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

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

Myocardial muscarinic acetylcholine receptor: Choline and tris unmask heterogeneity of antagonist binding sites

The binding properties of myocardial muscarinic acetylcholine receptors are altered in the presence of choline or Tris. The binding of the antagonist [³H]quinuclidinyl benzilate is reduced in the presence of choline or Tris buffer, when compared to parallel determinations in a physiologic salt solution or phosphate buffer. Scatchard analysis indicates the reduced binding is due to a decrease in the apparent number of receptor sites. Experiments with other organic buffers exclude the possibility that the reduced binding in Tris is due to the absence of sodium ions. In the presence of choline or Tris up to 45% of the receptors are not accessible to [³H]quinuclidinyl benzilate. The remaining sites maintain their high affinity for the antagonist. A heterogeneity of antagonist sites is evident.     

Thermic response of selective muscarinic agonists and antagonists in rat

Effect of some selective agonists and antagonists of cholinergic M receptor subtypes on rectal temperature was investigated in rats at an ambient temperature of 25 degrees +/- 2 degrees C. Centrally administered acetylcholine (ACh) induced transient hypothermia, whereas the muscarinic M1 receptor agonists, arecholine (ip) and McN-A-343 (McN) (icv), induced sustained and dose-related hypothermia. However, the nonspecific muscarinic receptor agonist, oxotremorine, and physostigmine, induced hypothermia at a lower dose and hyperthermia, accompanied by tremors, at higher doses. The muscarinic M2 receptor agonist, carbachol (icv) also produced a dose-related dual effect, hyperthermia and hypothermia being induced by the lower and higher doses, respectively. The M1 receptor antagonists, scopolamine (ip) and pirenzepine (icv), induced hyperthermia, whereas the M2 receptor antagonists, gallamine (icv) and AF-DX 116 (AFDX) (ip), produced hypothermia. The hypothermic effects of ACh. arecholine, McN, physostigmine, oxotremorine and carbachol were attenuated by scopolamine and pirenzepine. However, although scopolamine also inhibited the hyperthermic and tremorogenic effects of the higher dose of oxotremorine, it had a synergistic effect with the hyperthermia-inducing higher dose of physostigmine. AFDX attenuated the hyperthermic effect of the lower dose of carbachol, indicating that it was M2 receptor-mediated. Hemicholinium, an ACh synthesis inhibitor, had a transient hypothermic effect followed by slight hyperthermia. However, it markedly antagonized the hypothermic effects of gallamine and AFDX, indicating that their effects were dependent upon the availability of neuronal ACh. The results indicate that cholinergic hypothermia is a function of central muscarinic M1 receptors, with the M2 receptors serving as automodulators.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of mRNA and binding sites of adrenoceptors and muscarinic receptors in the rat heart

Since there exist some obscurities in the expression of mRNAs and their receptors in the heart, we have investigated the gene expression (mRNA levels) of adrenoceptors (alpha1A-, alpha1B-, beta1-, beta2-, beta3-) and muscarinic receptors (M2) and the density of receptor binding sites (alpha1A-, alpha1B-, beta1-, beta2-adrenoceptors, muscarinic receptors). Moreover, the heart regions consist of tissue rich in ganglion cells (that are of importance in heart neural circuits) and those virtually free of them (myocytes). Therefore, we have examined the differences in the distribution of mRNAs/receptor binding sites in the atrial samples of the heart rich in ganglion cells vs. those are virtually free of them. Binding sites and mRNAs of muscarinic receptors and alpha1B-adrenoceptors differ in their distribution in different heart regions. The mRNAs for beta1- and beta2-adrenoceptors were almost equally distributed herein, while the amount of beta-adrenoceptors significantly differs in the heart regions. The alpha1A- and beta3-adrenoceptors mRNAs were also found in all investigated heart regions, but at significantly lower level and have not shown region differences. This is a new finding, especially to beta3-adrenoceptors, as they were not regularly found in each heart regions. alpha1B-adrenoceptors have similar distribution of their mRNAs and binding sites in some heart parts. Thus, we can conclude that there are noticeable differences in the presence of receptors in heart regions that contain ganglion cells in comparison to those are virtually free of them.     

Effect of pH on binding of agonists and antagonists to rat heart muscarinic receptors.

Insulin, epidermal growth factor (EGF), platelet-derived growth factor, multiplication-stimulating activity and 10% foetal-calf serum each stimulated the phosphorylation of a cytosolic Mr-22000 acidic heat-stable protein in Swiss mouse 3T3-L1 adipocytes. Phosphorylation of this protein was not stimulated by isoprenaline or dibutyryl cyclic AMP. The effect of insulin was maximal (3-fold increase) by 10 min; half-maximal stimulation was observed at 70 pM-insulin. Both [32P]phosphoserine and [32P]phosphothreonine residues were present in the Mr-22000 protein after insulin- and growth-factor-stimulated phosphorylation, but no [32P]phosphotyrosine. The major site of insulin- and EGF-stimulated phosphorylation appeared to be a threonine residue, in contrast with previously studied insulin-stimulated phosphorylation of serine residues. Insulin treatment appeared to result in a shift of the protein toward the anode on isoelectric focusing. Insulin and EGF present simultaneously did not lead to phosphorylation beyond that seen with each hormone singly. We surmise that insulin, EGF and perhaps other growth factors may activate a common protein kinase or inhibit a common protein phosphatase in 3T3-L1 adipocytes which acts on the Mr-22000 protein.     

Pre-clinical and clinical pharmacology of selective muscarinic M3 receptor antagonists

Muscarinic M3 receptor antagonists have therapeutic potential for the treatment of disorders associated with altered smooth muscle contractility or tone. These include irritable bowel syndrome (IBS), chronic obstructive airways disease (COAD) and urinary incontinence. Zamifenacin is a potent muscarinic receptor antagonist on the guinea pig ileum (pA2 value 9.27) with selectivity over M2 receptors in the atria (135-fold) and M1/M4 receptors in the rabbit vas deferens (78-fold). In addition, zamifenacin had lower affinity for the M3 receptor in the salivary gland (pKi 7.97). In animals, zamifenacin potently inhibited gut motility in the absence of cardiovascular effects and with selectivity over inhibition of salivary secretion. In healthy volunteers, zamifenacin inhibited small and large bowel motility and increased the rate of gastric emptying over a dose range which was associated with minimal anticholinergic side effects. These data show that zamifenacin, a selective muscarinic M3 receptor antagonist, was well tolerated in man and was efficacious as an inhibitor of gut motility. Further studies in patients are required with muscarinic M3 receptor antagonists to confirm efficacy against symptoms in diseases associated with altered smooth muscle contractility.     

Diphenyl sulfoxides as selective antagonists of the muscarinic M2 receptor

Structure activity studies on [4-(phenylsulfonyl)phenyl]methylpiperazine led to the discovery of 4-cyclohexyl-alpha-[4-[[4-methoxyphenyl(S)-sufinyl]phenyl]-1-pi perazineacetonitrile, 1, an M2 selective muscarinic antagonist. Affinity at the cloned human M2 receptor was 2.7 nM; the M1/M2 selectivity is 40-fold.     

Isopropyl amide derivatives of potent and selective muscarinic M2 receptor antagonists

Low molecular weight amide derivatives were synthesized and evaluated as M(2) receptor antagonists for the treatment of Alzheimer's disease. Isopropyl amides 19 and 31 are highly potent, selective and low molecular weight M(2) receptor antagonists with structural features different from our clinical candidate 1.     

The design of novel methoctramine-related tetraamines as muscarinic receptor subtype selective antagonists

Several novel methoctramine-related tetraamines were designed, and their biological profiles at muscarinic receptor subtypes were assessed by functional experiments in isolated guinea pig and rat atria (M2) and smooth muscle (ileum and trachea, M3) and by binding assays in rat cortex (M1), heart (M2), and submaxillary gland (M3) homogenates and NG 108-15 cells (M4). Tripitramine, a nonsymmetrical tetraamine, resulted in the most potent and the most selective muscarinic M2 receptor antagonist of the series (pA2 = 9.14-9.85; pKi = 9.54). Spirotramine (FC 15-94), a symmetrical tetraamine, was able to differentiate between muscarinic M1 receptors (pKi = 7.88) and the other subtypes (M2, pKi = 6.20; M3, pKi = 5.81; M4, pKi = 6.27). Thus, tripitramine and spirotramine could be valuable tools for the pharmacological classification and characterization of muscarinic receptor subtypes.     

Effect of selective muscarinic receptor agonists and antagonists on active-avoidance learning acquisition in rats

Effect of some selective muscarinic receptor agonists and antagonists was investigated on learning acquisition in an active-avoidance paradigm in rats which records an anticipatory conditioned avoidance apart from the classical conditioned avoidance response. The muscarinic M1 agonists, arecholine, pilocarpine and McN-A-343, facilitated learning acquisition, which was attenuated by the selective M1 antagonist, pirenzepine. On the other hand, M2 receptor agonist, carbachol, and physostigmine, induced a dose-related dual response, with lower doses retarding and higher doses facilitating the learning acquisition. The former effect was attenuated by gallamine, a muscarinic M2 antagonist, while the latter response was inhibited by pirenzepine, indicating that these putative M2 receptor agonist lose their receptor specificity on dose increment. The selective M2 receptor antagonists, gallamine and AF-DX 116, facilitated learning acquisition, which was inhibited by pirenzepine and the acetylcholine synthesis inhibitor hemicholinium. The results support the cholinergic hypothesis of learning and memory and indicate that M1 receptor agonists and M2 receptor antagonists are likely to prove beneficial in memory deficits. The data also indicates that the clinical dose of some drugs, like physostigmine, needs to be carefully established for optimum therapeutic benefit.     

An endogenous inhibitor of GABA receptor binding

An endogenous inhibitor of GABA receptor binding was prepared from synaptic membrane of rat brain with 0.05% Triton X-100. The endogenous inhibitor was competitive with GABA for GABA binding sites. The inhibition of GABA receptor binding by the endogenous inhibitor was blocked by the allosteric effect of diazepam. In the presence of diazepam, specific [³H]GABA binding was greater in a medium containing the endogenous inhibitor than in one containing an equal inhibitory potency of GABA, whereas there was no difference in the absence of diazepam. This indicated that the endogenous inhibitor was not GABA itself.     

Molecular pharmacology of muscarinic receptor heterogeneity

Muscarinic receptors can be pharmacologically classified into 3 types at the present time, however, five genes for the receptor have been identified. The muscarinic receptor types have unique antagonist selectivity, distribution and are linked to specific second messenger systems. The interaction between the muscarinic receptor types and G proteins may depend on the systems in which the receptors are integrated. Expression of the cloned gene in mammalian cells will be useful in delineating the relationships between the pharmacological types of muscarinic receptors and their genes and studying the interactions between the receptor, G proteins, and second messenger coupling.     

Parasympathetic denervation decreases muscarinic receptor binding in rat parotid.

After unilateral postganglionic parasympathetic denervation of rat parotid glands for 3, 6 or 16 days, the binding of [³H] quinuclidinylbenzilate, a potent and specific muscarinic antagonist, was decreased by 28–45%. The largest part of the decrease was already present at 3 days, and thus appeared to coincide with the loss of parasympathetic terminals (as shown by a 94% reduction in choline acetyltransferase activity). The decrease in binding was expressed as a reduced number of membrane sites, with no significant change in affinity, and could be accounted for only in part by a reduction in the size of denervated glands. The rapid loss of binding suggests the existence of muscarinic receptors on parasympathetic terminals. The clear absence of any receptor increases at longer periods of denervation, when parotid glands are reported to give supersensitive secretory responses, suggests that mechanisms other than receptor increases are important in denervation supersensitivity in exocrine glands.     

Sulfide analogues as potent and selective M(2) muscarinic receptor antagonists

We have discovered highly potent, selective sulfide M(2) receptor antagonists with low molecular weight and different structural features compared with our phase I clinical candidate Sch 211803. Analogue 30 showed superior M(2) receptor selectivity profile over Sch 211803. More importantly, this study provided new leads for the discovery of M(2) receptor antagonists as potential drug candidates.     

Differential regulation of high-affinity agonist binding to muscarinic sites in the rat heart, cerebellum, and cerebral cortex

The muscarinic agonist [3H]cismethyldioxolane ([3H]CD) was used to characterize the effects of regulators upon high-affinity agonist binding sites of the rat heart, cerebral cortex and cerebellum. Comparative studies with sodium ions (Na+), magnesium ions (Mg++), N-ethylmaleimide (NEM) and the guanine nucleotide Gpp(NH)p revealed tissue-specific effects. Mg++ preferentially enhanced while Gpp(NH)p and NEM reduced high-affinity [3H]CD binding in the heart and cerebellum. By comparison NEM enhanced high-affinity agonist binding in the cerebral cortex while Gpp(NH)p and Mg++ had little or no effect. Kinetic studies support an allosteric mechanism for these effects and provide further evidence for muscarinic receptor subtypes in mammalian tissues.     

Synthesis and optimization of novel and selective muscarinic M(3) receptor antagonists

A series of constrained piperidine analogues were synthesized as novel muscarinic M(3) receptor antagonists. Evaluation of these compounds in binding assays revealed that they not only have high affinity for the M(3) receptor but also have high selectivity over the M(2) receptor.     

Molecular probes for muscarinic receptors: functionalized congeners of selective muscarinic antagonists

The muscarinic agonist oxotremorine and the tricyclic muscarinic antagonists pirenzepine and telenzepine have been derivatized using a functionalized congener approach for the purpose of synthesizing high affinity ligand probes that are suitable for conjugation with prosthetic groups, for receptor cross-linking, fluorescent and radioactive detection, etc. A novel fluorescent conjugate of TAC (telenzepine amine congener), an n-decylamino derivative of the m1-selective antagonist, with the fluorescent trisulfonated pyrene dye Cascade Blue may be useful for assaying the receptor as an alternative to radiotracers. In a rat m3 receptor mutant containing a single amino acid substitution in the sixth transmembrane domain (Asn507 to Ala) the parent telenzepine lost 636-fold in affinity, while TAC lost only 27-fold. Thus, the decylamino group of TAC stabilizes the bound state and thus enhances potency by acting as a distal anchor in the receptor binding site. We have built a computer-assisted molecular model of the transmembrane regions of muscarinic receptors based on homology with the G-protein coupled receptor rhodopsin, for which a low resolution structure is known. We have coordinated the antagonist pharmacophore (tricyclic and piperazine moieties) with residues of the third and seventh helices of the rat m3 receptor. Although the decylamino chain of TAC is likely to be highly flexible and may adopt many conformations, we located one possible site for a salt bridge formation with the positively charged -NH3+ group, i.e. Asp113 in helix II.     

Pertussis vaccine reduces agonist binding to the rat heart muscarinic receptor and its guanine nucleotide modulation

The injection ofBordetella pertussis, inactivated by merthiolate, causes a 2-fold increase in the IC50 of carbamylcholine (carbachol) in displacing [³H];-L(–) quinuclidinyl benzilate binding ([³H]QNB) to the receptor. In control animals, 50 M Gpp(NH)p causes a 6-fold decrease in the affinity of carbachol binding, whereas after vaccination the reduction is only 1.6-fold. After pertussis treatment there is no alteration in the affinity and number of [³H]QNB binding sites of to the muscarinic receptor.