BIOCHEMICAL CHARACTERISTICS OF MUSCARINIC CHOLINORECEPTORS IN SWINE TRACHEAL SMOOTH MUSCLE

• 1 The tritiated muscarinic cholinoreceptor antagonist quinuclidinyl benzilate, [³H]QNB, was used to characterize the muscarinic receptors associated with homogenized membrane of the smooth muscle from swine trachea. Based on receptor binding assays, the homogenate had specific, saturable, high-affinity receptors for [³H]QNB.
• 2 Specific binding was time- and temperature-dependent. The association of [³H]QNB with the muscarinic receptor reached equilibrium much sooner at 37°C than 25°C at a [³H]QNB concentration of 180 pM (30 min and 2 h, respectively). Equilibrium at both temperatures was attained within 5 min at a [³H]QNB concentration of 1800 pM. All remaining experiments were performed at 37°C.
• 3 Binding was saturable with respect to [³H]QNB and tissue concentrations. Analysis of binding isotherms yielded an apparent equilibrium dissociation constant (K_D) of 51±20 pM and a maximum receptor density (B_max) of 2.17±0.27 pmole/mg protein. The Hill coefficient for [³H]QNB binding was 1.07±0.16. The association (K₁) and dissociation (K_-1) rate constants were determined to be (5.51±0.16) × 10⁸ M^?1 min^?1 and (1.41±0.18) × 10^?2 min^?1, respectively. K_D calculated from the ratio of K₁ and K_-1 was 26.3±3.8 pM; this value is close to the value of K_D calculated from Scatchard plots of binding isotherms.
• 4 The density of muscarinic receptor binding sites was 10-fold greater in tracheal smooth muscle than in tracheal epithelium (0.20±0.03 pmole/mg protein). There is no difference between weanling and young adult swine in the density of muscarinic receptors in tracheal smooth muscle.
• 5 The nonselective muscarinic antagonists atropine, scopolamine and quinuclidinyl benzilate (QNB) competitively inhibited [³H]QNB binding to the homogenate with Hill coefficients of 0.9-1.0 and inhibition constants (K_i) of nanomolar range.
• 6 Competition with selective muscarinic antagonists pirenzepine and 3-quinuclidinyl xanthene-9-carboxylate (QNX) gave K_i values, 0.26 M and 0.78 nM, respectively, and Hill coefficients of approximately 1. There was a single population of [³H]QNB binding sites of the M₂ subtype for all tested muscarinic antagonists.
• 7 Competition with selective muscarinic agonists pilocarpine and carbachol yielded K_i values of micromolar range, Hill coefficients of less than 1, and revealed the existence of two binding sites (P < 0.01).

     

Interaction of antihistaminics with muscarinic receptor (III)

The muscarinic antagonist 1-[benzilic 4,4′-³H]quinuclidinyl benzilate ([³H] QNB) bound to a single class of muscarinic receptors with high affinity in rabbit ileal membranes. The K _D and B _max values for [³H]QNB calculated from analysis of saturation isotherms were 52.5 pM and 154 fmol/mg, respectively. Chlorpheniramine (CHP), histamine H₁ blocker, increased K _D value for [³H]QNB without affecting the binding site concentrations and Hill coefficient. The K _i value of CHP for inhibition of [³H]QNB binding in ileal membranes was 1.44μM and the pseudo-Hill coefficient for CHP was close to unit. In the functional assay carbachol, muscarinic agonist, increased the contractile force of ileum with ED₅₀ value of 0.11μM. CHP caused the rightward shift of the dose-response curve to carbachol. The pA₂ value of CHP determined from Schild analysis of carbachol-induced contraction was 5.77 and the slope was unity indicating competitive antagonism with carbachol. The dissociation constant (K _i ) of CHP obtained in competitive experiments with [³H]QNB was similar to the K _A value (1.69μM) of CHP as inhibitor of carbachol-induced contraction in rabbit ileum. This result suggests that the binding of H₁ blocker, CHP, vs [³H]QNB to muscarinic receptors in ileal membranes represents an interaction with a receptor of physiological relevance.     

Effect of bay K 8644, a calcium channel agonist,on dog cardiac muscarinic receptors

To investigate further whether the effects of the dihydropyridine (DHP) drugs on calcium channels are related to those of these drugs on muscarinic receptors, the binding characteristics of the DHP calcium channel agonist, Bay K 8644, on muscarinic receptors and calcium channels were compared to those of the DHP calcium channel antagonists, nicardipine and nimodipine in the dog cardiac sarcolemma. Bay K 8644, nicardipine and nimodipine inhibited the specific [³H]QNB binding with K _i values of 16.7μM, 3.5μM and 15.5μM respectively. Saturation data of [³H]QNB binding in the presence of these DHP drugs showed this inhibition to be competitive. Bay K 8644, like nicardipine and nimodipine, blocked the binding of [³H]nitrendipine to the high affinity DHP binding sites, but atropine did not, indicating that the muscarinic receptors and the DHP binding sites on calcium channels are distinct. The K _i value of Bay K 8644 for the DHP binding sites was 4 nM. Nicardipine and nimodipine (K _i :0.1–0.2 nM) were at least 20 times more potent than Bay K 8644 in inhibiting [³H]nitrendipine binding. Thus, the muscarinic receptors were about 4000 times less sensitive than these high affinity DHP binding sites to Bay K 8644. These results suggest that the DHP calcium agonist Bay K 8644 binds directly to the muscarinic receptors but its interaction with the muscarinic receptors is not related to its binding to the DHP binding sites on calcium channels.     

Interaction of agonists and selective antagonists with gastric smooth muscle muscarinic receptors

Summary The interaction of cholinergic agonists and antagonists with smooth muscle muscarinic receptors has been investigated by measurement of displacement of the muscarinic antagonist [³H]QNB (quinuclidinyl benzilate) in membranes prepared from toad stomach. The binding of [³H]QNB was saturable, reversible and of high affinity (K _D = 423 pM). The muscarinic receptor subtypes present in gastric smooth muscle were classified by determining the relative affinities for the selective antagonists pirenzepine (M₁), AF-DX 116 (M₂) and 4-DAMP (M₃). The results from these studies indicate the presence of a heterogeneous population of muscarinic receptor subtypes, with a majority (88%) exhibiting characteristics of M₃ receptors and a much smaller population (12%) exhibiting characteristics of M₂ receptors. The binding curve for the displacement of [³H]QNB binding by the agonist oxotremorine was complex and was consistent with presence of two affinity states: 24% of the receptors had a high affinity (K _D = 4.7 nM) for oxotremorine and 76% displayed nearly a 1,000-fold lower affinity (K _D = 4.4 M). When oxotremorine displacement of [³H]QNB binding was determined in the presence GTPS, high affinity binding was abolished, indicating that high affinity agonist binding may represent receptors coupled to G proteins. Moreover, pertussis toxin pretreatment of membranes also abolished high affinity agonist binding, indicating that the muscarinic receptors are coupled to pertussis toxin-sensitive G proteins. Reaction of smooth muscle membranes with pertussis toxin in the presence [³²P]NAD caused the [³²P]-labelling of a 40 kD protein that may represent the subunit(s) of G proteins that are known to be NAD-ribosylated by the toxin. We conclude that both M₃ and M₂ receptors may be coupled to G proteins in a pertussis-sensitive manner. Send offprint requests to T. W. Honeyman at the above address     

Binding of [3H]quinuclidinyl benzilate to intestinal mucus: An artifact in identification of epithelial cell muscarinic receptors

The widely used muscarinic receptor ligand [³H]quinuclidinyl benzilate ([³H]QNB) was found to bind in a site-specific but artifactual manner to rat intestinal mucus, obscuring specific binding to muscarinic receptors on intestinal epithelial cells. Atropine inhibited [³H]QNB binding to mucus with an apparent IC₅₀ of 2.1 × 10^?7 M, compared to an IC₅₀ of 1.4 × 10^?8 M obtained with a homogenate of intestinal epithelial cells. Unlabeled QNB also inhibited binding of [³H]QNB to mucus but the apparent IC₅₀(4 × 10^?7 M) was about 300-fold greater than the IC₅₀ determined with a control tissue, heart muscle (IC₅₀, 1.2 × 10^?9M). [³H]QNB binding was saturable over the concentration range of 1–7 nM in the heart, with an apparent k_D of 0.76 nM. As expected from the high IC₅₀ for QNB in the mucus binding experiments, binding to mucus was not saturable over the 1–15 nM concentration range. Based on pH profiles and temperature dependency of binding, it seems unlikely that mucin, the primary component of mucus, was responsible for [³H]QNB binding to the mucus. The findings have implications for studies which involve binding of [³H]QNB in particular and other ligands in general to mucus-secreting epithelial tissues.     

The influence of guanyl-5′-yl imidodiphosphate and sodium chloride on the binding of the muscarinic agonist, [3H] cis methyldioxolane

[³H]Quinuclydinyl benzylate([³H]QNB) binding was carried out on crude synaptosomal membranes isolated from cat cerebral cortex. The specific binding showed a single type of site with K_D 0.25 nM, Hill number 0.89 and B_max 114 pmol/g protein. The local anesthetics procaine, tetracaine, and the adrenergic antagonists phentolamine and propranolol, in concentrations between 1 nM and 500 μM, inhibited [³H]QNB binding with K_i varying between 9 μM for procaine and 80 μM for propranolol. The Hill coefficients obtained from logit/log plots suggested that there was no cooperativity between the binding sites for local anesthetics. At various concentrations the inhibition by procaine and propranolol may appear as competitive or non-competitive. The Hill numbers obtained from the saturation curves suggest that there was no cooperativity between anesthetics and [³H]QNB binding sites. Neither 1 mM Ca²⁺ nor Mg²⁺ affected [³H]QNB binding or the action of the drugs. The effect of local anesthetics and adrenergic antagonists was reversible and these drugs did not protect the muscarinic receptor from the deleterious effect of Triton X-100 as was the case with muscarinic agents. Our findings suggest that local anesthetics inhibit [³H]QNB binding to the muscarinic receptor by acting at some accessory site but not on the true receptor site. The possible mechanism of action of local anesthetics on synaptic transmission is discussed.     

Muscarinic Receptor Binding Sites of the M4 Subtype in Porcine Lung Parenchyma

Abstract: Muscarinic acetylcholine receptors regulate distal airway resistance and secretion. The subtype expressed in the lung in different species remains uncertain. It has recently become possible to identify the M₄ subtype by careful comparison of antagonist affinities. We characterized the binding of [³H]quinuclidinyl benzilate ([³H]QNB) to muscarinic receptors in cell membranes from lung parenchyma of 2–8 week old pigs in comparison to cloned human M₃ and M₄ receptors expressed in COS cells, to M₂ in rat atria and to M₄ in bovine adrenal medulla. In porcine lung, [³H]QNB bound with high affinity (K_d=95±9pM) to a single homogeneous population of muscarinic receptor sites (B_max=340±10 fmol/mg protein). Competition studies showed that the affinity (expressed as pK_i) of 3 selective blockers was in close agreement between pig lung and cloned human m₄ (r=0.996). A series of 10 blockers showed affinities closely matching reported values for M₄ receptors of the adrenal medulla (r=0.965). Conversely, affinity values in porcine lung differed significantly (P< 0.05, t-test) from those determined in parallel with either human cloned M₃ or with rat atria expressing the M₂ subtype. We conclude that pig lung muscarinic receptor binding sites most closely resemble the M₄ subtype, in contrast to the M₃ subtype typical of large airways in this species.     

Peripheral blood lymphocyte muscarinic cholinergic receptors in airway hyperresponsiveness: a marker of cholinergic dysfunction?

1 Muscarinic cholinergic receptors were assayed in human peripheral blood lymphocytes of healthy control and airway hyperresponsive subjects using a radioligand binding assay technique and the muscarinic cholinergic receptor antagonist [³H]-quinuclidinyl benzilate (QNB) as a radioligand. Subjects investigated were divided in four different groups based on threshold responses to methacholine inhaled as challenge test. 2 [³H]-QNB was bound to human peripheral blood lymphocytes in a manner consistent with the labelling of muscarinic cholinergic receptors. Dissociation constant (K_d) values of [³H]-QNB binding were similar in the different groups examined, whereas maximum density of binding sites (B_max) was increased in airway hyperresponsive subjects in comparison with healthy controls. 3 The above findings indicate that the density of muscarinic cholinergic receptors is increased in peripheral blood lymphocytes of airway hyperresponsive subjects. 4 This suggests that airway hyperresponsiveness is associated with cholinergic hyperreactivity and is probably a systemic cholinergic dysfunction since it is accompanied by changes in the density of muscarinic cholinergic receptors expressed by peripheral blood lymphocytes.     

AUTORADIOGRAPHIC EVIDENCE FOR A HETEROGENEOUS DISTRIBUTION OF α1- ADRENORECEPTORS LABELLED BY [3 H] PRAZOSIN IN RAT,DOG AND HUMAN KIDNEY

• 1 The selective α_radrenoreceptor antagonist radioligand [³ H] prazosin has been used to localize α₁ adrenoreceptors in slide mounted sections of rat, dog and human kidney.
• 2 The biochemical characteristics of [³ H] prazosin binding to rat kidney sections were examined and found to be saturable, reversible, stereoselective, with a K_D of 0.12 nM and B_max of 6.72 ± 0.2 fmoles/section.
• 3 ³ H Ultrofilm images of [³ H] prazosin binding to rat, dog, and human kidney revealed binding to the vasculature but in the rat additional receptors were confined to the renal cortex.
• 4 In the rat kidney autoradiography using emulsion coated coverslips showed that binding in the renal cortex was largely to proximal tubules.
• 5 In all three species the autoradiographic studies support a role for α₁-adrenoreceptors in control of renal blood flow. In the rat the location of α₁-adrenoreceptors suggests that they can also have an important influence on fluid and electrolyte balance, gluconeogenesis and production of prostanoids.

     

Role of phospholipids in muscarinic binding by neural membranes.

A role for acidic phospholipids in brain muscarinic receptors is suggested by their effect on the specific binding of a potent muscarinic antagonist, tritiated 3-quinuclidinyl benzilate ([³H]QNB), to neural membranes. The addition of phosphatidylserine (PS) enhances specific muscarinic binding up to 40 per cent, while phosphatidic acid (PA) and phosphatidylinositol (PI) are less effective, and neutral lipids are generally without effect. Unsaturated fatty acids, but neither saturated fatty acids nor the methyl esters of unsaturated fatty acids, inhibit [³H]QNB binding, the amount of inhibition being proportional to the degree of unsaturation. Unsaturation in the phospholipids is also inimical to muscarinic binding. Pretreatment of the membranes with phospholipases A and C decreases binding 100 and 60 per cent respectively. The addition of acidic phospholipids restores some binding activity after phospholipase treatment. Inhibition of [³H]QNB binding by low concentrations of cationic detergents is completely reversible by PS and PA. Ionic, pH and temperature effects on QNB binding indicate that the association of QNB with muscarinic receptors involves primarily hydrophobic interactions.     

Binding studies with [3H]cis-methyldioxolane in different tissues

Summary Special conditions - tricine buffer containing Ca²⁺ and Mg²⁺, 22°C (TCM) — allow to label a much higher proportion of muscarinic receptors by [³H]cis-methyldioxolane (CD) than hitherto described (Vickroy et al. 1984 a). Taking the maximum number of binding sites, B _max, of [³H]QNB as 100%, B _max of [³H]CD amounts to 83% in the rat heart instead of the reported 17%, 33% in the cerebral cortex instead of 6%, 20% in hippocampus and 55% in pons/medulla. In the salivary glands specific binding was negligible. The affinities of a number of muscarinic agonists and antagonists to [³H]CD and [³H]QNB binding sites in different tissues of the rat are compared. Apparent affinities of agonists are much higher in the [³H]CD system, affinities of antagonists are slightly higher in the [³H]QNB system. In both assay systems receptors of heart and pons/ medulla membranes seem to have similar drug specificity. They differ somewhat from those in the cortex. Receptors in the salivary glands, however, seem to be completely different from those in the other three tissues. In the heart [³H]CD binding can be abolished almost completely by GppNHp. In the cortex about half of the [³H]CD binding is susceptible to GppNHp. The reduction of binding in the cortex is due to a change in B _max and not in the dissociation constant K _D. Competition of unlabelled pirenzepine with [³H]CD: In heart and pons/medulla only low affinity sites for pirenzepine (M₂-receptors) are labelled by [³H]CD. In regions rich in M₁ receptors like hippocampus (80% M₁ receptors) or cortex (65–70% M₁ receptors) the proportion of M₁ receptors labelled by [³H]CD is smaller than expected considering the concentration of M₁ receptors present in these tissues. Thus [³H]CD, under the conditions described in this paper, seems to label preferentially but not exclusively M₂ receptors in their agonist high affinity form. Send offprint requests to A. Closse at the above address     

Autoradiographic localization of muscarinic acetylcholine receptors in the rat pulmonary vascular tree.

The pharmacological characteristics and the anatomical localization of muscarinic receptors in the pulmonary vascular tree were investigated in lung sections of Wister-Kyoto (WKY) and spontaneously hypertensive rats (SHR). [3H]Quinuclidinyl benzylate [( 3H]QNB) was bound by sections of rat lung in a manner consistent with the labeling of muscarinic acetylcholine receptors, with a dissociation constant value (Kd) of 0.41 +/- 0.3 nM in WKY rats and of 0.37 +/- 0.2 nM in SHR. The density of muscarinic acetylcholine receptors was higher in sections of lung of WKY rats than of SHR. In the pulmonary vasculature these sites were associated with the smooth muscle of the medial layer of different size branches of the pulmonary artery and vein. No [3H]QNB binding sites were found within the endothelium in the blood vessels of either WKY rats or SHR. The density of [3H]QNB binding sites was significantly lower in the smooth muscle of pulmonary vein and its branches in SHR. There were no significant hypertension-dependent changes in the density and pattern of muscarinic receptors of pulmonary artery smooth muscle.     

3H]Quinuclidinyl benzilate binding to muscarinic receptors and [3H]WB-4101 binding to alpha-adrenergic receptors in rabbit iris. Comparison of results in slices and microsomal fractions

The binding characteristics of [³H]-(l)-quinuclidinyl benzilate (QNB) and [³H]WB-4101 to microsomal fractions and slices from rabbit iris muscle were compared. [³H] QNB binding to both microsomal fractions and muscle slices was of high affinity and low capacity and was displaced by muscarinic ligands. The equilibrium dissociation constants (K_D) for [³H]QNB binding to microsomes and slices were 0.069 nM and 1.97nM, respectively. This shift to a higher value for the K_p of the microsomal fraction compared with that of the slices was also observed lor the association rate constants (K_I) and inhibition constants (K_J), but not for the dissociation rate constants (K_?1). Kinetic studies on the binding characteristics of [³H]WB-4101 revealed high affinity sites with K_D values of 2.33 and 10.19 nM for microsomal fractions and slices, respectively. The findings of comparable binding patterns for [³H]QNB and [³H]WB-4101 binding to microsomal fractions and intact muscle slices argue against the possibility of alterations in receptor properties following tissue disruption. It is proposed that the differences in receptor-mediated biochemical responses that are seen between intact tissue and cell-free homogenates, such as the ‘phosphoinositide effect’, are more likely to be due to alterations in receptor function, e.g. changes in ionic permeabilities, rather than to actual changes in receptor properties.     

Muscarinic cholinergic binding in striatal and mesolimbic areas of the rat: reduction by 6-hydroxydopa.

The pilocarpine-induced catalepsy and the specific binding of [³H]quinuclidinyl benzilate ([³H]QNB) in synaptic membranes were investigated in the adult rat neonatally treated with 6-hydroxydopa (6-OHDOPA). An i.p. administration of pilocarpine (75–150 mg/kg) elicited less catalepsy in the 6-OHDOPA-treated rats than in the controls. The specific binding of [³H]QNB in the striatal or mesolimbic homogenates proved to be saturable. In addition, treatment with 6-OHDOPA caused a significant reduction in B_max values of [³H]QNB binding in both regions but did not alter the K_D values as compared with those of the controls. These results suggest that neonatal treatment with 6-OHDOPA induces a muscarinic-cholinergic hyposensitivity in the rat brain which is probably due to the decrease in the number of muscarinic-cholinergic receptors in the striatal and/or mesolimbic areas.     

Differential alterations of cholinergic muscarinic receptors during chronic and acute tolerance to organophosphorus insecticides

Male mice treated for 2 weeks with the anticholinesterase insecticide disulfoton (O,O-diethylS-[2-(ethylthio)-ethyl] phosphorodithioate; 10 mg per kg per day) became tolerant to the hypothermic and antinociceptive effects of disulfoton itself and of oxotremorine, a muscarinic cholinergic agonist. Homogenates of brain and ileum from tolerant animals exhibited reduced binding of the specific muscarinic antagonist [³H]quinuclidinyl benzilate ([³H]QNB). In forebrains of tolerant animals, the number of receptors (B_max) was decreased 40% with no change in the affinity constant. Acetylcholinesterase (AChE) activity was 15% of control. Forty-eight hours after a single injection of disulfoton (10 mg/kg) mice were more resistant than their controls to the hypothermic and antinociceptive effects of a second administration of the same insecticide and of oxotremorine. Tolerance was not present 96 hr after a single administration of disulfoton. A single injection of disulfoton produced 74, 65 and 27% inhibition of AChE activity after 4, 48 and 96 hr respectively. Four hours after a second injection at 48 or 96 hr, 73 or 72% inhibition was found. [³H]QNB binding of animals treated with a single injection of disulfoton and of controls did not differ at either time point. An increase in the K_i for inhibition of [³H]QNB binding by unlabeled oxotremorine was observed in forebrain from mice killed 48 hr after a single injection of disulfoton, indicating a decreased affinity of the muscarinic receptor for agonists. Binding of [³H]oxotremorine-M was decreased significantly 48 hr after a single injection of disulfoton and after chronic treatment. It is suggested that a differential down-regulation of muscarinic receptors occurs in acute and chronic tolerance, involving agonist and antagonist binding sites and depending on duration of exposure.     

Interactions of D600 (methoxyverapamil) and local anesthetics with rat brain α-adrenergic and muscarinic receptors

D600 (methoxyverapamil) was found to inhibit the specific binding assayed in rat brain homogenates of the antagonist agents [³H]WB 4101 and [³H]QNB to the α-adrenergic and muscarinic receptors respectively. The IC₅₀ concentrations of D600 in standard binding experiments were 1.7 × 10_?6 M and 1.4 × 10 _?5M, with calculated k₁ values of 0.98 × 10_?6 M and 8.83 × 10_?6M.Scatchard analyses showed these inhibitions to be competitive. Lidocaine and tetracaine also inhibited radioligand binding to these receptors, with K₁ values of 5.25 × 10^?4M and 4.85 × 10^?5 M for the α-receptor and 8.2 × 10^t-5 M and 6.94 × 10^?6 M for the muscarinic receptor; these inhibitions also appeared to be competitive. Increasing the Ca²⁺ concentration in the assays to 10 mM did not influence the effects of D600 or the anesthetics. Analyses of inhibitions of muscarinic receptor binding produced by D600 and lidocaine over a range of pH indicated that the inhibitory species of D600 is the uncharged form, whereas the charged form of lidocaine is inhibitory. Interactions of D600 and lidocaine with the agonist site on the muscarinic receptor were studied by measuring the effects of these agents on the displacement of [³H]QNB by the muscarinic agonist carbachol. Comparison of these results with a theoretical model indicates that carbachol, [³H]QNB, and D600 or lidocaine competitively displace one another at the same agonist site. The binding of labeled naloxone to the opiate receptor was also inhibited by D600, the IC₅₀g being 4 × 10^t?6 M. These inhibitory effects of D600 and the local anesthetics on different receptors suggest that these agents may act by a common mechanism, namely by perturbing membrane structures. These results suggest caution in interpreting experiments in which D600 and verapamil are used analytically as Ca antagonists to assess the involvement of Ca in a biological system.     

Properties of the muscarinic cholinergic receptors in rat atrium

Summary 1. Properties of the muscarinic cholinergic receptor sites in the rat atrial homogenate and microsomal fraction were studied by the use of tritium labelled 3-quinuclidinyl benzilate ([³H]-QNB), a potent muscarinic antagonist. 2. The specific [³H]-QNB binding to the receptor sites displayed saturability, stereospecificity as well as reversibility. 3. The competition studies showed that muscarinic antagonists were more potent than muscarinic agonists. 4. Certain neuromuscular blocking agents, antipsychotics, antiarrhythmics and antihistamines also were capable of interacting with the [³H]-QNB binding sites. However, - and -adrenergic agents, calcium antagonist (D-600) and ionophore (A-23187) failed to show any effect. 5. Analyses by the double reciprocal plot, Hill plot and Scatchard plot of the dependence of the specific [³H]-QNB binding on the concentration of QNB suggested that binding was occurring to a single population of receptor sites in the atrial homogenate or microsomal fraction. Further, there was no evidence of any detectable site to site interactions (positive or negative cooperative type). From the Scatchard plot, the equilibrium dissociation constant (K_D) of 1.1 nM was calculated and the Hill coefficient was close to 1.0 6. Interaction of the muscarinic antagonists with the [³H]-QNB sites showed the Hill coefficients close to 1.0 whereas for the agonists, the coefficients were much less than 1.0 indicating that agonist-receptor site interactions have some different characteristics from those following antagonist-receptor site interaction. 7. The rate and the maximal level of QNB binding to the receptor sites was markedly influenced by the temperature; various cations, on the other hand, displayed no effect either on the association or dissociation of QNB binding. The specific QNB binding exhibited a broad pH optimum from pH 6.0–8.5. 8. Treatment of the membrane fraction (or homogenate) with either phospholipase A or C and with p-chloromercuribenzoate caused significant inhibition of [³H]-QNB binding. 9. The QNB binding site was resistant to tryptic digestion. Even when about 40% of the membrane proteins were removed by the tryptic proteolysis, the [³H]-QNB binding ability of the membrane remained unaffected; in fact, the removal of tryptic proteolytic products by centrifugation markedly increased the specific QNB binding to the membrane.     

Novel alkoxy-oxazolyl-tetrahydropyridine muscarinic cholinergic receptor antagonists

The purpose of the present studies was to compare a novel series of alkoxy-oxazolyl-tetrahydropyridines (A-OXTPs) as muscarinic receptor antagonists. The affinity of these compounds for muscarinic receptors was determined by inhibition of [³H]pirenzepine to M1 receptors in hippocampus, [³H]QNB to M2 receptors in brainstem, and [³H]oxotremorine-M to high affinity muscarinic agonist binding sites in cortex. All of the compounds had higher affinity for [³H]pirenzepine than for [³H]QNB or [³H]oxotremorine-M labeled receptors, consistent with an interpretation that they are relatively selective M1 receptor antagonists, although none were as selective as pirenzepine. In addition, dose-response curves were determined for antagonism of oxotremorine-induced salivation (mediated by M3 receptors) and tremor (mediated by non-M1 receptors) in mice. In general, the A-OXTPs were equipotent and equieffective in antagonizing both salivation and tremor, although there were modest differences for some compounds. Dose-response curves also were determined on behavior maintained under a spatial-alternation schedule of food presentation in rats as a measure of effects on working memory. The A-OXTPs produced dose-related decreases in percent correct responding at doses three- to ten-fold lower than those which decreased rates of responding. However, only one compound, MB-OXTP, produced effects on percent correct responding consistent with a selective effect on memory as opposed to non-memory variables. The present results provide evidence that these alkoxy-oxazolyltetrahydropyridines are a novel series of modestly M1-selective muscarinic receptor antagonists, and that one member of the series, MB-OXTP, appears to be more selective in its effects on memory than previously studied muscarinic antagonists.     

The oxime HGG-12 as a muscarinic acetylcholine receptor antagonist without intrinsic activity in cardiac membranes

Direct interactions of the bispyridinium oxime HGG-12 with muscarinic acetylcholine receptors were investigated in porcine cardiac atrial membranes. Competition binding experiments using the radiolabeled muscarinic receptor antagonist (³H)QNB revealed specific binding of HGG-12 to muscarinic acetylcholine receptors of porcine atrial membranes with a dissociation constant of 3.8×10^–7 mol/l. Muscarinic acetylcholine receptor-stimulated binding of the radiolabeled GTP analog (³⁵S)GTP[S] to guanine nucleotide binding proteins (G-proteins) was used to study antagonistic and possible agonistic effects of HGG-12 at muscarinic acetylcholine receptors. HGG-12 completely inhibited carbachol- and oxotremorine-stimulated (³⁵S)GTP[S] binding to pertussis toxin sensitive and insensitive G-proteins in a competitive manner. Inhibition constants (K_I) of HGG-12 for blockade of carbachol- and oxotremorine-stimulated GTP[S]-binding (9.7×10^–7 mol/l and 1.7×10^–6 mol/l, respectively) were higher by about a factor of 100 than those of the muscarinic acetylcholine receptor antagonist atropine. In the absence of muscarinic acetylcholine receptor agonists, HGG-12 by itself had no stimulatory effect on (³⁵S)GTP[S] binding in porcine atrial membranes. The results of this study show that the oxime HGG-12 is a competitive antagonist without intrinsic activity at porcine atrial muscarinic acetylcholine receptors. The stimulatory action of HGG-12 on muscarinic acetylcholine receptors which has been described by several authors is, therefore, suggested to be due to partial inhibition of acetylcholinesterase by the oxime rather than to direct agonism at muscarinic acetylcholine receptors.     

Heterogeneity of Guinea-Pig Lung Muscarinic Receptors Revealed by [3H]4-DAMP-binding

Summary: Muscarinic receptors present in guinea-pig lung were characterized using the M₃ selective radioligand [³H]4-diphenylacetoxy-N-methyl-piperidine methiodide ([³H]4-DAMP). In saturation studies, [³H]4-DAMP identified two populations of binding sites with approximately 4% of the sites displaying high affinity (K_d=0.21 nM and B_max= 10 fmol/mg prot.) while the remaining sites were low affinity ones (K_d=18.11 nM and B_max=269 fmol/mg prot.). In competition studies with [³H]4-DAMP (0.35 nM), methoctramine and hexahydro-siladifenidol (HHSiD) identified 50 and 70% of high affinity binding sites displaying the pharmacological profile of the M₂ and the M₃ receptors, respectively. No evidence was found for high affinity [³H]pirenzepine binding sites in guinea-pig lung. However, pirenzepine/[³H]4-DAMP competition experiments suggested that pirenzepine recognized an equal proportion of [³H]4-DAMP binding sites with intermediate and low affinity binding constants. The intermediate affinity binding constant was inconsistent with the presence of M₁ receptors and reflected more the presence of M₄ or a mixture of M₃ and M₄ receptors. The low affinity pirenzepine binding sites may represent M₂ receptors. These results provide further evidence for the occurrence of M₂ and M₃ receptors and suggest the presence of the M₄ muscarinic receptor subtype in guinea-pig lung.