Spinal muscarinic receptors are activated during low or high frequency TENS-induced antihyperalgesia in rats

Transcutaneous electrical nerve stimulation (TENS) is a non-pharmacological modality used clinically to relieve pain. Central involvement of serotonin and endogenous opioids are implicated in TENS-induced analgesia. Activation of spinal cholinergic receptors is antinociceptive and these receptors interact with opioid and serotonin receptors. In the current study, the possible involvement of spinal cholinergic receptors in TENS analgesia was investigated in rats. Hyperalgesia was induced by inflaming one knee joint with 3% kaolin–carrageenan and assessed by measuring paw withdrawal latency (PWL) to heat before and 4 h after injection. The non-selective nicotinic antagonist mecamylamine (50 μg), non-selective muscarinic antagonist atropine (30 μg) or one of the muscarinic subtype antagonists: pirenzepine (M₁, 10 μg), methoctramine (M₂, 10 μg), 4-DAMP (M₃, 10 μg), or saline was administered intrathecally just prior to TENS treatment. Low or high frequency TENS was then applied to the inflamed knee and PWL was determined again. Atropine, pirenzepine and 4-DAMP significantly attenuated the antihyperalgesic effects of low and high frequency TENS while mecamylamine and methoctramine had no effects, compared to saline control. The results show that TENS-induced antihyperalgesia is mediated partially by activation of spinal muscarinic receptors but not spinal nicotinic receptors. Further, the results also indicate that spinal M₁ and M₃ muscarinic receptor subtypes mediate the muscarinic component of TENS antihyperalgesia.     

Epithelial muscarinic M1 receptors contribute to carbachol-induced ion secretion in mouse colon

Cholinergically induced intestinal anion secretion is generally believed to be caused by stimulation of epithelial muscarinic M₃ receptors, whereas muscarinic M₁ receptors are thought to be localized primarily on enteric neurons. In order to test this assumption, carbachol-stimulated Cl⁻ secretion across distal colon, measured as increase in short-circuit current (I_sc), was compared between M₁-knockout (M₁R-KO) and M₃-knockout (M₃R-KO) mice. Surprisingly, the maximal increase in I_sc evoked by carbachol was more than twice as large in M₃R-KO compared to M₁R-KO mice. This difference was not due to a reduced secretory capacity of the epithelium from M₃R-KO animals, as forskolin stimulated a similar maximal I_sc in both types of animals. The neurotoxin tetrodotoxin diminished, but did not abolish the secretory response evoked by carbachol in M₃R-KO distal colon, suggesting the existence of epithelial muscarinic receptors other than the type M₃. Furthermore, in muscarinic receptor wild-type animals, the muscarinic M₁ receptor antagonist pirenzepine inhibited the carbachol-stimulated I_sc by more than 70% suggesting the presence of epithelial muscarinic M₁ receptors; a conclusion, which was confirmed by the identification of mRNA for muscarinic M₁ receptors in isolated crypts from wild-type colon. Consequently, epithelial muscarinic receptors from the type M₁ contribute to cholinergically induced ion secretion in mouse colon.     

Compensation of muscarinic bronchial effects of talsaclidine by concomitant sympathetic activation in guinea pigs

The aim of the present investigation was to determine the reasons why the muscarinic receptor agonist talsaclidine (WAL 2014 FU, 1-azabicyclo[2.2.2] octane,3-(2-propynyloxy)-, (R)-,(E)-2-butenedioate) is devoid of bronchospastic effects in anaesthetized guinea pigs but causes contracture in isolated tracheal muscle from this species. Effects on airway resistance were assessed with a modified Konzett–Rössler method in guinea pigs anaesthetized with urethane. Intravenous injection of 1–64 mg/kg talsaclidine did not cause substantial bronchospasm in control animals. After blockade of β-adrenoceptors, the muscarinic receptor agonist induced dose-dependent bronchospasm which could be blocked by atropine. In despinalized animals and in animals with spinal transection, talsaclidine was bronchospastic but ED₅₀ values were higher and maximal effects were smaller than in intact animals after β-adrenoceptor blockade. In adrenalectomized guinea pigs, talsaclidine was nearly as bronchospastic as after blockade of β-adrenoceptors. In contrast, the muscarinic ganglion stimulant McN-A-343, 4-(m-chlorophenylcarbamoyloxy)-2-butyn-trimethyl-ammonium chloride, (2–32 mg/kg i.v.), which has a muscarinic receptor profile similar to that of talsaclidine, i.e., full muscarinic agonism and highest affinity at muscarinic M₁ receptors, partial agonism at muscarinic M₃ receptors, but in contrast to talsaclidine does not penetrate the blood–brain barrier, caused dose-dependent bronchospasm in control animals. These results indicate that talsaclidine has bronchospastic potential which, however, does not become evident in vivo because of functional antagonism via β-adrenoceptors resulting from concomitant activation of the sympathetic nervous system in general and the adrenals in particular. It can be concluded that the unique profile of action of talsaclidine is due to partial agonism at bronchial muscarinic M₃ receptors, a prerequisite for susceptibility to functional antagonism, and to its ability to penetrate the blood–brain barrier readily and to induce sympathetic activation as a result of full agonism at peripheral ganglionic and adrenal as well as central muscarinic M₁ receptors.     

Muscarinic receptor subtype in porcine coronary artery

The specific binding of (-)-[³H]QNB (quinuclidinyl benzilate) in membrane fractions of porcine coronary artery was saturable, of high affinity and stereoselective. It has been shown that there exist (-)-[³H]QNB binding sites with high (K_i = 12 nM)- and low(K_i = 1010 nM)-affinity for pirenzepine in the coronary artery but predominantly low-affinity sites in cardiac muscle. AF-DX 116 and gallamine showed a lower affinity to ( - )-[³H]QNB binding sites in the coronary artery compared to cardiac muscle. Thus, the present study suggests that porcine coronary artery contains a significant number of muscarinic receptors, probably both M₁ and M₂ subtypes.     

Pharmacological characterization and distribution of muscarinic receptors in human placental syncytiotrophoblast brush-border and basal plasma membranes

Based on the existence of choline acetyltransferase and acetylcholine in human placenta, we have investigated the presence of muscarinic acetylcholine receptors in brush-border and basal plasma membranes from human term placenta. Radioligand binding assay, using [³H]N-methyl-scopolamine as tracer, showed the existence of acetylcholine muscarinic receptors in brush-border (K_d 0.28±0.04 nM; B_max 9.4±1.6 fmol/mg protein) and basal plasma membranes (K_d 0.24±0.05 nM; B_max 34.3±6.3 fmol/mg protein). In order to perform a pharmacological characterization of these receptors, competition binding experiments were carried out using the muscarinic receptor antagonists pirenzepine, (11(2-diethyl-amino)methyl)-1-piperidinylacetyl-5-11-dihydro-6H-pyrido(14)benzodiazepine (AF-DX 116), himbacine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), dicyclomine and hexahydro-sila-difenidol (HHSD). The results obtained showed that the muscarinic receptors in brush-border and basal plasma membranes belong to different subtypes. In brush-border membranes, the receptor found match in terms of affinity for the antagonists with the muscarinic M₁ receptor subtype (K_i pirenzepine, 13.6±8.2 nM; K_i AF-DX 116, 1680±271 nM; K_i himbacine, 212±6.5 nM; K_i 4-DAMP, 1.5±0.4 nM; K_i dicyclomine, 5.1±0.8 nM; K_i HHSD, 34.3±7.3 nM), whereas the receptor in basal plasma membrane seems to be of the muscarinic M₂ receptor subtype (K_i pirenzepine, 202±48 nM; K_i AF-DX 116, 124±60 nM; K_i himbacine, 20.6±4.8 nM; K_i 4-DAMP, 4.5±1.2 nM; K_i dicyclomine, 54.6±22 nM; K_i HHSD, 89.2±15.8 nM). The results obtained show the existence of muscarinic acetylcholine receptors in brush-border and basal plasma membranes from human term placenta with a different distribution pattern in terms of number of receptors and distribution of different subtypes. The functional significance of these findings is as yet unknown, but these receptors probably mediate different functions as they belong to different subtypes and are coupled to different second messengers.     

The role of muscarinic receptor antagonism in antipsychotic-induced hippocampal acetylcholine release

Olanzapine and clozapine produce robust increases in hippocampal acetylcholine release during acetylcholinesterase inhibition, while other antipsychotics, including thioridazine, have only small effects. Since thioridazine binds with similar high affinities to muscarinic receptors as olanzapine and clozapine, muscarinic autoreceptor blockade was ruled out as a primary mechanism [Neuropsychopharmacology 26 (2002) 583]. This study compared in vitro binding affinities and functional activities of olanzapine, clozapine, thioridazine, ziprasidone, risperidone, chlorpromazine and scopolamine at muscarinic M2 receptors with their in vivo potencies to increase acetylcholine release in the rat hippocampus. We found that scopolamine, olanzapine and clozapine, but also high doses of thioridazine and chlorpromazine, markedly increase acetylcholine release. The reduced in vivo potencies of thioridazine and chlorpromazine are consistent with their significantly weaker functional antagonist activity at human muscarinic M2 receptors, while thioridazine's reduced binding affinity for rat muscarinic M2 receptors and lower brain exposure, may further contribute to its weak in vivo potency compared to olanzapine. The excellent correlation between in vitro antagonist activities of antipsychotics at muscarinic M2 receptors and their in vivo potencies to increase acetylcholine release, suggests that olanzapine, clozapine, as well as thioridazine and chlorpromazine, increase acetylcholine release via blockade of terminal muscarinic M2 autoreceptors.     

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 effect of zotepine, risperidone, clozapine and olanzapine on MK-801-disrupted sensorimotor gating

Dizocilpine (MK-801; 0.3 mg/kg i.p.)-induced disruption in prepulse inhibition of the acoustic startle response (PPI) can be preferentially restored by “atypical” antipsychotics. In contrast, some findings indicate that not all of the “atypical” antipsychotics, such as clozapine and risperidone, are effective in restoring the NMDA antagonist-induced deficits in PPI.

In our study, we evaluated the effect of four different “atypical” antipsychotic drugs on deficits in PPI induced by MK-801. Zotepine and risperidone have high affinities to D2-like and 5-HT_2A receptors, while clozapine and olanzapine have multipharmacological profiles with the highest affinities to serotonin 5-HT_1A,2A/2C receptors and muscarinic receptors.

Results have shown that MK-801 disrupted PPI and increased the ASR in rats. Our results showed no effect of zotepine (1 and 2 mg/kg) and risperidone (0.1 and 1 mg/kg) on disrupted PPI by MK-801. Administration of clozapine (5 and 10 mg/kg) and olanzapine (2.5 and 5 mg/kg) restored the deficits in PPI induced by MK-801. Additionally, we found a decrease of approximately 46% in PPI after administration of clozapine (5 mg/kg) and olanzapine (2.5 and 5 mg/kg) without MK-801 treatment.

In summary, the four “atypical” antipsychotics had different efficacies to restore the disrupted PPI by MK-801. Only clozapine and olanzapin restored the MK-801-induced deficits in PPI.     

Potent antagonism of 5-HT(3) and 5-HT(6) receptors by olanzapine.

The interaction of the psychotropic agent olanzapine with serotonin 5-HT₃ and 5-HT₆ receptors was investigated. Olanzapine did not contract the isolated guinea pig ileum, but blocked contractions induced by the 5-HT₃ receptor agonist 2-methyl serotonin (2-CH₃ 5-HT) with a pK_B value of 6.38±0.03, close to the affinity of the 5-HT₃ receptor antagonist ondansetron. The atypical antipsychotic risperidone (1 μM) did not significantly inhibit 2-CH₃ 5-HT-induced contractions. Olanzapine had high affinity (pK_i=8.30±0.06) for human 5-HT₆ receptors in radioligand binding studies. Olanzapine did not stimulate [³⁵S]guanosine-5′-O-(3-thio)triphosphate ([³⁵S]GTPγS) binding to the G protein G_s in cells containing human 5-HT₆ receptors, but inhibited 5-HT-stimulated [³⁵S]GTPγS binding (pK_B=7.38±0.16). Among other antipsychotics investigated, clozapine antagonized 5-HT₆ receptors with a pK_B=7.42±0.15, ziprasidone was three-fold less potent, and risperidone, quetiapine and haloperidol were weak antagonists. Thus, olanzapine was not an agonist, but was a potent antagonist at 5-HT₆ receptors and had marked antagonism at 5-HT₃ receptors.     

The role of muscarinic M1 and M2 receptors in airway constriction in the cat.

The role of prejunctional inhibitory and facilitatory muscarinic receptors was investigated in cats with tracheal hyperresponsiveness to vagal stimulation. Intrathoracic airway caliber (total lung resistance (R_L) and dynamic compliance (C_dyn)) and the diameter of tracheal ring 4 were measured during vagal stimulation and local acetylcholine (ACh) injection before and after administration of the M₁ receptor antagonist pirenzepine or the M₂ receptor antagonist gallamine. The responses of tracheal ring 4, R_L, and C_dyn to ACh were unaltered by gallamine or pirenzepine. Changes in R_L and C_dyn during vagal stimulation were enhanced by gallamine, but the magnitude of tracheal constriction was unchanged. Vagally induced tracheal constriction was decreased by pirenzepine in hyperresponsive but not in control cats. The M₂ receptors limit intrathoracic airway constriction, but a functional role for M₂ receptors in the cervical trachea could not be demonstrated. However, these data suggest that M₁ excitatory receptors may play a role in vagally mediated tracheal hyperreactivity.     

Comparison of the in-vivo muscarinic cholinergic receptor availability in patients treated with clozapine and olanzapine

Clozapine and olanzapine are two atypical antipsychotics that bind to a broad range of receptors in vitro. Our group previously reported on the binding of clozapine and olanzapine to muscarinic receptors in vivo. Based on these data, a direct comparison of the muscarinic receptor availability in vivo under treatment with these atypical antipsychotics was performed. [123I]IQNB SPECT scans were obtained in seven subjects treated with a high dose (20 mg) of olanzapine and seven subjects treated with a moderate dose (mean 275.0 mg, range 200-450 mg) of clozapine. Muscarinic receptor-binding indices were determined for basal ganglia, cortex, thalamus and pons. When comparing moderate-dose clozapine with high-dose olanzapine, significantly lower muscarinic receptor availability was found for clozapine in all four cortical regions of interest. Our results suggest that treatment with clozapine results in a stronger blockade of the muscarinic cholinergic receptors than with olanzapine. These results are compatible with the higher rates of anticholinergic side-effects seen with clozapine in clinical practice.     

Clozapine interaction with the M2 and M4 subtypes of muscarinic receptors.

Available evidence indicates that the antipsychotic drug clozapine acts as a partial agonist at the muscarinic M4 and as an antagonist at the M2 receptors. We wondered whether there is indeed a fundamental difference between its action on these two receptor subtypes, and whether it interacts with their classical or allosteric binding sites. In experiments on Chinese hamster ovary cells stably expressing the M2 or M4 receptors, clozapine inhibited the binding of the specific muscarinic ligand [3H]N-methylscopolamine to either receptor subtype. The affinity of the high-affinity sites for clozapine was diminished by GTP in the way expected for agonists on both the M2 and the M4 receptor subtypes. Arunlakshana-Schild plots of data obtained in saturation binding experiments with [3H]N-methylscopolamine at different concentrations of clozapine were linear with a slope of unity. Clozapine did not alter the time course of [3H]N-methylscopolamine dissociation from muscarinic M2 or M4 receptors. It inhibited the synthesis of cyclic AMP in cells expressing the M4 receptor subtype, but did not measurably inhibit the synthesis of cyclic AMP in cells expressing the M2 receptor subtype. We conclude that clozapine has a high affinity for muscarinic M2 and M4 receptor subtypes, that it associates with the classical and not with the allosteric binding site, and that it acts as a partial agonist on both the M2 and the M4 receptor subtype.     

Muscarinic interactions of bisindolylmaleimide analogues

We have used radioligand binding studies to determine the affinities of seven bisindolylmaleimide analogues, six of which are selective inhibitors of protein kinase C, at human muscarinic M₁–M₄ receptors. The compounds were most potent at M₁ receptors, and Ro-31-8220 was the most potent analogue, with a K_d of 0.6 μM at M₁ receptors. The weakest compounds, bisindolylmaleimide IV and bisindolylmaleimide V, had K_d values of 100 μM. If it is necessary to use protein kinase C inhibitors at concentrations of 10 μM or more in studies involving muscarinic receptors then bisindolylmaleimide IV may be the most appropriate inhibitor to use.     

Involvement of nicotinic and muscarinic acetylcholine receptors on striatal HgCl2-induced dopamine release in freely moving rats

The possible role of acetylcholine receptors on the HgCl₂-induced dopamine (DA) release from rat striatum was investigated by using in vivo brain microdialysis technique after administration of selective nicotinic and muscarinic receptor antagonists, mecamylamine and atropine, respectively. Intrastriatal infusion of 1 mM HgCl₂ increased striatal DA to 1717.2 ± 375.4% respect to basal levels. Infusion of 1 mM HgCl₂ in 1 mM mecamylamine pretreated animals produced an increase on striatal DA levels 58% less than that induced in non-pretreated animals. In the case of atropine, this treatment reduced 62% the effect produced by HgCl₂ as compared to non-pretreated rats. These data show that acetylcholine receptors could participate on HgCl₂-induced dopamine release since administration of nicotinic and muscarinic receptor antagonists reduces HgCl₂ effects on DA release.     

Angiotensin receptor subtypes in the uterine artery during ovine pregnancy

This study was undertaken to determine if changes in receptor density or affinity could account for the reduced vascular sensitivity to angiotensin II seen during pregnancy. Angiotensin receptor subtypes in the uterine arteries of non-pregnant, pregnant and postpartum ewes were investigated using saturation and competition receptor binding techniques with the specific receptor antagonists, losartan (DuP-753) and PD-123319 (S)1-[[4-(dimethylamino)-3-methylphenyl]-methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo(4,5-c)pyridine-6-carboxylic acid, ditrifluoroacetate, monohydrate). Receptor density and affinity of total angiotensin receptors, as well as the angiotensin AT₁ and AT₂ receptor subtypes in uterine arteries were compared with those in the mesenteric artery and aorta. The uterine artery contains both AT₁ and AT₂ receptor subtypes, whereas the mesenteric artery and aorta contain primarily the AT₁ receptor subtype. In uterine arteries from pregnant sheep, angiotensin receptor density was increased because AT₂ receptors were increased. AT₁ receptor density was not altered. This change was not seen in the aorta. In the uterine artery, receptor affinity for [Sar¹,Ile⁸]angiotensin II decreased in mid-gestation (IC₅₀ 7.7±1.2×10⁻⁹ M) compared with non-pregnant ewes (IC₅₀ 3.0±0.6×10⁻⁹ M, P=0.006), and there was decreased affinity of angiotensin AT₁ receptors for losartan during pregnancy (IC₅₀ 2.8±1.0×10⁻⁴ M) compared with non-pregnant ewes (IC₅₀ 2.2±1.3×10⁻⁶ M, P=0.025). Our results show changes in the density and affinity of the angiotensin receptor subtypes in the uterine artery which could explain its reduced responsiveness to circulating angiotensin II during pregnancy.     

Bethanechol-induced responses in mudpuppy parasympathetic neurons

The effect of bethanechol on membrane potential and excitability was determined in mudpuppy parasympathetic postganglionic neurons. Bethanechol induced a large amplitude hyperpolarization, which was followed by a smaller amplitude depolarization, in 115 out of 135 cells tested. In approximately 20% of these cells, a brief depolarization preceded the hyperpolarization. During the bethanechol-induced hyperpolarization, the membrane input resistance decreased markedly, whereas the input resistance was increased during the subsequent depolarization. The hyperpolarization and depolarization were blocked by atropine and were unaffected by d-tubocurarine, thus, both appeared to be mediated by muscarinic receptors. The bethanechol-induced hyperpolarization was inhibited by the M₂ muscarinic receptor antagonist AF-DX 116, whereas the bethanechol-induced depolarization was unaffected.

Both a nonselective increase in membrane conductance and a decrease in membrane potassium conductance appeared to be involved in the generation of the bethanechol-induced depolarization. Evidence for the first mechanism was obtained in barium-treated cells in which bethanechol initiated a rapid onset depolarization, which was reversed at membrane potentials near 0 mV. Evidence for the second mechanism was obtained when the hyperpolarization was inhibited by AF-DX 116. In AF-DX 116-treated cells, the membrane input resistance was increased during most of the bethanechol-induced depolarization.

Mudpuppy neurons initiate repetitive action potential activity in response to long depolarizing current pulses. Following application of bethanechol, with the hyperpolarization negated electrotonically, the number of action potentials produced by a depolarizing current pulse was greater than that produced prior to application of bethanechol.

It is suggested that activation of muscarinic receptors on mudpuppy cardiac neurons influences multiple conductance systems and determines the excitability of these neurons.     

Mixed agonist-antagonist properties of clozapine at different human cloned muscarinic receptor subtypes expressed in Chinese hamster ovary cells.

We recently reported that clozapine behaves as a partial agonist at the cloned human m4 muscarinic receptor subtype. In the present study, we investigated whether the drug could elicit similar effects at the cloned human m1, m2, and m3 muscarinic receptor subtypes expressed in the Chinese hamster ovary (CHO) cells. Clozapine elicited a concentration-dependent stimulation of [3H]inositol phosphates accumulation in CHO cells expressing either the m1 or the m3 receptor subtype. Moreover, clozapine inhibited forskolin-stimulated cyclic AMP accumulation and enhanced [35S] GTP gamma S binding to membrane G proteins in CHO cells expressing the m2 receptor. These agonist effects of clozapine were antagonized by atropine. The intrinsic activity of clozapine was lower than that of the full cholinergic agonist carbachol, and, when the compounds were combined, clozapine potently reduced the receptor responses to carbachol. These data indicate that clozapine behaves as a partial agonist at different muscarinic receptor subtypes and may provide new hints for understanding the receptor mechanisms underlying the antipsychotic efficacy of the drug.     

Developing chemical genetic approaches to explore G protein-coupled receptor function: validation of the use of a receptor activated solely by synthetic ligand (RASSL)

Molecular evolution and chemical genetics have been applied to generate functional pairings of mutated G protein-coupled receptors (GPCRs) and nonendogenous ligands. These mutant receptors, referred to as receptors activated solely by synthetic ligands (RASSLs) or designer receptors exclusively activated by designer drugs (DREADDs), have huge potential to define physiological roles of GPCRs and to validate receptors in animal models as therapeutic targets to treat human disease. However, appreciation of ligand bias and functional selectivity of different ligands at the same receptor suggests that RASSLs may signal differently than wild-type receptors activated by endogenous agonists. We assessed this by generating forms of wild-type human M(3) muscarinic receptor and a RASSL variant that responds selectively to clozapine N-oxide. Although the RASSL receptor had reduced affinity for muscarinic antagonists, including atropine, stimulation with clozapine N-oxide produced effects very similar to those generated by acetylcholine at the wild-type M(3)-receptor. Such effects included the relative movement of the third intracellular loop and C-terminal tail of intramolecular fluorescence resonance energy transfer sensors and the ability of the wild type and evolved mutant to regulate extracellular signal-regulated kinase 1/2 phosphorylation. Each form interacted similarly with β-arrestin 2 and was internalized from the cell surface in response to the appropriate ligand. Furthermore, the pattern of phosphorylation of specific serine residues within the evolved receptor in response to clozapine N-oxide was very similar to that produced by acetylcholine at the wild type. Such results provide confidence that, at least for the M(3) muscarinic receptor, results obtained after transgenic expression of this RASSL are likely to mirror the actions of acetylcholine at the wild type receptor.     

Secoverine is a non-selective muscarinic antagonist on rat heart and brain receptors

In an attempt to determine if the selectivity of secoverine observed in vivo and in isolated tissues might be due to selective association with muscarinic receptor subtypes, we analyzed the binding of secoverine to three different receptors with specific radioligands: rat cardiac receptors (M2 receptors with low affinity for atropine), and rat cerebral cortical M1 receptors and M2 receptors with high affinity for atropine. At concentrations up to 10(-6) M, secoverine interaction with muscarinic receptors was competitive and of high affinity (Ki 4.10(-9) M) for cardiac and brain receptors. A detailed analysis using, in addition to [3H]N-methylscopolamine, the agonist [3H]oxotremorine-M (selective for high affinity binding sites at cardiac receptors) and the M1-selective antagonist [3H]pirenzepine at brain receptors, revealed identical affinities towards both receptor types, making it unlikely that secoverine distinguished the different muscarinic receptor subtypes. At concentrations between 10(-6) and 10(-3) M, secoverine interaction with an additional receptor site resulted in profound changes of tracer kinetics, suggesting the formation of a ternary complex (secoverine-radioligand-muscarinic receptor). The potency of secoverine for provoking this allosteric interaction was both tracer- and tissue-dependent. It is concluded that secoverine does not differentiate between M1, brain M2 and cardiac M2 receptors or between cardiac receptors with high, low and very low affinity for agonists. At very high concentrations secoverine recognized an allosteric site on the muscarinic receptors and reduced the dissociation rates of the 3H-ligands.     

Central muscarinic M2 cholinoceptors involved in cholinergic hypertension

Cholinomimetic agents increase blood pressure and heart rate via central muscarinic cholinoceptors in various aspects. It was reported that i.c.v. injection of the muscarinic M₁ and M₃ cholinoceptor selective antagonist, 4-DAMP (4-diphenylacetoxy-N-methyl-piperidine methiodide, inhibited the pressor response to physostigmine, while the M₁ selective antagonist, pirenzepine, was ineffective. In the present study, the involvement of muscarinic M₂ cholinoceptors in central cholinergic hypertension and tachycardia was investigated. Physostigmine (10–80 μg/kg i.v.), a cholinesterase inhibitor, and oxotremorine (20–40 μg/kg i.v.), a direct muscarinic cholinoceptor agonist, caused a dose-dependent increase in blood pressure. Additionally, physostigmine induced dose-dependent tachycardiac responses. I.c.v. administration of the muscarinic M₂ cholinoceptor antagonists, AF-DX 116 and methoctramine, inhibited both physostigmine (60 μg/kg) and oxotremorine (20 μg/kg)-induced pressor responses at their lower doses used in this study (100 nmol/rat and 10 nmol/rat, respectively). These findings indicate the partial involvement of postsynaptic muscarinic M₂ cholinoceptors. The higher doses of the antagonists (AF-DX 116 300 nmol/rat and methoctramine 30 nmol/rat) potentiated the blood pressure increase due to physostigmine but did not affect that due to oxotremorine. The physostigmine-induced tachycardiac responses were influenced similarly by these antagonists. These results suggest the presence and tonic influence of presynaptic inhibitory muscarinic M₂ cholinoceptors.