Radioligand Dissociation Research Articles

Characterization of a folate receptor in parotid gland and a folate binding protein in saliva from humans. Epitope relatedness to human milk folate binding protein.

The present study was performed to establish the antigenic identity and origin of the folate binding protein in human saliva. We identified a folate receptor in human parotid and submandibular gland which immunoreacted with antibodies against human milk folate binding protein, as evidenced by ELISA and immunostaining of ductal epithelium and secretory glandular material. The receptor concentration was 0.4-1.4 nmol 3H-folate bound/g protein. Ligand binding was of a high-affinity (K=10(10) M(-1)) type, exhibited positive cooperativity, a slow radioligand dissociation at pH 7.4, and inhibition by folate analogues. The concentration of immunoreactive folate binding protein in saliva as determined by ELISA with antibodies against human milk folate binding protein was several fold higher than that determined by radioligand binding (nil - 1 nM). This indicates that a major fraction of the immunoreactive material does not bind 3H-folate, and could represent a precursor form of the protein. In conclusion, the folate binding protein in human saliva seems to be a secretory product of the salivary glands. The protein is also epitope-related to folate binding proteins in other human mucosal secretions.

Evidence for a Tandem Two-site Model of Ligand Binding to Muscarinic Acetylcholine Receptors

After short preincubations with N-[(3)H]methylscopolamine ([(3)H]NMS) or R(-)-[(3)H]quinuclidinyl benzilate ([(3)H]QNB), radioligand dissociation from muscarinic M(1) receptors in Chinese hamster ovary cell membranes was fast, monoexponential, and independent of the concentration of unlabeled NMS or QNB added to reveal dissociation. After long preincubations, the dissociation was slow, not monoexponential, and inversely related to the concentration of the unlabeled ligand. Apparently, the unlabeled ligand becomes able to associate with the receptor simultaneously with the already bound radioligand if the preincubation lasts for a long period, and to hinder radioligand dissociation. When the membranes were preincubated with [(3)H]NMS and then exposed to benzilylcholine mustard (covalently binding specific ligand), [(3)H]NMS dissociation was blocked in wild-type receptors, but not in mutated (D99N) M(1) receptors. Covalently binding [(3)H]propylbenzilylcholine mustard detected substantially more binding sites than [(3)H]NMS. The observations support a model in which the receptor binding domain has two tandemly arranged subsites for classical ligands, a peripheral one and a central one. Ligands bind to the peripheral subsite first (binding with lower affinity) and translocate to the central subsite (binding with higher affinity). The peripheral subsite of M(1) receptors may include Asp-99. Experimental data on [(3)H]NMS and [(3)H]QNB association and dissociation perfectly agree with the predictions of the tandem two-site model.

Ligand binding characteristics of a glycosylphosphatidyl inositol membrane-anchored HeLa cell folate receptor epitope-related to human milk folate binding protein.

The folate receptor (FR) in HeLa cells was characterized as to ligand binding mechanism, antigenic properties and membrane anchor in order to obtain information to be used for the design of biological agents targeting FR in malignant tumors. The receptor displayed the following binding characteristics in equilibrium dialysis experiments (37 degrees C, pH 7.4) with [3H] folate: a high-affinity type of binding that exhibited positive cooperativity with a Hill coefficient > 1.0 and an upward convex Scatchard plot, a slow radioligand dissociation at pH 7.4 becoming rapid at pH 3.5 and inhibition in the presence of other folates. The molecular size of the receptor was 100 kDa on gel filtration with Triton X-100, or similar to that of high molecular weight human milk folate binding protein (FBP). The latter protein represents a 25 kDa molecule which equipped with a hydrophobic glycosylphosphatidyl inositol (GPI) membrane anchor susceptible to cleavage by phosphatidylinositol specific phospholipase C (PI-PLC) forms micelles of 1kDa size with Triton X-100. The HeLa cell FR immunoreacted with antibodies against purified human milk FBP in ELISA, and in a fluorescence activated cell sorting system, where HeLa cells exposed to increasing concentrations of antibody showed a dose-dependent response. Exposure to PI-PLC decreased the fraction of immunolabeled cells indicating a linkage of FR to cell membranes by a GPI anchor. HeLa cells incubated with radiofolate showed a continuous uptake with time, however, with a complete suppression of uptake in the presence of an excess of cold folate. Prewash of cells at acidic pH to remove endogenous folate increased the uptake. Binding and uptake of [3H] folate was increased in cells grown in a folate-deprived medium. The HeLa FR seems to be epitope related to human milk FBP.

Further evidence for the heterogeneity of functional muscarinic receptors in guinea pig gallbladder

Previous studies have suggested the presence of multiple muscarinic receptor subtypes in guinea pig gallbladder smooth muscle, although the relative abundance and functional role of these subtypes remains an area of significant research efforts. The present study utilized both radioligand kinetic and functional experiments to further probe the nature of the muscarinic receptors in gallbladder smooth muscle and their mode of coupling to intra- and extra-cellular Ca 2+ sources. Dissociation kinetic studies using [ 3H] N-methylscopolamine ([ 3H]NMS) indicated that the binding profile in guinea pig gallbladder smooth muscle could not be reconciled with that expected for a single muscarinic receptor subtype, the latter determined in parallel experiments conducted on the cloned muscarinic M 1–M 5 subtypes in Chinese hamster ovary (CHO) cells. Furthermore, comparison of the gallbladder data with the dissociation characteristics of [ 3H]NMS in guinea pig urinary bladder revealed a significantly different kinetic profile, with the urinary bladder, but not the gallbladder, demonstrating biphasic radioligand dissociation kinetics. In functional experiments, carbachol caused a concentration-dependent contraction of guinea pig gallbladder smooth muscle strips in Ca 2+-free or 5 mM Sr 2+-substituted physiological salt solutions (PSS) with amplitudes of the maximal contractions corresponding to 45.8±8.0% and 33.2±6.6% of control responses in normal PSS, respectively. Furthermore, the stimulus–response characteristics of carbachol-mediated contraction appeared significantly altered in Ca 2+-free PSS relative to normal or Sr 2+-substituted PSS. The antagonist, methoctramine (1×10 −7–3×10 −5 M), exerted only a slight inhibition of carbachol (10 −5 M)-induced contractions in 5 mM Sr 2+-substituted medium, whereas it was significantly more potent in antagonizing gallbladder contractions in response to 10 −5 M carbachol in the absence of extracellular Ca 2+. Both atropine and tripitramine were equipotent in antagonizing carbachol-induced contractions in Ca 2+-free (pIC 50: 6.85±0.11 for atropine and 5.75±0.32 for tripitramine) and Sr 2+-substituted media (pIC 50: 6.88±0.25 for atropine and 5.70±0.16 for tripitramine), and pirenzepine was only slightly more potent in Ca 2+-free PSS (pIC 50: 5.66±0.23) than in Sr 2+-substituted PSS (pIC 50: 5.33±0.21). Taken together, our data indicate that carbachol contracts guinea pig gallbladder by stimulating two distinct muscarinic receptor subtypes linked to extracellular Ca 2+ influx and intracellular Ca 2+ release. These two subtypes may represent the muscarinic M 3 and M 4 receptors, although the presence of the muscarinic M 2 receptor subtype is also suggested from the binding data.

High-Affinity Folate Receptor in Human Ovary, Serous Ovarian Adenocarcinoma, and Ascites: Radioligand Binding Mechanism, Molecular Size, Ionic Properties, Hydrophobic Domain, and Immunoreactivity

High-affinity folate receptors are expressed in normal ovaries and ovarian carcinomas. Binding of [3H]folate in human ovary, serous ovarian carcinoma tissue, and ascites is a complex process that has not been well characterized. This study shows changes in binding affinity and mechanism of binding with decreasing receptor concentration, inhibition by folate derivatives, and a slow radioligand dissociation at pH 7.4 becoming rapid and complete at pH 3.5. The receptor seems to be positively charged since it elutes in the front effluent of a DEAE-Sepharose CL-6B ion-exchange column at pH 6.3. The gel filtration profile of Triton X-100-solubilized tissue and ascites contained two peaks of radioligand-bound receptor (25 and 100 kDa). Exposure of ascites to cleavage by phosphatidylinositol-specific phospholipase C resulted in a partial conversion of the 100-kDa peak to a 25-kDa peak. This suggests that the receptor may be anchored to the membrane by a glycosylphosphatidyl residue that inserts into Triton X-100 micelles, resulting in a large molecular size on gel filtration. The receptor in ovarian carcinoma tissue immunoreacts with antibodies against purified human milk folate receptor protein as shown by enzyme-linked immunosorbent assay, immunoprecipitation, sodium dodecyl sulfate–polyacrylamide gel electrophoresis immunoblotting (a single band of 45 kDa), and immunohistochemistry. In only three of seven ovarian carcinomas did expression of radioligand-bound receptors exceed levels found in five normal ovaries. However, only receptors in ovarian carcinoma specimens showed a high degree of immunoreactivity. Hence, even without elevations of the total receptor level, a folate receptor isoform homologous to human milk folate receptor protein seemed to prevail in serous ovarian carcinomas.

Evidence for a multiple binding mode of bispyridinium-type allosteric modulators of muscarinic receptors

The ligand binding properties of muscarinic receptors can be modulated by allosterically acting compounds. Here, a set of novel bispyridinium-type compounds was investigated which were designed to study structure-activity relationships and to provide more insight into the molecular events underlying the allosteric delay of the dissociation of [ 3H] N-methylscopolamine from muscarinic M 2 receptors in porcine cardiac membranes. The parent compound, a non-substituted bispyridinium oxime, displayed a weak allosteric potency and was unable to prevent radioligand dissociation at maximum concentrations. Introduction of either a phthalimidomethyl-moiety or a dichlorobenzyl-moiety at one end of the parent compound led to a considerable increase of the allosteric activity with regard to both the potency and the maximum effect. In these unilaterally ring-substituted bispyridiniums, homologous contralateral non-aromatic modifications were accompanied by divergent potency shifts depending on whether the unilateral ring was phthalimidomethyl or dichlorobenzyl. The findings point to a multiple binding mode of bispyridinium compounds at M 2 receptors in the [ 3H] N-methylscopolamine-occupied state, i.e., different orientations of the compounds at the allosteric binding area or even an interaction with distinct allosteric recognition sites.

Two-point kinetic experiments to quantify allosteric effects on radioligand dissociation.

Allosteric modulation of a receptor may be proved experimentally by demonstrating an altered radioligand dissociation in the presence of the allosteric modulator. Two-point kinetic experiments provide a screening-type approach to determine the delay of radioligand dissociation caused by allosteric modulation. In this article, Evi Kostenis and Klaus Mohr describe a pitfall in the data analysis that may lead to a suboptimum determination of the allosteric potency in the case of monophasic dissociations, and suggest how this problem may be resolved.

Presynaptic Inhibition of [3H]Nitrendipine Binding and Modification of Membrane Properties by Insecticidal Dihydropyrazoles in Mammalian Brain

RH-3421 and RH-5529 displace specifically bound [3H]nitrendipine in mouse brain synaptosomes, suggesting that dihydropyrazoles act at presynaptic L-type calcium channels in mammalian brain. Scatchard analysis revealed that both compounds interfere with binding of [3H]nitrendipine through a mechanism which reduces the number of available radioligand binding sites (Bmax) without altering the affinity (Kd) for remaining sites. In addition, there was no discernible change in either the rate of association of [3H]nitrendipine or in the rate of radioligand dissociation when the binding preparation was exposed to dihydropyrazoles. Such a binding profile indicates that dihydropyrazoles operate as noncompetitive blockers of [3H]nitrendipine binding without allosteric involvement. The effects of RH-3421 and RH-5529 on the physical properties of synaptic plasma membranes prepared from mouse brain were also evaluated using the fluorescence probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethyl-ammonium phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), which monitor the physical state of lipids in the membrane core and membrane surface, respectively. Exposure of synaptic membranes to RH-3421 and RH-5529 resulted in a reduction in fluorescence polarization of both probes, confirming that these lipid domains are accessible to dihydropyrazoles. Estimates of static and dynamic parameters show that lipid order and fluidity are modified within surface and core regions by dihydropyrazoles; however, the changes to regions probed by TMA-DPH appear more closely correlated with binding data. Overall, our results are consistent with the proposal that dihydropyrazoles reduce the availability of [3H]nitrendipine binding sites, possibly by modifying the physicostructural properties of the surface membrane. However, we do not exclude the possibility that dihydropyrazoles are irreversible inhibitors of [3H]nitrendipine binding or cause more subtle interference with regulatory biomolecules in the channel environment.

Positive allosteric interactions on cardiac muscarinic receptors: effects of chemical modifications of disulphide and carboxyl groups

Changes in the allosteric effects of alcuronium on rat cardiac muscarinic receptors were investigated after chemical modifications of S-S bonds or free carboxyl groups. In membranes pretreated with dithiothreitol, alcuronium lost its positive action on the binding of [ 3 H] methyl-N- scopolamine while its inhibitory effect on radioligand dissociation was preserved. In membranes pretreated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), known to modify free carboxyl groups in proteins, the ability to bind [ 3 H] methyl-N- scopolamine was preserved if the pretreatment had been performed in the presence of alcuronium, methyl- N-scopolamine or carbachol, while the positive cooperative effect of alcuronium on [ 3 H] methyl-N- scopolamine binding was only preserved in membranes that had been exposed to EDC in the presence of alcuronium. Methyl- N-scopolamine, carbachol and alcuronium differed in their ability to protect (against EDC) the action of alcuronium on the rate of [ 3 H] methyl-N- scopolamine dissociation. The results suggest that the disulphide bridge connecting the first two extracellular loops of muscarinic receptors is important for the positive allosteric action of alcuronium and that three carboxyl groups (presumably aspartate residues) are involved in receptor interactions with alcuronium and methyl- N-scopolamine. The first group is important for the effect of alcuronium on the affinity for methyl- N-scopolamine, the second is critical for the effect of alcuronium on the rate of methyl- N-scopolamine dissociation, and the third is critical for methyl- N-scopolamine binding. Presumably, the two charged nitrogens of alcuronium associate with the first and the second of the three groups involved.

The high-affinity folate receptor of normal and malignant human colonic mucosa.

The hypothesis that folate depletion is a risk factor for development of colonic neoplasia prompted us to study the presence of a putative folate receptor in human colon mucosa. Binding of 3H-folate to normal and malignant mucosa studied by equilibrium dialysis was of high-affinity type (K = 10(10) L/mol) and displayed apparent positive cooperativity. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. As compared to methotrexate, 5-formyltetrahydrofolate was a potent inhibitor of binding. Gel filtration revealed a 25 kDa and a 100 kDa peak of folate-binding activity. Immunoreactivity studies were performed with rabbit antibodies against human 25 kDa milk folate-binding protein. Immunoblotting showed a single band at 65 kDa, and tissue sections exhibited immunostaining of mucosal areas. The present folate receptor with characteristics similar to those of other high-affinity folate-binding proteins may serve as a regulator of intracellular folate concentration in colon mucosa.

Folate receptor of human mammary adenocarcinoma.

Binding of 3H-folate to human mammary tumor homogenate was of a high-affinity type (K = 10(10) M-1) and displayed apparent positive cooperativity. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. As compared to methotrexate, 5-formyltetrahydrofolate acted as a strong inhibitor of radioligand binding. Gel chromatography of radioligand-labeled homogenate of tumor tissue revealed three peaks: a small > or = 110 kDa peak and two major peaks of folate-binding activity (M(r) approximately 25 kDa and M(r) approximately 100 kDa). Mammary tumor tissue showed immunostaining with rabbit antibodies against human milk folate binder. A parallel elevation in the concentrations of folate-binding protein and triglyceride in tumor tissue as compared to normal tissue adjacent to the tumor was compatible with the localization of folate-binding protein in the triglyceride-rich fraction of mammary gland homogenate.

High-affinity folate binding in human prostate

Binding of 3H-folate in Triton X-100 solubilized human prostate homogenate was of a high-affinity type and displayed apparent positive cooperativity typical of specific folate binding. Radioligand dissociation was slow at pH 7.4, but rapid at pH 3.5. Gel chromatography revealed two major folate binding proteins (M(r) approximately 100 and 25 kDa), but only one single band (M(r) approximately 65-70 kDa) was detectable on SDS-PAGE and immunoblotting with rabbit-anti human milk folate binding protein. Concentration of folate binding protein in prostate homogenate expressed as maximum 3H-folate binding was 1.10 nmol/g protein, and the cross-reactivity with rabbit-anti human milk folate binding protein serum was 15% as determined by an enzyme-linked immunosorbent assay (median values; n = 6).

High-affinity folate binding in human mammary gland.

High-affinity 3H-folate binding in Triton X-100 solubilized human mammary gland tissue displayed characteristics, e.g. apparent positive cooperativity and increasing affinity with decreasing concentration of folate binding protein, shown to be typical of specific folate binding. Radioligand dissociation was slow at pH 7.4. A major fraction of the bound radioligand dissociated rapidly at pH 3.5, while a residual binding of 20% persisted even after prolonged dialysis at pH 3.5. Gel chromatography revealed two major folate binding proteins (M(r) approximately 100 kDa and 25 kDa). However, only one single band was detectable on SDS-PAGE immunoblotting. The highest folate binding activity per g protein was associated with the upper triglyceride-containing layer of the 1000 g supernatant of the homogenate. The folate binding protein extracted from this layer had a low cross-reactivity (< 5%) with rabbit antibodies against 25 kDa human milk folate binding protein. The folate binding protein in the 1000 g pellet and the aqueous phase of the 1000 g supernatant was present at a low concentration and had a cross-reactivity of 100%.

The effect of orthotopic transplantation on total, beta 1- and beta 2-adrenoceptors in the human heart.

1. [125I]-(-)pindolol binding was used to determine beta-adrenoceptor density in homogenate preparations of right ventricular endomyocardial biopsies from 43 non-rejecting patients over the first 13 months following cardiac transplantation. The selective beta 1 subtype antagonist ligand CGP 20712A was used to determine the subtype density in 32 of the patients. Biopsy specimens from 15 donor hearts were used as controls. 2. beta-adrenoceptor density (expressed in terms of fmol mg-1 protein) was increased in the group of transplanted hearts as a whole compared with the donor hearts with respect to total (35 +/- 2 vs 23 +/- 2) and the beta 1 subtype (25 +/- 2 vs 16 +/- 2) whereas the beta 2 subtype and radioligand dissociation constant did not differ. 3. Non-parametric analysis of variance of total receptor density over time revealed significant heterogeneity which appears to be due to a discrete increase in beta-adrenoceptor density during the 4th post operative month. 4. These results indicate that beta-adrenoceptor density is not constant following transplantation. Furthermore, the increase in receptor density following transplantation is due mainly to an increase in the beta 1 subtype without a significant change in the beta 2 subtype.

A high-affinity folate binding protein in human semen

The presence of a folate binding protein of high-affinity type (affinity constant 3.10(10) M-1, maximum folate binding 1.4 nM) in human semen was demonstrated in equilibrium dialysis experiments (37 degrees C, pH 7.4) with the radioligand 3H-folate. Radioligand dissociation from the binding protein was slow at pH 7.4, but rapid at pH 3.5. By use of rabbit antibodies against 25 kDa human milk folate binding protein we determined the concentration of folate binding protein in 16 specimens of human semen in an enzyme-linked immunosorbent assay. The concentration of immunoreactive folate binding protein was independent of the number of spermatozoa in individual specimens. Gel filtration showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one of 100 kDa and a minor one of 25 kDa.

On the interaction of gallamine with muscarinic receptor subtypes

The interaction of gallamine with muscarinic receptor subtypes was examined using radioligand binding studies. In competition studies using [ 3H]N-methylscopolamine ([ 3H]NMS), gallamine displayed high affinity for the rat cardiac and guinea-pig uterine M 2 muscarinic receptors and for the atypical muscarinic receptor present in chicken heart. Gallamine displayed low affinity for rat glandular and human 1321 N1 astrocytoma cell M 3 receptors and also for the M 4 receptors of NG108-15 and PC12 cells. The compound displayed intermediate affinity for M 1 receptors of rat cortex labeled using [ 3H]pirenzepine. The interaction of gallamine with the M 1 and M 2 receptors appeared to be competitive at the low concentrations required to determine affinity estimates. Thus, gallamine inhibited the binding of [ 3H]pirenzepine to M 1 receptors and [ 3H]NMS to M 2 receptors at concentrations that were 263- and 23-fold lower, respectively, than those required to decrease radioligand dissociation kinetics. Furthermore, gallamine, at a concentration that inhibited between 63 and 71% of specific radioligand binding, had no effect on the observed rate of association of the radioligand with either the M 1 or the M 2 receptor. At the M 3 glandular receptor, there was little separation between the concentrations of gallamine that produced inhibition of binding and those that decreased the association and dissociation rates of [ 3H]NMS. It is therefore difficult to determine if the inhibition of binding seen in competition studies on the M 3 receptor was produced through a competitive or an allosteric mechanism. Despite its possible allosteric properties at the M 3 receptor, gallamine can be used to detect heterogeneity of muscarinic receptor subtypes in several tissues and therefore represents a useful tool for defining muscarinic receptor subtypes.

A new class of calcium entry blockers defined by 1,3-diphosphonates: Interactions of SR-7037 (belfosdil) with receptors for calcium channel ligands

Abstract Tetrabutyl-2(2-phenoxyethyl)-1,3-propylidene diphosphonate (SR-7037) completely displaced dihydropyridine [( 3H]PN200-110), phenylalkylamine [( 3H]D888), and benzothiazepine [( 3H]diltiazem) ligands from brain L-type calcium channels. Half-maximal inhibition of [3H]PN200-110 binding occurred at 19 nM with a Hill coefficient of 0.96. SR-7037 primarily decreased the affinity for [3H]PN200-110 with a small, but significantly, effect on the maximal binding capacity. Kinetic studies showed that this was due to an increased radioligand dissociation rate from 0.04 min-1 to 0.43 min-1 in the presence of the diphosphonate. Displacement of [3H]D888 by SR-7037 was biphasic with respective IC50 of 44 and 8400 nM. Likewise, unlabeled (-)-D888 identified two sites with IC50 values of 0.9 and 27 nM. Both SR-7037 (1000 nM) and D888 (200 nM) accelerated radioligand dissociation about 2-fold. [3H]Diltiazem binding was inhibited by SR-7037 with an IC50 value of 29 nM. The inhibition of dihydropyridine binding by SR-7037 is enhanced by most divalent cations at millimolar concentrations with the following potency: Mn2+ greater than Mg2+ greater than Ca2+ greater than Co2+. Barium has the opposite effect. The half-maximal effect of calcium occurred at 6 microM free ion. Specific binding of [3H]D888 was antagonized in the presence of 1 mM CaCl2. It is concluded that SR-7037 has allosteric interactions with the dihydropyridine receptor of the L-type calcium channel. The differential effect of Ca2+ on the potency of D888 and diltiazem relative to that of SR-7037 indicates that the three drugs may bind to nonequivalent sites. These results support specific calcium channel inhibition, possibly at a novel site, as the primary mechanism of the diphosphonate's pharmacological actions.

Characterization of guinea-pig cardiac muscarinic receptors by radioligand dissociation kinetics

The question of cardiac muscarinic receptor heterogeneity was studied in guinea-pig auricles and ventricle. Radioligand dissociation kinetics were analyzed in the absence and presence of alinidine and AQ-A 39, two muscarinic agents and allosteric modulators of radiotracer dissociation. The dissociation kinetics were monophasic with all 3 radiotracers used and in both auricles and ventricle. [ 125I]3-Quinuclidinyl 4-iodobenzilate ([ 125I]QNB) dissociated with an identical half-life ( t 1 2 off ) in ventricle and in left and right auricle, respectively. Alinidine (1 mM) decreased t 1 2 off by 50% in ventricle but had no significant influence in auricles. AQ-A 39 (= falipamil) uniformly increased t 1 2 off 4-fold in all 3 tissues. The binding of tritium-labelled quinuclidinyl benzilate ([ 3H]QNB) dissociated equally rapidly from both ventricular and auricular receptors under control conditions but was not affected by alinidine. AQ-A 39 (1 mM) slowed [ 3H]QNB dissociation 6.5-fold in ventricle but only 3-fold in both auricles. [ 3H]N-Methyl scopolamine ([ 3H]NMS) dissociation was uniform in both auricles but was somewhat slower in the ventricle, both in the absence and presence of alinidine. AQ-A 39 was without effect. These results demonstrate differences in ventricular versus auricular receptors detected by [ 125I]QNB in the presence of alinidine and by [ 3H]QNB in the presence of AQ-A 39. The quaternary ligand [ 3H]NMS was unable to detect receptor heterogeneity. No differences were found between right and left auricular receptors. The results presented provide no evidence for the existence of different subtypes of muscarinic receptors for the negative chronotropic and inotropic actions of muscarinic agonists.