H3 receptor antagonists were fitted best to a one-site model, showing pKi-values in general accordance with the pA2-values obtained in mouse cerebral cortex. Displacement of [125I]iodophenpropit by the H3 receptor agonists (R)-alpha-methylhistamine, immepip, imetit and histamine were fitted best to a two-site model. Competition binding curves of (R)-alpha-methylhistamine showed a rightward shift upon incubation with GTPgammaS (10 microM), indicating the involvement of G-proteins in H3 agonist binding. In contrast, competition binding curves of the antagonists iodophenpropit, thioperamide and burimamide were not affected by GTPgammaS (10 microM). Autoradiographic experiments showed that [125I]iodophenpropit binding sites were heterogeneously distributed, similarly to the distribution of histamine H3 receptors reported in rat brain. Highest densities were observed in the cerebral cortex, the striatum, the nucleus accumbens, the globus pallidus and the substantia nigra. In conclusion, we have demonstrated that in mouse brain, [125I]iodophenpropit selectively binds to histamine H3 receptors. We also observed that the mouse brain H3 receptors labelled by [125I]iodophenpropit displayed binding characteristics and a distribution similar to rat brain.     

Is Ro 03-7894 an irreversible antagonist at beta-adrenoceptor sites?

Ro 03-7894 (0.6 mM) produced a non-parallel shift to the right of dose-response curves to (-)-isoprenaline in K+ depolarized uterine preparations from the guinea-pig. The displacement of the curves was readily reversed by washing. A rightward shift of similar magnitude was also produced by Ro 03-7894 in transmurally stimulated ileal preparations. The relaxant effects of fenoterol in carbachol-contracted guinea-pig tracheal preparations (in the presence of 2 microM atenolol) were not altered by 0.6 mM Ro 03-7894. In the three tissues there was no evidence of a reduction in the maximal inhibitory response to the agonists. In uterine and tracheal preparations, Ro 03-7894 (0.6 mM) depressed contractile responses to exogenous calcium. The depression of responses was enhanced after washout of Ro 03-7894 for 80 min. Contractile responses of ileal preparations to transmural stimulation were also depressed by Ro 03-7894. Concentration-effect curves for the positive inotropic effects of (-)-isoprenaline in guinea-pig left atrial preparations were markedly shifted to the right and the maximum response depressed by 0.6 mM Ro 03-7894. Although the rightward shift of the curves was fully reversed during the 120 min washout period, the maximal responses remained depressed. In similar experiments, Ro 03-7894 produced a washout-resistant depression of inotropic responses to histamine and calcium. The results of radioligand binding studies in left atria using (-)-[125I]-iodocyanopindolol indicated that, when compared to the untreated atria, there was no reduction in the maximal density of binding sites 120 min after washout of 0.6 mM Ro 03-7894.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-adrenoceptor-binding studies of the cardioselective beta blockers bisoprolol, H-I 42 BS, and HX-CH 44 BS to heart membranes and intact ventricular myocytes of adult rats: two beta 1-binding sites for bisoprolol.

beta-Adrenoceptor binding of cardioselective drugs to intact ventricular myocytes was performed using [3H]CGP-12177, a hydrophilic non-beta 1/beta 2-selective antagonist radioligand. The beta-adrenoceptor density on the intact cardiomyocytes was about 2 x 10(5) molecules/cell. The beta 1-selective antagonists H-I 42 BS and HX-CH 44 BS competed for [3H]CGP-12177 binding sites on the ventricular myocytes in an essentially monophasic manner with Ki = 72.6 nM and Ki = 76.7 nM, respectively. This is in contrast to the results of binding data from heart membranes, where these beta 1-antagonists bind in a biphasic manner with about 30% of an additional low affinity site, presumably corresponding to the beta 2-adrenoceptor. The beta 1-selective antagonist bisoprolol revealed two binding sites at the heart membranes and at the myocytes as well with Ki(1) = 34.2 and 20.0 nM and Ki(2) = 3,014 and 918 nM, respectively. Our results suggest that viable adult rat ventricular myocytes may contain two beta 1-adrenoceptor binding sites exhibiting different affinities for bisoprolol, whereas beta 2-adrenergic receptors are completely absent.     

Chronic treatment with (+/-)DOI, a psychotomimetic 5-HT2 agonist, downregulates 5-HT2 receptors in rat brain

(+/-)DOI (2,5-dimethoxy-4-iodo-phenylisopropylamine) is a hallucinogenic phenylalkylamine that has been characterized as a 5-HT2-selective agonist. Chronic treatment with (+/-)DOI [1.0 mg/kg/day (2.8 mumol/kg) for 8 days] significantly reduced the binding of [3H]ketanserin, [125I]LSD, and [125I]R-DOI as measured at single ligand concentrations in rat cortical homogenates. In saturation studies, chronic DOI treatment significantly lowered the Bmax of [3H]ketanserin binding and the high-affinity binding of [125I]R-DOI without altering the Kd values. In rats treated acutely with a single dose of (+/-)DOI, binding of [125I]R-DOI, [125I]LSD, and [3H]ketanserin was not significantly different from controls in membranes preincubated at 37 degrees C for 60 minutes. In all experiments nonspecific binding was determined by incubation with 1 microM ritanserin. This work demonstrates that chronic treatment with a 5-HT2-selective agonist hallucinogen reduces the number of binding sites for 5-HT2 agonists as well as for 5-HT2 antagonists.     

p-[125I]iodoclonidine is a partial agonist at the alpha 2-adrenergic receptor

The binding properties of p-[125I]iodoclonidine [( 125I]PIC) to human platelet membranes and the functional characteristics of PIC are reported. [125I]PIC bound rapidly and reversibly to platelet membranes, with a first-order association rate constant (kon) at room temperature of 8.0 +/- 2.7 x 10(6) M-1 sec-1 and a dissociation rate constant (koff) of 2.0 +/- 0.8 x 10(-3) sec-1. Scatchard plots of specific [125I]PIC binding (0.1-5 nM) were linear, with a Kd of 1.2 +/- 0.1 nM. [125I]PIC bound to the same number of high affinity sites as the alpha 2-adrenergic receptor (alpha 2-AR) full agonist [3H] bromoxidine (UK14,304), which represented approximately 40% of the sites bound by the antagonist [3H]yohimbine. Guanosine 5'-(beta, gamma-imido)triphosphate greatly reduced the amount of [125I]PIC bound (greater than 80%), without changing the Kd of the residual binding. In competition experiments, the alpha 2-AR-selective ligands yohimbine, bromoxidine, oxymetazoline, clonidine, p-aminoclonidine, (-)-epinephrine, and idazoxan all had Ki values in the low nanomolar range, whereas prazosin, propranolol, and serotonin yielded Ki values in the micromolar range. Epinephrine competition for [125I]PIC binding was stereoselective. Competition for [3H]bromoxidine binding by PIC gave a Ki of 1.0 nM (nH = 1.0), whereas competition for [3H]yohimbine could be resolved into high and low affinity components, with Ki values of 3.7 and 84 nM, respectively. PIC had minimal agonist activity in inhibiting adenylate cyclase in platelet membranes, but it potentiated platelet aggregation induced by ADP with an EC50 of 1.5 microM. PIC also inhibited epinephrine-induced aggregation, with an IC50 of 5.1 microM. Thus, PIC behaves as a partial agonist in a human platelet aggregation assay. [125I]PIC binds to the alpha 2B-AR in NG-10815 cell membranes with a Kd of 0.5 +/- 0.1 nM. [125I]PIC should prove useful in binding assays involving tissues with a low receptor density or in small tissue samples and in studies of cloned and expressed alpha 2-AR.     

Selective labelling of beta 1-adrenoceptors in rabbit lung membranes by (-)[3H]bisoprolol

The binding properties of a newly developed, highly selective beta 1-adrenoceptor antagonist radioligand, (-)[3H]bisoprolol (EMD 33512) were investigated in rabbit lung membranes containing a mixture of 80% beta 1-and 20% beta 2-adrenoceptors. The binding of (-)[3H]bisoprolol at 25 degrees C was saturable, of high affinity (KD = 4.7 +/- 0.6 nM, N = 4), rapid and readily reversible. The maximal number of (-)[3H]bisoprolol binding sites (244 +/- 31 fmol bound/mg protein, N = 4), however, was only 80% of the number of sites labelled by the non-selective beta-adrenoceptor radioligand (-)[125I]iodocyanopindolol (299 +/- 36 fmol bound/mg protein, N = 4). beta-Adrenoceptor antagonists (non-selective: propranolol, alprenolol; beta 1-selective: metoprolol, practolol, bisoprolol; beta 2-selective: ICI 118,551) inhibited (-)[3H]bisoprolol binding with monophasic displacement curves and pseudo-Hill coefficients of 1.0 indicating that in rabbit lung membranes (-)[3H]bisoprolol labels a homogeneous class of beta-adrenoceptors. Agonists inhibited binding with an order of potency: (-)-isoprenaline greater than (-)-noradrenaline = (-)-adrenaline, which is a typical one for beta 1-adrenoceptors. It is concluded that in rabbit lung membranes (-)[3H]bisoprolol selectively labels beta 1-adrenoceptors. (-)[3H]Bisoprolol therefore seems to be a suitable ligand for direct determination of the properties of beta 1-adrenoceptors in those tissues where both beta-adrenoceptor subtypes coexist.     

Identification of both NK1 and NK2 receptors in guinea-pig airways.

1. NK1 and NK2 receptors have been characterized in guinea-pig lung membrane preparations by use of [125I-Tyr8]-substance P and [125I]-neurokinin A binding assays in conjunction with tachykinin-receptor selective agonists ([Sar9Met(O2)11]substance P for NK1 and [beta Ala8]neurokinin A (4-10) for NK2) and antagonists (CP-99,994 for NK1 and SR48968 for NK2). 2. The presence of high affinity, G-protein-coupled NK1 receptors in guinea-pig lung parenchymal membranes has been confirmed. The rank order of affinity for competing tachykinins was as predicted for an NK1 receptor: substance P = [Sar9Met(O2)11]substance P > substance P-methyl ester = physalaemin > neurokinin A = neurokinin B >> [beta Ala8]neurokinin A (4-10). The novel NK1 antagonist CP-99,994 has a Ki of 0.4 nM at this NK1 site. 3. In order to characterize [125I]-neurokinin A binding to guinea-pig lung, the number of [125I]-neurokinin A specific binding sites was increased 3-4 fold by purification of the parenchymal membranes over discontinuous sucrose gradients. The rank order of affinity determined for NK1- and NK2-receptor agonists and antagonists in competition for these sites showed that the majority (80%) of [125I]-neurokinin A specific binding was also to the NK1 receptor. 4. Under conditions where the guinea-pig lung parenchymal NK1 receptor was fully occupied by a saturating concentration of either [Sar9Met(O2)11]substance P (1 microM) or CP-99,994 (2.7 microM), residual [125I]-neurokinin A specific binding was inhibited in a concentration-dependent manner by both [beta Ala8]neurokinin A and SR48968. This result shows that the NK2 receptor is also present in these preparations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of beta-adrenoceptor subtype on rat isolated ventricular myocytes by use of highly selective beta-antagonists.

1. The relative proportions of beta 1- and beta 2-adrenoceptors were determined by radioligand binding studies in three different rat myocardial preparations: membranes prepared from rat ventricle (ventricular membranes), membranes prepared from rat isolated ventricular myocytes (myocyte membranes), and myocytes isolated from rat ventricle (myocytes). 2. Competition experiments using CGP 20712A or ICI 118,551 with [125I]-iodocyanopindolol ([125I]-ICYP) revealed high- and low-affinity binding sites in ventricular membranes. The concentration at which each beta-antagonist occupied 100% of its high-affinity binding sites was 300 nM for CGP 20712A (beta 1-adrenoceptor) and 50 nM for ICI 118,551 (beta 2-adrenoceptor). 3. The density of high-affinity (beta 1-adrenoceptor) and low-affinity (beta 2-adrenoceptor) binding sites for CGP 20712A was measured by a saturation experiment using [125I]-ICYP in the presence and absence of 300 nM CGP 20712A. In ventricular membranes, the proportions of high-affinity and low-affinity binding sites for CGP 20712A were 73% and 27%, respectively, whereas in myocyte membranes, the corresponding figures were 90% and 10%, respectively. The density of low-affinity binding sites for CGP 20712A in ventricular membranes, defined as [125I]-ICYP-specific binding in the presence of 300 nM CGP 20712A, was decreased by addition of 50 nM ICI 118,551, whereas that in myocyte membranes was not affected. 4. In myocytes, specific binding of [125I]-ICYP and [3H]-CGP 12177 was not detected by saturation experiments performed in the presence of 300 nM CGP 20712A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha-adrenoceptor antagonistic action of amiloride

1. In isolated perfused rat liver, the effects of alpha-adrenergic stimulation by phenylephrine (2 microM), such as an increase of portal pressure, glucose output, Ca2+ release into the perfusate and the characteristic K+ flux changes across the hepatocyte plasma membrane were almost completely abolished in the presence of amiloride (0.5 mM). 2. When the phenylephrine concentration was raised to about 100 microM, the effects of the alpha-adrenergic agonist on hepatic metabolism, Ca2+ and K+ fluxes, but not on the portal venous pressure, were restored, suggesting a competitive antagonism by amiloride. 3. Amiloride antagonized in a concentration-dependent manner noradrenaline-induced isometric contractions of strips of the rabbit pulmonary artery. The concentration-response curve of noradrenaline was shifted to the right, and the maximal response obtained was also depressed, suggesting a mixed competitive and non-competitive antagonism. The estimated amiloride-adrenoceptor-dissociation constant was 8 microM. 4. The affinity of amiloride to the alpha- and beta-adrenoceptor subtypes was determined by radioligand binding assays using [125I]BE 2254 binding to rat liver plasma membranes (alpha 1-subtype), [3H]yohimbine binding to human platelet membranes (alpha 2-subtype), (-)-[125I]iodocyanopindolol (ICYP) binding to rabbit lung membranes in presence of the beta 2-adrenoceptor antagonist ICI 118,551 (beta 1-subtype) and ICYP binding to rat lung membranes in presence of the beta 1-blocker atenolol (beta 2-subtype). In all systems, amiloride inhibited specific ligand binding concentration-dependently, the Ki values for amiloride were about 25, 52, 148 and 161 microM for alpha 1- alpha 2-, beta 1- and beta 2-adrenoceptor subtypes, respectively. 5. It is concluded that amiloride in concentrations below those required for inhibition of the Na+/H+ exchanger is a potent antagonist of alpha- and beta-adrenoceptors in a variety of experimental systems. Whether the adrenergic antagonism of amiloride is important for antihypertensive therapy, remains to be elucidated.     

Multiple affinity forms of the calcitonin gene-related peptide receptor in rat cerebellum.

Binding of 125I-calcitonin gene-related peptide (125I-CGRP) to rat cerebellum membranes and the sensitivity to guanine nucleotides of binding were investigated. Cerebellum binding sites labeled by 125I-CGRP appear to be highly specific, inasmuch as CGRP inhibited binding with an IC50 of 100 pM but other peptides were inactive or much less active in displacing 125I-CGRP from these sites. 125I-CGRP binding sites in cerebellum membranes were saturable and of high affinity. Scatchard analysis of the saturation binding data revealed a homogeneous population of binding sites, with a KD of 224 +/- 28 pM and Bmax of 131 +/- 15 fmol/mg of protein. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) (100 microM), a single population of binding sites, with a KD of 464 +/- 77 pM and Bmax of 100 +/- 14 fmol/mg of protein, was observed. The kinetics of association of 125I-CGRP with cerebellum membranes were monophasic at all ligand concentrations tested. However, the observed association rate constant (kobs) was not dependent on [125I-CGRP] in a linear fashion in either the absence or the presence of GTP gamma S (100 microM). The kinetics of dissociation of 125I-CGRP from cerebellum membranes were multiexponential, with fast and slow dissociating components having rate constants of 0.34 +/- 0.01 and 0.025 +/- 0.001 min-1, respectively. The fast dissociating component represented 60 +/- 2% of the total specific binding sites. Dissociation of 125I-CGRP from cerebellum sites was much faster in the presence of GTP gamma S (100 microM) but still exhibited dissociation from two affinity components. The rate constants for these components of dissociation were 0.67 +/- 0.03 and 0.077 +/- 0.007 min-1, with the faster dissociating component representing 66 +/- 1% of the total specific binding sites. These findings provide the first evidence that CGRP receptors exist in multiple affinity states and that cerebellum CGRP receptors are regulated by guanine nucleotides. Our results also suggest the existence of two affinity states of the CGRP-receptor-guanine nucleotide-binding protein ternary complex.     

Characterization of peripheral benzodiazepine binding sites in human term placenta

Peripheral benzodiazepine binding sites were characterized in human term placental membranes using [3H]PK 11195, which is a ligand specific for peripheral benzodiazepine binding sites. Binding of [3H]PK 11195 to human term placental membranes was found to be saturable. Scatchard analysis revealed a single population of binding sites (r = 0.98). Equilibrium dissociation constant (KD) was 2.1 +/- 0.3 nM, and density of binding sites (Bmax) was 920 +/- 105 fmol/mg protein. The KD value calculated from kinetic experiments was 3.6 +/- 0.2 nM. The ability of various drugs to displace [3H]PK 11195 from human term placental binding sites was tested: the inhibition constants (KI) for PK 11195, Ro 5-4864, and diazepam were 2.9, 11.8, and 177 nM, respectively, whereas clonazepam, methyl-beta-carboline-3-carboxylate, Ro 15-1788, chlordiazepoxide, atropine, and estradiol were inefficient in displacing [3H]PK 11195 (KI greater than 10(-5) M).     

Molecular pharmacology of 5-HT1 and 5-HT2 recognition sites in rat and pig brain membranes: radioligand binding studies with [3H]5-HT, [3H]8-OH-DPAT, (-)[125I]iodocyanopindolol, [3H]mesulergine and [3H]ketanserin

The pharmacological characteristics of the binding of [3H]8-OH-DPAT ([3H]8-hydroxy-2(di-n-propylamino)tetralin, [125I]CYP ((-)[125I]iodocyanopindolol) (in the presence of 30 microM (-)isoprenaline) and [3H]mesulergine to 5-HT1 recognition sites were studied in rat and pig brain membranes. [3H]8-OH-DPAT bound in rat and pig cortex to the 5-HT1A recognition site characterized by high affinity for 5-CT (5-carboxamido-tryptamine), 8-OH-DPAT, 5-HT (5-hydroxytryptamine, serotonin) and low affinity for pirenperone, ketanserin and mesulergine. [125I]CYP bound in rat but not in pig cortex to the 5-HT1B site which shows high affinity for (-)21-009 (4[3-ter-butyl-amino-2-hydroxy-propoxy]indol-2-carbonic acid isopropyl ester), (+/-)ICYP (3-I-cyanopindolol), 5-HT, RU 24969 (5-methoxy-3-[1,2,3,6-tetrahydropyridon-4-yl]1H-indole) and low affinity for 8-OH-DPAT, mesulergine and pirenperone. [3H]Mesulergine bound in pig choroid plexus and in rat cortex (besides binding to 5-HT2 sites in rat cortex) to the 5-HT1C recognition site characterized by high affinity for metergoline, mesulergine, 5-HT and methergine and low affinity for (-)21-009, ICYP, 8-OH-DPAT and spiroperidol. The pharmacological profile of 5-HT1A sites in rat and pig cortex appears to be identical; 5-HT1C sites in pig choroid plexus and rat cortex show no differences. In contrast, it was not possible to label 5-HT1B sites with [125I]CYP in pig brain membranes indicating that like 5-HT2 receptors, 5-HT1 recognition sites show species differences. The pharmacological profiles of the three 5-HT1 recognition sites are clearly different from one another. Furthermore, the pharmacological profile of each individual 5-HT1 recognition site is also different from that of the 5-HT2 receptors labelled with [3H]ketanserin in rat cortex membranes although some similarities exist between 5-HT2 and 5-HT1C sites. Finally, the beta-adrenoceptor antagonist (-)21-009 which has different affinities for 5-HT1A, 5-HT1B and 5-HT1C recognition sites, yielded triphasic competition curves for [3H]5-HT binding in rat cortex membranes providing evidence that [3H]5-HT labels three distinct 5-HT1 sites in these membranes.     

Comparison of [125I]beta-endorphin binding to rat brain and NG108-15 cells using a monoclonal antibody directed against the opioid receptor

The properties of [125I]beta h-endorphin-binding sites from rat brain membranes and membranes from the NG108-15 cell line were compared using a monoclonal antibody directed against the opioid receptor and opioid peptides as probes. The binding of [125I]beta h-endorphin to both rat brain and NG108-15 membranes yielded linear Scatchard plots with Kd values of 1.2 nM and 1.5 nM, respectively, and Bmax values of 865 fmol/mg rat brain membrane protein and 1077 fmol/mg NG108-15 membrane protein. A monoclonal antibody, OR-689.2.4, capable of inhibiting mu and delta binding but not kappa binding to rat brain membranes, noncompetitively inhibited the binding of 1 nM [125I]beta h-endorphin to rat brain and NG108-15 membranes with an IC50 value of 405 nM for rat brain membranes and 543 nM for NG108-15 membranes. The monoclonal antibody also inhibited the binding of 3 nM [3H] [D-penicillamine2, D-penicillamine5] enkephalin to NG108-15 membranes with an IC50 value of 370 nM. In addition to blocking the binding of [125I]beta h-endorphin to brain membranes, the antibody also displaced [125I]beta h-endorphin from membranes. Site-specific opioid peptides had large variations in their IC50 values depending on whether they were inhibiting [125I]beta h-endorphin binding to rat brain or the NG108-15 membranes. When the peptides were tested with the monoclonal antibody for their combined ability to inhibit [125I]beta h-endorphin binding to both membrane preparations, the peptides and antibody blocked binding as though they were acting at allosterically coupled sites, not two totally independent sites. These studies suggest that mu-, delta-, and beta-endorphin-binding sites share some sequence homology with the 35,000-dalton protein that the antibody is directed against.