Affinity profiles of various muscarinic antagonists for cloned human muscarinic acetylcholine receptor (mAChR) subtypes and mAChRs in rat heart and submandibular gland.

A family of five subtypes of muscarinic acetylcholine receptors (mAChR) has been identified based on their molecular structures and second signal transduction pathways. In the present study, we examined the antagonist binding profiles of 9 muscarinic antagonists (atropine, 4-DAMP, pirenzepine, oxybutynin, tiquizium, timepidium, propiverine, darifenacin and zamifenacin) for human muscarinic acetylcholine receptor subtypes (m1, m2, m3, m4 and m5) produced by using a baculovirus infection system in Sf9 insect cells, and rat tissue membrane preparations (heart and submandibular gland). In a scopolamine methyl chloride [N-methyl-3H]- ([3H]NMS) binding assay, pirenzepine and timepidium displayed the highest affinities for the m1 and m2 subtypes, respectively, and both zamifenacin and darifenacin had the highest affinities for the m3 subtype, although the selectivities among the five subtypes were less than 10-fold. Propiverine showed a slightly higher affinity for the m5 subtype, whereas none of the drugs used in this study was uniquely selective for the m4 subtype. The binding affinities of muscarinic antagonists for rat heart and submandibular gland strong correlated with those for human cloned m2 and m3 subtypes, respectively. These data suggest that [3H]NMS binding studies using rat heart and submandibular gland might be useful methods which predict the affinities of test drugs for human muscarinic M2 and M3 receptor subtypes.     

Heterogeneity of the M1 muscarinic receptor subtype between peripheral lung and cerebral cortex demonstrated by the selective antagonist AF-DX 116

Recent studies have demonstrated that the majority of muscarinic receptors in rabbit peripheral lung homogenates bind pirenzepine with high affinity (putative M1 subtype). In experiments of AF-DX 116 inhibiting [3H](-)quinuclidinyl benzilate or [3H]pirenzepine, we found similar inhibitory constants for AF-DX 116 binding in rat heart and rabbit peripheral lung that were 4-fold smaller (i.e. of higher affinity) than the inhibitory constant for rat cerebral cortex. This result demonstrates heterogeneity of the M1 muscarinic receptor subtype between peripheral lung and cerebral cortex.     

In vivo demonstration of M3 muscarinic receptor subtype selectivity of darifenacin in mice

A novel muscarinic receptor antagonist, darifenacin, inhibited specific binding of [N-methyl-(3)H]scopolamine ([(3)H]NMS) in the mouse bladder, submaxillary gland and heart in a concentration-dependent manner. The inhibitory effect was most potent in the submaxillary gland, followed by the bladder and heart. In addition, darifenacin inhibited specific [(3)H]NMS binding in the membranes of CHO-K1 cell lines expressing muscarinic M(2) and M(3) receptor subtypes, and the potency was significantly (22-fold) greater at the M(3) than at the M(2) subtype. At 0.5 to 12 h after oral administration of darifenacin, a significant increase in K(d) values for specific [(3)H]NMS binding was seen in the bladder, submaxillary gland and lung of mice, compared with control values. Also, there was a sustained decrease in the B(max) values in the submaxillary gland. These data suggest that muscarinic receptor binding of oral darifenacin is rapid in onset and of a long duration. On the other hand, oral darifenacin exerted only temporary or little binding of muscarinic receptors in the heart and colon. Pilocarpine-induced salivary secretion in mice was continuously suppressed by oral darifenacin. The time-course of suppression coincided well with that for the muscarinic receptor binding in the submaxillary gland. The antagonistic effect of darifenacin against the dose-response curves for pilocarpine appeared to be insurmountable. In conclusion, the present study has shown that oral darifenacin may exert a pronounced and long-lasting binding of muscarinic receptors in tissues expressing the M(3) subtype.     

Pharmacologic characterization of muscarinic receptors of insect brains.

Muscarinic receptors in brain membranes from honey bees, houseflies, and the American cockroach were identified by their specific binding of the non-selective muscarinic receptor antagonist [3H]quinuclidinyl benzilate ([3H]QNB) and the displacement of this binding by agonists as well as subtype-selective antagonists, using filtration assays. The binding parameters, obtained from Scatchard analysis, indicated that insect muscarinic receptors, like those of mammalian brains, had high affinities for [3H]QNB (KD = 0.47 nM in honey bees, 0.17 nM in houseflies and 0.13 nM in the cockroach). However, the receptor concentration was low (108, 64.7, and 108 fmol/mg protein for the three species, respectively). The association and dissociation rates of [3H]QNB binding to honey bee brain membranes, sensitivity of [3H]QNB binding to muscarinic agonists, and high affinity for atropine were also features generally similar to muscarinic receptors of mammalian brains. In order to further characterize the three insect brain muscarinic receptors, the displacement of [3H]QNB binding by subtype-selective antagonists was studied. The rank order of potency of pirenzepine (PZ), the M1 selective antagonist, 11-[2-[dimethylamino)-methyl)1-piperidinyl)acetyl)-5,11- dihydro-6H-pyrido(2,3-b)-(1,4)-benzodiazepin-6 one (AF-DX 116), the M2-selective antagonist, and 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) the M3-selective antagonist, was also the same as that of mammalian brains, i.e., 4-DAMP greater than PZ greater than AF-DX 116. The three insect brain receptors had 27-50-fold lower affinity for PZ (Ki 484-900 nM) than did the mammalian brain receptor (Ki 16 nM), but similar to that reported for the muscarinic receptor subtype cloned from Drosophila. Also, the affinity of insect receptors for 4-DAMP (Ki 18.9-56.6 nM) was much lower than that of the M3 receptor, which predominates in rat submaxillary gland (Ki of 0.37 nM on [3H]QNB binding). These drug specificities of muscarinic receptors of brains from three insect species suggest that insect brains may be predominantly of a unique subtype that is close to, though significantly different from, the mammalian M3 subtype.     

A Point Mutation in the Coding Sequence of the β-Hexosaminidase α Gene Results in Defective Processing of the Enzyme Protein in an Unusual GM2-Gangliosidosis Variant

The M1-selective (high affinity for pirenzepine) muscarinic acetylcholine receptor (mAChR) antagonist pirenzepine displaced both N-[3H]methylscopolamine [( 3H]NMS) and [3H]quinuclidinylbenzilate from intact human SK-N-SH neuroblastoma cells with a low affinity (Ki = 869-1,066 nM), a result indicating the predominance of the M2 or M3 (low affinity for pirenzepine) receptor subtype in these cells. Whereas a selective M2 agent, AF-DX 116 [11-2[[2-[(diethylamino)methyl]-1-piperidinyl]- acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) bound to the mAChRs with a very low affinity (Ki = 6.0 microM), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), an agent that binds with high affinity to the M3 subtype, potently inhibited [3H]NMS binding (Ki = 7.2 nM). 4-DAMP was also 1,000-fold more effective than AF-DX 116 at blocking stimulated phosphoinositide (PPI) hydrolysis in these cells. Covalent labeling studies (with [3H]propylbenzilycholine mustard) suggest that the size of the SK-N-SH mAChR (Mr = 81,000-98,000) distinguishes it from the predominant mAChR species in rat cerebral cortex (Mr = 66,000), an M1-enriched tissue. These results provide the first demonstration of a neural M3 mAChR subtype that couples to PPI turnover.     

Muscarinic receptor subtypes in bovine adrenal medulla

Catecholamine secretion in the bovine adrenal medulla is evoked largely by nicotinic receptor activation. However, bovine adrenal medulla also contain muscarinic receptors that mediate several cell responses. To understand the physiological role of muscarinic receptors in the bovine adrenal medulla it is important to identify the pharmacological subtypes present in this tissue. For this, we analyzed the abilities of differnt selective muscarinic antagonists in displacing the binding of the non-selective antagonist [3H] quinuclidinyl benzylate to an enriched plasma membrane fraction prepared from bovine adrenal medulla. All the selective antagonists bind at least two bindings sites with different affinities. The binding profile of the sites with high proportion is similar to the M2 subtype and those present in low proportion have a M1 profile. However, some variation in the proportion of the sites for the different ligands suggest the presence of the third pharmacological subtype (M3). We conclude that the sites in high proportion (60–80%) correspond to M2 muscarinic subtypes, and the rest is constitute by M1 plus M3 subtypes. The presence of multiplicity of subtypes in the adrenal medulla membranes suggests a diversity of functions of muscarinic receptors in the adrenal gland.Abbreviations [3H]QNB [3H]quinuclidinyl benzylate - HHSiD hexahydro-siladifenidol-hydrochloride - AF-DX 116 11-[[2-(diethylamino)methyl]]-1-piperidinyl]-5,11-dihydro-6H-pyrido[2,3,-b][1,4]benzodiazepin-6-one - 4-DAMP 4-diphenylacetoxy-N-methyl piperidine methobromide     

Discovery of a muscarinic M3 receptor antagonist with high selectivity for M3 over M2 receptors among 2-[(1S,3S)-3-sulfonylaminocyclopentyl]phenylacetamide derivatives

In the course of developing a metabolically stable M3 receptor antagonist from the prototype antagonist, J-104129 (1), introduction of certain substituents into the cyclopentane ring of 1 was found to be effective not only in improving metabolic stability but also in greatly enhancing the subtype selectivity. Among the cyclopentane analogues, sulfonamide derivatives (10f) and (10g) displayed 160- and 310-fold selectivity for M3 over M2 receptors, and both were significantly more selective than the prototype antagonist (120-fold). Subsequent derivatization of the sulfonamide series led to the highly selective M3 receptor antagonists (10h, 10i and 10j) with >490-fold selectivity for M3 over M2 receptors. Among them, p-nitrophenylsulfonamide (J-107320, 10h) exhibited 1100-fold selectivity for M3 receptors (Ki = 2.5 nM) over M2 receptors (Ki = 2800 nM) in the human muscarinic receptor binding assay using [3H]-NMS as a radio ligand.     

Differential effects of paraoxon on the M3 muscarinic receptor and its effector system in rat submaxillary gland cells

The effects of the organophosphorus anticholinesterase paraoxon on the binding of radioactive ligands to the M₃ subtype of the muscarinic receptor and receptor-coupled synthesis of second messengers in intact rat submaxillary gland (SMG) cells were investigated. The binding of [³H]quinuclidinyl benzilate ([³H]QNB) was most sensitive to atropine and the M₃-specific antagonist 4-DAMP followed by pirenzepine and least sensitive to the cardioselective M₂ antagonist AFDX116. This, and the binding characteristics of [³H]4-DAMP, confirmed that the muscarinic receptors in this preparation are of the M₃ subtype. Activation of these muscarinic receptors by carbamylcholine (CBC) produced both stimulation of phosphoinositide (PI) hydrolysis and inhibition of cAMP synthesis, suggesting that this receptor subtype couples to both effector systems. Paraoxon (100 μM) reduced B_max of [³H]4-DAMP binding from 27 ± 4 to 13 ± 3 fmol/mg protein with nonsignificant change in affinity, suggesting noncompetitive inhibition of binding by paraoxon. Like the agonist CBC, paraoxon inhibited the forskolininduced cAMP formation in SMG cells with an EC₅₀ of 200 nM, but paraoxon was > 500 fold more potent than CBC. However, while the inhibition by CBC was counteracted by 2 μM atropine, that by paraoxon was unaffected by up to 100 μM atropine. It suggested that this effect of paraoxon was not via binding to the muscarinic receptor. Paraoxon did not affect β-adrenoreceptor function in the preparation, since it did not affect the 10 μM isoproterenol-induced cAMP synthesis, which was inhibited totally by 10 μM propranolol and partially by CBC. Paraoxon had a small but significant effect on CBC-stimulated PI metabolism in the SMG cells. It is suggested that paraoxon binds to two different sites in these SMG cells. One is an allosteric site on the M₃ muscarinic receptor which affects ligand binding and may modulate receptor function. The other site may be on the G_i proteinadenylyl cyclase system, and produces CBC-like action, that is, inhibition of the forskolin-stimulated [³H]cAMP synthesis, and is unaffected by atropine inhibition of the muscarinic receptor. This adds to the complexity of paraoxon actions on muscarinic receptors and their effector systems.     

Subacute reserpine treatment reveals preferential coupling between the M3 muscarinic receptor subtype and phosphatidylinositol turnover.

Muscarinic receptors in the rat cerebral cortex, cardiac atria and vas deferens were identified, quantitated, and characterized relative to phosphatidylinositol (PI) turnover as the functional response to stimulation of specific receptor subtypes. Receptor densities as determined by 3H-QNB binding were ranked: cerebral cortex greater than vas deferens greater than heart. Using displacement of 3H-QNB binding by the selective M1 and M2 muscarinic receptor antagonists pirenzepine and 11[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro- 6H-pyrido [2,3-b] [1,4] benzodiazepine-6-one (AF-DX 116) respectively, heterogeneous populations were found in the cerebral cortex and vas deferens. The M1 receptor subtype predominated in the former and the M2 predominated in the latter. An homogeneous M2 receptor population was present in the heart. Methacholine-stimulated accumulation of 3H inositol-1-phosphate was greater in the vas deferens than in the cerebral cortex, whereas PI turnover was not enhanced in cardiac atria. Reserpine treatment of rats (0.5 mg kg-1 day-1 for 7 days) increased muscarinic receptor density in the vas deferens coincident with a shift in the low affinity pKi for AF-DX 116 to a value comparable to high affinity binding, and abolished the enhanced PI hydrolysis. In the cerebral cortex, reserpine treatment shifted only the early portion of the methacholine dose-response curve to the right. These results are judged to be supportive of preferential coupling between the M3 muscarinic receptor subtype and PI turnover.     

Characterization of muscarinic receptor subtypes in human tissues

The affinities of selective, pirenzepine and AF-DX 116, and classical, N-methylscopolamine and atropine, muscarinic cholinergic receptor antagonists were investigated in displacement binding experiments with [3H]Pirenzepine and [3H]N-methylscopolamine in membranes from human autoptic tissues (forebrain, cerebellum, atria, ventricle and submaxillary salivary glands). Affinity estimates of N-methylscopolamine and atropine indicated a non-selective profile. Pirenzepine showed differentiation between the M1 neuronal receptor of the forebrain and the receptors in other tissues while AF-DX 116 clearly discriminated between muscarinic receptors of heart and glands. The results in human tissues confirm the previously described selectivity profiles of pirenzepine and AF-DX 116 in rat tissues. These findings thus reveal the presence also in man of three distinct muscarinic receptor subtypes: the neuronal M1, the cardiac M2 and the glandular M3.     

Identification of three muscarinic receptor subtypes in rat lung using binding studies with selective antagonists

Heterogeneity of the muscarinic receptor population in the rat central and peripheral lung was found in competition binding experiments against [3H]quinuclidinyl benzilate [( 3H]QNB) using the selective antagonists pirenzepine, AF-DX 116 and hexahydrosiladifenidol (HHSiD). Pirenzepine displaced [3H]QNB with low affinity from preparations of central airways indicating the absence of M1 receptors in the trachea and bronchi. Muscarinic receptors in the central airways are comprised of both M2 and M3 receptors since AF-DX 116, an M2-selective antagonist, bound with high affinity to 70% of the available sites while HHSiD, an M3-selective antagonist bound with high affinity to the remaining binding sites. In the peripheral lung, pirenzepine bound with high affinity to 14% of the receptor population, AF-DX 116 bound with high affinity to 79% of the binding sites while HHSiD bound with high affinity to 18% of the binding sites. The presence of M1 receptors in the peripheral airways but not in the central airways was confirmed using [3H]telenzepine, an M1 receptor ligand. [3H]Telenzepine showed specific saturable binding to 8% of [3H]QNB labeled binding sites in homogenates of rat peripheral lung, while there was no detectable specific binding in homogenates of rat trachea or heart. The results presented here demonstrate that there are three muscarinic receptor subtypes in rat lungs, and that the distribution of the different subtypes varies within the lungs. Throughout the airways, the dominant muscarinic receptor subtype is M2. In the trachea and bronchi the remaining receptors are M3, while in the peripheral lungs, the remaining receptors are both M1 and M3.     

Molecular probes for muscarinic receptors: derivatives of the M1-antagonist telenzepine.

Functionalized congeners of the M1-selective muscarinic antagonist telenzepine (4,9-dihydro-3-methyl-4-[(4-methyl-1-piperazinyl)acetyl]-10H- thieno[3,4-b][1,5]benzodiazepin-10-one) were developed and found to bind to the receptor with affinities (Ki values) in approximately the nanomolar range. The derivatives contain a 10-aminodecyl group, which provides a nucleophilic functionality for further derivatization. The attachment of a spacer chain to the distal piperazinyl nitrogen was based on previous findings of enhanced affinity at muscarinic receptors in an analogous series of alkylamino derivatives of pirenzepine [J. Med. Chem. (1991) 34, 2133-2145]. The telenzepine derivatives contain prosthetic groups for radioiodination, protein cross-linking, photoaffinity labeling, and fluorescent labeling and biotin for avidin complexation. The affinity for muscarinic receptors in rat forebrain (mainly m1 subtype) was determined in competitive binding assays vs [3H]-N-methylscopolamine. A (p-aminophenyl)-acetyl derivative for photoaffinity labeling had a Ki value of 0.29 nM at forebrain muscarinic receptors (16-fold higher affinity than telenzepine). A biotin conjugate displayed a Ki value of 0.60 nM at m2-receptors and a 5-fold selectivity versus forebrain. The high affinity of these derivatives makes them suitable for the characterization of muscarinic receptors in pharmacological and spectroscopic studies, for peptide mapping, and for histochemical studies.     

Alpha adrenergic drugs inhibit [3H]-QNB binding to muscarinic receptors of rat heart, brain and parotid gland membranes

Alpha adrenergic agonists and antagonists as clonidine, guanfacine, yohimbine, phenylephrine and prazosin inhibited the [3H]-QNB binding to rat brain cortex muscarinic acetylcholine receptor (mAChR, M-1 subtype), heart (M-2 subtype) and parotid gland homogenate (M-3 subtype) in a dose-dependent competitive fashion. Ki values were between 10(-6) and 10(-3) M. Hill coefficients were about 1. No correlation was found between mAChR inhibiting capacity of these drugs and their activity on alpha adrenergic receptors. In contrast, other transmitters, as dopamine, GABA, glutamic acid, histamine, serotonin, isoproterenol and platelet activating factor (PAF) did not affect the QNB binding.     

The Mr 90,000 heat shock protein-free androgen receptor has a high affinity for steroid, in contrast to the glucocorticoid receptor

Transformed and bacterially expressed glucocorticoid receptors free from Mr 90,000 heat shock protein (hsp90) have a 100-fold lower steroid-binding affinity than the hsp90-bound nontransformed receptor, suggesting that hsp90 is needed for high-affinity steroid binding [Nemoto, T., Ohara-Nemoto, Y., Denis, M., & Gustafsson, J.-A. (1990) Biochemistry 29, 1880-1886]. To investigate whether or not this phenomenon is common to all steroid receptors, we investigated the steroid-binding affinities of bacterially expressed and transformed androgen receptors. The C-terminal portion of the rat androgen receptor containing the putative steroid-binding domain was expressed as a fusion protein of protein A in Escherichia coli. The recombinant protein bound a synthetic androgen, [3H]R1881, with high affinity (Kd = 0.8 +/- 0.3 nM). Glycerol gradient analysis revealed that the recombinant protein sedimented at around the 3S region irrespective of the presence of molybdate, indicating that the receptor is present in monomeric form. The steroid-free transformed androgen receptor was obtained by exposure of rat submandibular gland cytosol to 0.4 M NaCl in the absence of steroid. High-performance ion-exchange liquid chromatography analysis showed that the transformed androgen receptor bound to [3H]R1881 with high affinity. Thus these observations indicate that, in contrast to the glucocorticoid receptor, hsp90 is not required for the high-affinity steroid binding of the androgen receptor. In addition, the hsp90-free androgen receptor prebound with radioinert R1881 was efficiently relabeled with [3H]R1881, while the triamcinolone acetonide-bound, transformed glucocorticoid receptor failed in ligand exchange. The inability to achieve ligand exchange probably reflects the low steroid-binding affinity of this entity.     

Two polypeptides from Dendroaspis angusticeps venom selectively inhibit the binding of central muscarinic cholinergic receptor ligands.

Two new polypeptides were isolated and purified from the venom of the snake Dendroaspis angusticeps, which also contains other neuroactive peptides such as Dendrotoxins and Fasciculins. The amino acid composition of the peptides was determined and the first 10 amino acids from the MTX2 N-terminal fragment were sequenced. The so-called muscarinic toxins (MTX1 and MTX2) have been shown to inhibit the specific binding of [3H]QNB (0.15 nM), [3H]PZ (2.5 nM) and [3H]oxoM (2 nM) to bovine cerebral cortex membranes by 60, 88 and 82% respectively. In contrast, they caused only a 30% blockade of the [3H]QNB specific binding to similar membrane preparations from the brainstem. The Hill number for the [3H]PZ binding inhibition by the putative muscarinic toxin MTX2 was 0.95 suggesting homogeneity in the behaviour of the sites involved. The data from [3H]oxoM binding gave a Hill number of 0.83. The decreases in the specific binding involved increases in KD for the three different ligands (8-fold for [3H]QNB, 4-fold for [3H]PZ and 3.5-fold for [3H]oxoM) without significant changes in Bmax, except for a slight decrease in the [3H]oxoM binding sites (-19%); such results suggest that there may be a competitive inhibition between the MTXs and these ligands. The Ki for MTX2/[3H]PZ was 22.58 +/- 3.52 nM; for MTX2/[3H]oxoM, 144.9 +/- 21.07 nM and for MTX2/[3H]QNB, 134.98 +/- 18.35 nM. The labelling of MTX2 with 125I allowed direct demonstration of specific and saturable binding to bovine cerebral cortex synaptosomal membranes. In conclusion, the results reported in this study strongly support the hypotheses that the two polypeptides isolated from D. angusticeps venom selectively inhibit specific ligand binding to central muscarinic receptors, in a competitive manner at least for the antagonist [3H]PZ and that the MTX2 specifically binds to a central site that is suggested to be a muscarinic receptor of the M1 subtype.     

Subtype of muscarinic receptor coupled to the attenuation of hormone-stimulated cAMP accumulation in NG108-15 neuroblastoma x glioma hybrid cells.

The subtype of muscarinic receptor which mediates cAMP attenuation is not established. Therefore, several selective muscarinic antagonists were used to characterize the subtype of muscarinic receptor coupled to the inhibition of hormone-stimulated cAMP accumulation using NG108-15 neuroblastoma x glioma hybrid cells. These cells were prelabeled with [2-3H]-adenine, washed, and resuspended in a culture medium containing the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM). The labeled cells were preincubated with the different antagonists 12-15 min. before they were challenged with agonists. The formation of [3H]-cAMP was activated by PGE1 (1 microM) or forskolin (1 microM). In all cases, [3H]-cAMP formed was separated and measured. Carbachol (100 microM) and McN-A343 (10 mM) were used as standard muscarinic agonists. These studies gave the following results: a) McN-A343 (10 mM), an M1 receptor agonist, was only a partial agonist causing 40% inhibition of cAMP accumulation indicating that this effect was not mediated by an M1 receptor; b) The M1-selective antagonist, pirenzepine, exhibited low affinity (pA2 6.2) further suggesting that an M1 receptor was not coupled to the attenuation of cAMP accumulation; c) Two selective M2 antagonists (AF-DX 116 and methoctramine) and M3 antagonist (HHSiD) were used to further characterize these muscarinic receptors. The order of all antagonists based on their affinities (pA2 values) could be arranged in the following order: atropine (9.0) > methoctramine (7.6) > HHSiD (6.9) > AF-DX 116 (6.6) > pirenzepine (6.2). HHSiD exhibits the same degree of affinity to M2 receptors of other tissues as it does to those of NG cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of two radiolabeled quinuclidinyl benzilate ligands for the characterization of the human peripheral lung muscarinic receptor

Quinuclidinyl benzilate, a muscarinic antagonist, has previously been used in its tritiated form ([3H]-QNB) to study the lung muscarinic receptor. We investigated whether a newer iodinated form of QNB ([125I]-QNB) of higher specific activity would be an appropriate ligand to study the human peripheral lung muscarinic receptor. Both the tritiated and iodinated ligands bound specifically to human lung at 23 degrees C. At 37 degrees C the specific binding of [3H]-QNB increased slightly, but no specific binding of [125I]-QNB was found. The data from multiple equilibrium binding experiments covering a wide range of radiolabeled QNB concentrations were combined and analyzed using the computer modeling program, LIGAND. The tritiated QNB identified a single affinity human lung binding site with a Kd of 46 +/- 9 pM and a receptor concentration of 34 +/- 3 fmol/mg protein. The iodinated QNB identified a single higher affinity human lung binding site (Kd = 0.27 +/- 0.32 pM) of much smaller quantity (0.62 +/- 0.06 fmol/mg protein). Competition studies comparing the binding of unlabeled QNB relative to labeled QNB indicated that unlabeled QNB had the same Kd as that measured for [3H]-QNB, but a 5 log greater Kd than that measured for [125I]-QNB. Other muscarinic receptor agonists and antagonists competed with [3H]-QNB, but not [125I]-QNB for binding to muscarinic receptors with the expected magnitude and rank order of potency. We conclude that of the 2 radiolabeled forms of QNB available, only the tritiated form should be used to study the human peripheral lung muscarinic receptor.     

Beta adrenergic and muscarinic receptor densities of rat submandibular main duct

[3H]DHA binding studies show that main duct of rat submandibular gland has both beta 1 and beta 2 adrenoceptors, with the percentages of each being 69 and 31%, respectively, whereas whole submandibular gland has 90% beta 1 and 10% beta 2 adrenoceptors. Muscarinic receptors of main duct are 25% less than that of whole submandibular gland.