Binding characteristics and functional G protein coupling of muscarinic acetylcholine receptors in rat duodenum smooth muscle membranes

Summary The non-selective labelled antagonist [³H]N-methyl-scopolamine ([³H]NMS) was used to identify muscarinic acetylcholine receptors in rat duodenum smooth muscle membranes. Saturation and kinetic experiments revealed a binding site with a K_D-value of 0.2–0.3 nmol/l and a receptor concentration (B_max) of 100 fmol/mg protein. The affinities of eight selective muscarinic antagonists were determined and compared with those at M₁ (rat cerebral cortex), M₂ (rat heart), M₃ (rat submandibular gland) and M₄ (data from Dörje et al. 1991) receptors. The M₂-selective agent AF-DX 116, the group of M₂/M₄-selective compounds himbacine, AF-DX 384, AQ-RA 741 and methoctramine but also the M3-selective HHSiD showed affinities corresponding to M₂ and/or M₄ sites. The intermediate affinity of 4-DAMP favours a mixed M₂/M₄ receptor population mainly containing M₂ receptors. Two compounds, pirenzepine and AQ-RA 741, displayed biphasic displacement curves indicating the presence of a small population of putative M1 receptors. The rat duodenum antagonist binding profile, however, is not consistent with the presence of M3 receptors. We further demonstrate a concentration-dependent stimulation of [³⁵S]GTP[S] binding to duodenal G proteins by the muscarinic agonist oxotremorine. Estimation of the binding parameters of GTP[S] in absence and presence of oxotremorine provided evidence for a catalytic activation of G proteins by agonist-activated muscarinic receptors in rat duodenal membranes and a strong signal amplification on the G protein level. Send offprint requests to C. Liebmann at the above address     

EFFECT OF THYROID STATUS ON β-ADRENORECEPTORS AND MUSCARINIC RECEPTORS IN THE RAT LUNG

• 1 The effects of thyroid status on the specific binding of the muscarinic ligand (–)-[³H] quinuclidinyl benzilate (QNB) and of the β-adrenoreceptor ligand (–)-[³H] dihydroalprenolol (DHA) in the adult rat lung were investigated.
• 2 The specific binding of (–)-[³H] quinuclidinyl benzilate (QNB) to lung membranes was saturable and the equilibrium dissociation constant (K_D) determined from Scatchard analysis was 54 pM. Kinetic analysis of the binding of [³H] QNB yielded a K_D of 42 pM. [³H] QNB binding was inhibited by muscarinic agonists and antagonists, the order of their potency was l-hyoscyamine>atropine>scopolamine>oxotremorine>carbachol. These data were consistent with [³H] QNB binding to the muscarinic receptor.
• 3 Adult male rats treated for 2 weeks with the antithyroid agent 3-amino-1,2,4-triazole (ATZ) showed a 52% and 80% reduction in the serum concentration of triiodothyronine (T₃) and thyroxine (T₄) respectively. These hypothyroid rats also had a 39% decrease in the concentration of lung β-adrenoreceptors and a 37% decrease in the concentration of lung muscarinic receptors as compared to euthyroid controls. Concurrent treatment of rats with ATZ and T₄ for 2 weeks resulted in a reduction of 15% and 20% in the concentration of lung β-adrenoreceptors and muscarinic receptors respectively. The K_D values for [³H] DHA and [³H] QNB binding did not change with the ATZ or ATZ + T₄ treated groups.
• 4 Administration of T₄ (500 μg/kg/day) to male rats for 12 days did not result in any significant change in the concentration of either β-adrenoreceptors or muscarinic receptors compared to euthyroid controls. No change in the K_K values for [³H] DHA or [³H] QNB binding were detected.
• 5 The results show that hypothyroid rats have a reduced lung concentration of both β-adrenoreceptors and muscarinic receptors whereas in hyperthyroid rats these receptors do not significantly change from euthyroid controls.

     

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).

     

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     

New insight into active muscarinic receptors with the novel radioagonist [H]iperoxo

Activation of G protein-coupled receptors involves major conformational changes of the receptor protein ranging from the extracellular transmitter binding site to the intracellular G protein binding surface. GPCRs such as the muscarinic acetylcholine receptors are commonly probed with radioantagonists rather than radioagonists due to better physicochemical stability, higher affinity, and indifference towards receptor coupling states of the former. Here we introduce tritiated iperoxo, a superagonist at muscarinic M₂ receptors with very high affinity. In membrane suspensions of transfected CHO-cells, [³H]iperoxo – unlike the common radioagonists [³H]acetylcholine and [³H]oxotremorine M – allowed labelling of each of the five muscarinic receptor subtypes in radioagonist displacement and saturation binding studies. [³H]iperoxo revealed considerable differences in affinity between the even- and the odd-numbered muscarinic receptor subtypes with affinities for the M₂ and M₄ receptor in the picomolar range. Probing ternary complex formation on the M₂ receptor, [³H]iperoxo dissociation was not influenced by an archetypal allosteric inverse agonist, reflecting activation-related rearrangement of the extracellular loop region. At the inner side of M₂, the preferred G_i protein acted as a positive allosteric modulator of [³H]iperoxo binding, whereas G_s and G_q were neutral in spite of their robust coupling to the activated receptor. In intact CHO-hM₂ cells, endogenous guanylnucleotides promoted receptor/G protein-dissociation resulting in low-affinity agonist binding which, nevertheless, was still reported by [³H]iperoxo. Taken together, the muscarinic superagonist [³H]iperoxo is the best tool currently available for direct probing activation-related conformational transitions of muscarinic receptors.     

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.     

Divergence of allosteric effects of rapacuronium on binding and function of muscarinic receptors

Background

Many neuromuscular blockers act as negative allosteric modulators of muscarinic acetylcholine receptors by decreasing affinity and potency of acetylcholine. The neuromuscular blocker rapacuronium has been shown to have facilitatory effects at muscarinic receptors leading to bronchospasm. We examined the influence of rapacuronium on acetylcholine (ACh) binding to and activation of individual subtypes of muscarinic receptors expressed in Chinese hamster ovary cells to determine its receptor selectivity.

Results

At equilibrium rapacuronium bound to all subtypes of muscarinic receptors with micromolar affinity (2.7-17 μM) and displayed negative cooperativity with both high- and low-affinity ACh binding states. Rapacuronium accelerated [³H]ACh association with and dissociation from odd-numbered receptor subtypes. With respect to [³⁵S]GTPγS binding rapacuronium alone behaved as an inverse agonist at all subtypes. Rapacuronium concentration-dependently decreased the potency of ACh-induced [³⁵S]GTPγS binding at M₂ and M₄ receptors. In contrast, 0.1 μM rapacuronium significantly increased ACh potency at M₁, M₃, and M₅ receptors. Kinetic measurements at M₃ receptors showed acceleration of the rate of ACh-induced [³⁵S]GTPγS binding by rapacuronium.

Conclusions

Our data demonstrate a novel dichotomy in rapacuronium effects at odd-numbered muscarinic receptors. Rapacuronium accelerates the rate of ACh binding but decreases its affinity under equilibrium conditions. This results in potentiation of receptor activation at low concentrations of rapacuronium (1 μM) but not at high concentrations (10 μM). These observations highlight the relevance and necessity of performing physiological tests under non-equilibrium conditions in evaluating the functional effects of allosteric modulators at muscarinic receptors. They also provide molecular basis for potentiating M₃ receptor-mediated bronchoconstriction.     

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.     

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.     

Characterization of porcine coronary muscarinic receptors

Summary To determine the muscarinic receptor subtype involved in the contractile response of coronary smooth muscle, we investigated the profiles of various muscarinic receptor antagonists competing for [³H]N-methyl-scopolamine ([³H]NMS) binding to membrane preparations from porcine coronary arteries. [³H]NMS binds to a single population of muscarinic binding sites with a K_D of 135 pM and a B_max of 57 fmol/mg. The affinity profiles of AF-DX 116 [11-2((–((diethylamino)methyl)-1-piperidinyl)acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)-benzodiazepin-6-one], atropine, 4-DAMP [4-diphenylacetoxy-N-methylpiperidine methiodide], methoctramine [N,N-bis (6-((2-methoxybenzyl) amino)hexyl)-1,8-octane-diamine tetrahydrochloride], HHSiD [hexahydrosiladi-fenidol] and pirenzepine are consistent with binding to a mixed population of muscarinic binding sites, namely of the M₂ and M₃ subtype.Binding curves for AF-DX 116 and methoctramine are shallow with Hill-coefficients significantly less than unity. Comparison of data from binding studies with results obtained in functional experiments, i.e. antagonism of methacholine induced contraction of porcine coronary artery rings, it was found that only the low-affinity pK_i values of AF-DX 116 (6.26) and methoctramine (6.51) correlated well with functional pA₂ values.It is concluded that a mixed population of the M₂ and M₃ muscarinic receptor subtypes is present in porcine coronary arteries. Functional experiments do not support the contribution of the M₂ subtype to the contractile response. Cholinergic induced contractions of porcine coronary arteries appear to be evoked via stimulation of the muscarinic M₃ receptor subtype. However, since the compounds investigated here do not markedly discriminate between cloned m₃, m₄ and m₅ receptors the involvement of muscarinic receptors different from M₁, M₂ and M₃ cannot be excluded. Send offprint requests to M. Entzeroth at the above address     

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.     

Site-directed mutagenesis of the putative human muscarinic M2 receptor binding site

Experimental probing of the model of the muscarinic M₂ receptor binding site proposed by Hibert et al. [Hibert, M.F., Trumpp-Kallmeyer, S., Bruinsvels, A., Hoflak, K., 1991. Three-dimensional models of neurotransmitter G-binding protein-coupled receptors. Mol. Pharmacol. 40, 8–15.] was achieved by mutating each amino-acid proposed to interact with muscarinic ligands. Pharmacological analysis of the different mutant receptors transiently expressed in human embryonic kidney (HEK/293) cells was performed with a variety of agonists and antagonists. D103A, Y403A and N404A mutations prevented binding of [³H] N-methylscopolamine and [³H] quinuclidinyl benzilate with a reduction in affinity greater than 100-fold, indicating essential contributions of these residues to the binding site for the radioligands. W400A and W155A mutations had very large effects on the binding of [³H] N-methylscopolamine (150-fold, 960-fold) but modest effects on the binding of [³H] quinuclidinyl benzilate (4-fold, 17-fold). In addition, binding of oxotremorine-M, oxotremorine, arecoline and pilocarpine to W155A resulted in a greater than 100-fold decrease in affinity. Threonine mutations (T187A and T190A) alter binding of most agonists but not of antagonists. W99 makes little contribution (<10-fold) to the binding site of the M₂ receptor. D103, W155, W400, Y403 and N404 are likely to be part of the binding site for N-methylscopolamine and also to contribute to the binding site for quinuclidinyl benzilate. Some of the predicted residues do not seem to be part of the M₂ receptor binding site but W155 is important for proper ligand binding on the muscarinic M₂ receptor, as predicted by the proposed model.     

The effects of the neurosteroids: pregnenolone,progesterone and dehydroepiandrosterone on muscarinic receptor-induced responses in <Emphasis Type="Italic">Xenopus</Emphasis> oocytes expressing M<Subscript>1</Subscript> and M<Subscript>3</Subscript> receptors

The neurosteroids pregnenolone, progesterone, and dehydroepiandrosterone (DHEA) occur naturally in the nervous system. They act on neural tissues, participate in neuronal signaling, and are reported to alter neuronal excitability via nongenomic mechanisms. Muscarinic receptors have important roles in neuronal functions in the brain and autonomic nervous system. In this study, we investigated the effects of pregnenolone, progesterone, and DHEA on M₁ and M₃ muscarinic receptors using the Xenopus oocyte expression system. Pregnenolone and progesterone inhibited the acetylcholine (ACh)-mediated responses of M₁ and M₃ receptors expressed in Xenopus oocytes, whereas DHEA did not. The half-maximal inhibitory concentrations (IC₅₀) for pregnenolone inhibition of M₁ receptor- and M₃ receptor-mediated currents were 11.4 and 6.0 M respectively; the IC₅₀ values for progesterone inhibition of M₁ receptor- and M₃ receptor-mediated currents were 2.5 and 3.0 M respectively. The selective protein kinase C (PKC) inhibitor GF109203X had little effect on the pregnenolone or progesterone inhibition of the ACh-induced currents in Xenopus oocytes expressing M₁ or M₃ receptors. The inhibitory effects of pregnenolone and progesterone were overcome at higher concentrations of ACh. Pregnenolone and progesterone inhibited the [³H]quinuclidinyl benzilate (QNB) binding to M₁ and M₃ receptor expressed in Xenopus oocytes, and Scatchard plot analysis of [³H]QNB binding revealed that pregnenolone and progesterone altered the K_d value and the B_max, indicating noncompetitive inhibition. In conclusion, pregnenolone and progesterone inhibited M₁ and M₃ receptor functions noncompetitively by the mechanism independent of PKC and by interfering with ACh binding to the receptors.     

Signal transduction pathway of the muscarinic receptors mediating gallbladder contraction

In gallbladder smooth muscle, carbachol interacts with M₃ receptors to mediate contraction. To examine components of the intracellular second messenger system that is coupled to these receptors we have tested whether carbachol stimulates the formation of inositol phosphates (IP) to cause contraction. Guinea pig gallbladder muscle strips were prelabeled with [³H]inositol and were incubated with 0.1 mmol/l carbachol, a concentration causing maximal contraction. [³H]inositol monophosphates, [³H]inositol bisphosphates and [³H]inositol trisphosphates and contraction were measured at various times (0–90 s). To examine whether a pertussis toxin-sensitive guanine nucleotide binding protein is coupled to the muscarinic receptors, guinea pigs were pretreated with pertussis toxin (180 g/kg i.v./24 h). The effectiveness of pertussis toxin treatment was determined by measuring [³²P]ADP-ribosylation of a –40/41 kDa protein from gallbladder homogenates. Carbachol caused a significant time-dependent increase in the formation of [³H]inositol monophosphates, [³H]inositol bisphosphates and [³H]inositol trisphosphates. The time course of [³H]inositol trisphosphate turnover caused by carbachol was biphasic, and was detectable at 15 s and maximal at 60 s; at 75 s and 90 s formation of [³H]inositol trisphosphates decreased, whereas the time course of carbachol-induced contraction of the gallbladder smooth muscle strips reached a plateau after 90 s. The effects of carbachol on [³H]inositol trisphosphates and on contraction were abolished by atropine. Pretreatment with pertussis toxin resulted in ADP-ribosylation of a 40/41 kDa protein from gallbladder cell membranes but did not affect the concentration-response or time course of carbachol-induced contraction. These results indicate that carbachol-induced contraction of gallbladder smooth muscle cells is accompanied by the activation of inositol phosphate turnover and does not involve a pertussis toxin-sensitive G-protein.This article is based in part on the doctoral thesis of Burkhard Mackensen at the Faculty of Medicine, University of Hamburg, Germany. Some of the results were presented at the meeting of the American Gastroenterological Association (AGA) in San Francisco 1992 (von Schrenck et al. 1992) Correspondence to: T. von Schrenck at the above address     

Inhibition of transmitter release from rat sympathetic neurons via presynaptic M1 muscarinic acetylcholine receptors

Background and purpose:

M₂, M₃ and/or M₄ muscarinic acetylcholine receptors have been reported to mediate presynaptic inhibition in sympathetic neurons. M₁ receptors mediate an inhibition of K_v7, Ca_V1 and Ca_V2.2 channels. These effects cause increases and decreases in transmitter release, respectively, but presynaptic M₁ receptors are generally considered facilitatory. Here, we searched for inhibitory presynaptic M₁ receptors.

Experimental approach:

In primary cultures of rat superior cervical ganglion neurons, Ca²⁺ currents were recorded via the perforated patch-clamp technique, and the release of [³H]-noradrenaline was determined.

Key results:

The muscarinic agonist oxotremorine M (OxoM) transiently enhanced ³H outflow and reduced electrically evoked release, once the stimulant effect had faded. The stimulant effect was enhanced by pertussis toxin (PTX) and was abolished by blocking M₁ receptors, by opening K_v7 channels and by preventing action potential propagation. The inhibitory effect was not altered by preventing action potentials or by opening K_v7 channels, but was reduced by PTX and ω-conotoxin GVIA. The inhibition remaining after PTX treatment was abolished by blockage of M₁ receptors or inhibition of phospholipase C. When [³H]-noradrenaline release was triggered independently of voltage-activated Ca²⁺ channels (VACCs), OxoM failed to cause any inhibition. The inhibition of Ca²⁺ currents by OxoM was also reduced by ω-conotoxin and PTX and was abolished by M₁ antagonism in PTX-treated neurons.

Conclusions and implications:

These results demonstrate that M₁, in addition to M₂, M₃ and M₄, receptors mediate presynaptic inhibition in sympathetic neurons using phospholipase C to close VACCs.     

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.     

Prevention of guanine nucleotide-induced reductions in muscarinic agonist binding to rabbit ileal submucosal membranes by lidamidine and tetracaine

Summary The effects of low concentrations (< = 1 mM) of the anti-diarrhoeal drug lidamidine and sub-anaesthetic concentrations (< = 1 M) of tetracaine on the binding of the muscarinic antagonist [³H] N-methylscopolamine ([³H]NMS) and the high affinity muscarinic agonist, [³H] oxotremorine-M ([³H]oxo-M) to broken cell preparations of rabbit ileal submucosal membranes were investigated. High affinity [3H]NMS binding (K _D = 0.79 ± 0.05 nM, B _max = 1.75 ± 0.13 pmoles · mg^–1) was unaffected by either lidamidine or tetracaine. Inhibition of NMS binding by the muscarinic agonist carbachol was reduced by the nonhydrolyzable analogue of GTP, GppNHp. This effect of GppNHp was partially prevented by lidamidine. Analysis of equilibrium binding of the muscarinic agonist [³H] oxotremorine-M revealed that the binding of oxotremorine-M could be best described by the presence of the least two populations of sites with affinities of 0.9 ± 0.2 and 27.7 ± 0.9 nM respectively. High affinity [³H] oxotremorine-M binding was markedly reduced by GppNHp, GDPS and fluoride (5 mM). Neither lidamidine, nor tetracaine had any effect on the binding of oxotremorine-M when added alone. However, lidamidine and tetracaine prevented GppNHp, GDPS and fluoride-induced reductions in oxotremorine-M binding. The present findings are consistent with an allosteric interaction between lidamidine or tetracaine and GppNHp-induced reductions in oxotremorine-M binding to submucosal muscarinic receptors. These effects are discussed in relation to the observed action of lidamidine in potentiating presynaptic inhibition of acetylcholine release by muscarinic agonists. Send offprint requests to D. Bleakman     

Muscarinic cholinergic receptors in the human right coronary artery: a receptor binding and autoradiographic study

Summary We used a combination of radioreceptor binding and autoradiographic techniques to study the pharmacological characteristics and anatomical localization of [³H]-quinuclidinyl benzilate (QNB) binding sites in the human right coronary artery. The ligand was bound to sections of the human right coronary artery in a manner consistent with the labelling of muscarinic receptors. The addition of pirenzepine or of carbachol to the incubation medium to generate displacement curves was indicative of the presence of M₁ and M₂ receptors in the right coronary artery. Autoradiography showed the localization of M₁ sites primarily in the medial layer of the right coronary artery. M₂ sites were located primarily in the adventitia. No [³H]-QNB binding sites were observed in the endothelium. A possible role of muscarinic receptors in the pathogenesis of coronary vasospasm is discussed.     

Allosteric site in M2 acetylcholine receptors: evidence for a major conformational change upon binding of an orthosteric agonist instead of an antagonist

Muscarinic acetylcholine receptors contain two distinct ligand binding sites, i.e. the orthosteric site for acetylcholine and other conventional ligands, and an allosteric site located at the entrance of the ligand binding pocket. We used a set of allosteric agents to probe whether muscarinic M₂ receptors whose orthosteric site is occupied by an agonist still reveal the common allosteric site that has been identified in M₂ receptors being occupied by an orthosteric antagonist (N-methylscopolamine, NMS). Equilibrium and dissociation binding experiments were carried out in porcine heart homogenates using either the agonist [³H]oxotremorine M ([³H]OxoM) or the antagonist [³H]NMS. The affinities of the allosteric agents were determined for the radioligand-occupied receptor states and, additionally, for the radioligand-free (ground state) M₂ receptor. The archetypal agent W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropyl-ammonium bromide] and its bispyridinio middle chain analogue WDuo3 (1,3-bis[4-(phthalimidomethoxyimino-methyl)-pyridinium-1-yl]propane dibromide) had a clearly lower affinity for [³H]OxoM-liganded receptors compared with [³H]NMS-liganded and ground state receptors. In contrast, a derivative resembling only one half of W84 had equal affinities for both radioligand-occupied receptor states. Also, the agents gallamine and obidoxime did not discriminate between [³H]OxoM- and [³H]NMS-occupied receptors. The allosteric antagonistic tool obidoxime inhibited WDuo3 action in [³H]OxoM-liganded receptors with the same potency as in [³H]NMS-liganded receptors. We conclude that the common allosteric site is still present in OxoM-liganded M₂ receptors, but its spatial conformation is considerably altered compared with NMS-liganded receptors.     

Synthesis of 2-(3-Substituted-1,2,4-oxadiazol-5-yl)-8-methyl-8-azabicyclo[3.2.1]octanes and 2α-(3-Substituted-1,2,4-oxadiazol-5-yl)-8-methyl-8-azabicyclo[3.2.1]oct-2-enes as Potential Muscarinic Agonists

Radioligand binding affinities of seven muscarinic receptor ligands which possess an oxadiazole ring side chain have been determined in rat heart, rat brain, and m₁- or m₃-transfected CHO cell membrane preparations to determine the selectivity for subtypes of muscarinic receptor. The ratios of binding constants in brain membranes were measured as an indicator of potential agonist activity against [³H]QNB and [³H]Oxo-M. These muscarinic ligands did not discriminate the subtypes of muscarinic receptors. Six muscarinic ligands which have a 3-amino- or 3-methyl-1,2,4-oxadiazol-5-yl groups attached to the 8-methyl-8-azabicyclo[3.2.1]oct-2-ene or 8-methyl-8-azabicyclo[3.2.1]octane head group show binding constants between 2.04 x 10^–6 and 1.79 x 10^–5 M in rat heart, rat brain, and m₁- or m₃-transfected CHO cell membrane preparations. 1-Methyl-2-[3-amino-1,2,4-oxadiazol-5-yl]piperidine shows low binding constants of approximately 10^–4 M in rat heart and rat brain. (1R,5S)-2-[3-Amino-1,2,4-oxadiazol-5-yl]-8-methyl-8-azabicyclo-[3.2.1]oct-2-ene [(1R,5S)-17] was the most active compound.