Differential effects of volatile anesthetics on M3 muscarinic receptor coupling to the Galphaq heterotrimeric G protein

BACKGROUND: Halothane inhibits airway smooth muscle contraction in part by inhibiting the functional coupling between muscarinic receptors and one of its cognate heterotrimeric G proteins, Galphaq. Based on previous studies indicating a more potent effect of halothane and sevoflurane on airway smooth muscle contraction compared with isoflurane, the current study hypothesized that at anesthetic concentrations of 2 minimum alveolar concentration (MAC) or less, halothane and sevoflurane but not isoflurane inhibit acetylcholine-promoted Galphaq guanosine nucleotide exchange. METHODS: Galphaq guanosine nucleotide exchange was measured in crude membranes prepared from COS-7 cells transiently coexpressing the human M3 muscarinic receptor and human Galphaq. A radioactive, nonhydrolyzable analog of guanosine-5'-triphosphate, [35S]GTPgammaS, was used as a reporter for nucleotide exchange at Galphaq. RESULTS: Acetylcholine caused a concentration-dependent increase in Galphaq [35S]GTPgammaS-GDP exchange. Neither anesthetic affected constitutive Galphaq [35S]GTPgammaS-GDP exchange in the absence of acetylcholine. Conversely, each anesthetic caused a concentration-dependent and reversible inhibition of Galphaq [35S]GTPgammaS-GDP exchange when promoted by acetylcholine. At concentrations of 3 MAC or less, the effect of halothane and sevoflurane were significantly greater than that of isoflurane, with only a minimal inhibition by isoflurane observed at 2 MAC. CONCLUSION: The differential effects of volatile anesthetics on acetylcholine-promoted guanosine nucleotide exchange at Galphaq are consistent with the apparent more potent direct effect of halothane and sevoflurane compared with isoflurane on muscarinic receptor-mediated contraction of isolated airway smooth muscle. These differential effects also suggest a mode of anesthetic action that could be due to anesthetic-protein interactions and not simply anesthetic accumulation in the lipid membrane.     

M3 muscarinic receptor expression on suburothelial interstitial cells

OBJECTIVE

To identify the cells expressing the M₃ muscarinic receptor subtype in the lamina propria of the bladder.

MATERIALS AND METHODS

The bladders from five normal guinea pigs were isolated and fixed in 4% paraformaldehyde. Tissues sections (10 µm) were then cut and stained with antibodies to the type 3 muscarinic receptor (M₃), the interstitial cell marker vimentin and the nonspecific nerve marker PGP 9.5. The specificity of the antibody to the M₃ receptor was established using the complementary blocking peptide and Western blot analysis of human embryonic kidney (HEK) cells transfected to express the M₃ receptor protein.

RESULTS

The M₃ antibody pre‐incubated with its blocking peptide showed no immunohistochemical staining. Investigating this antibody using HEK cells transfected to express the M₃ receptor protein and control HEK cells showed a single band in the transfected cells and no band in the control cells. M₃ receptor immunoreactivity (M₃‐IR) was detected primarily on a dense network of vimentin‐positive (vim⁺) cells lying immediately below the urothelium, i.e. the suburothelial interstitial cells (Su‐ICs). The M₃‐IR was punctate and appeared to be located on the cell surface. The diffuse network of cells in the remaining regions of the lamina propria showed no M₃‐IR. Few nerve fibres were associated with the M₃‐IR Su‐ICs. The M₃‐IR Su‐ICs were most numerous and prominent in the lateral wall. The number of M₃‐IR/vim⁺ cells diminished towards the bladder base and were absent in the bladder urethral junction. In the base and urethral junction there were vim⁺ cells that were not M₃‐IR. A population of umbrella cells in the lateral wall also showed weak punctate M₃‐IR.

CONCLUSIONS

Using a well‐characterized M₃ antibody, these results show for the first time that the M₃ muscarinic receptor in the lamina propria is located specifically on the Su‐ICs. The physiological role of these cells is unknown and consequently the significance of what appears to be a cholinergic signalling system is unclear. Previously published data showed that these cells respond to nitric oxide and atrial natriuretic peptide with an increase in cGMP and possibly prostaglandin. All of these observations, taken together, suggest that the Su‐ICs receive multiple inputs and that they must be part of a complex signalling system in this region of the bladder wall.     

M2、M3受体与膀胱逼尿肌关系的研究进展

M2、M3受体与膀胱逼尿肌收缩的关系密切,近年来,由于分子生物学和药理学的发展,对于M2、M3受体在正常膀胱中的收缩机制以及病理状态的机制有了新的认识,对于这些新机制的认识有助于发现潜在的治疗靶点和开发新的药物。     

隔姜灸对大鼠脊髓损伤后神经源性膀胱M2、M3乙酰胆碱mRNA及P2X3受体表达的影响

目的 探讨隔姜灸改善脊髓损伤大鼠神经源性膀胱症状的作用靶点,为脊髓损伤神经源性膀胱患者临床干预提供理论基础。 方法 选取SD大鼠为实验动物,随机分组后,按照改良Allen′s法建立T10脊髓损伤模型,根据评估标准,筛选符合脊髓损伤后神经源性膀胱标准的动物模型纳入模型组和隔姜灸组;假手术组仅切除T10椎板,不破坏脊髓;各组均纳入10只SD大鼠。三组术后均给予抗感染、创口护理及Crede手法辅助排尿。隔姜灸组同时行隔姜灸治疗,即术后第1天开始,每日1次,每次每个穴位灸10 min,连续12 d。 结果 隔姜灸组治疗后嘌呤能P2X3受体的相对表达水平及M2、M3乙酰胆碱mRNA的相对表达水平显著高于模型组（均P＜0.05）。 结论 隔姜灸治疗可提高脊髓损伤大鼠脊髓神经节嘌呤能P2X3受体及膀胱组织中M2、M3乙酰胆碱mRNA的表达水平,从而有利于改善神经源性膀胱功能障碍。     

Interaction of edrophonium with muscarinic acetylcholine M2 and M3 receptors

BACKGROUND: It has been reported that edrophonium can antagonize the negative chronotropic effect of carbachol. This study was undertaken to evaluate in detail the interaction of edrophonium with muscarinic Mz and M3 receptors. METHODS: A functional study was conducted to evaluate the effects of edrophonium on the concentration-response curves for the negative chronotropic effect and the bronchoconstricting effect of carbachol in spontaneously beating right atria and tracheas of guinea pigs. An electrophysiologic study was conducted to compare the effects of edrophonium on carbachol-, guanosine triphosphate (GTP)gama S-, and adenosine-induced outward K+ currents in guinea pig atrial cells by whole cell voltage clamp technique. A radioligand binding study was conducted to examine the effects of edrophonium on specific [3HIN-methylscopolamine (NMS) binding to guinea pig atrial (M2) and submandibular gland (M3) membrane preparations, and on atropine-induced dissociation of [3H]NMS. RESULTS: Edrophonium shifted rightward the concentration-response curves for the negative chronotropic and bronchoconstricting effects of carbachol in a competitive manner. The pA2 values for cardiac and tracheal muscarinic receptors were 4.61 and 4.03, respectively. Edrophonium abolished the carbachol-induced outward current without affecting the GTPgamma S- and adenosine-induced currents in the atrial cells. Edrophonium inhibited [3H]NMS binding to M2 and M3 receptors in a concentration-dependent manner. The pseudo-Hill coefficient values and apparent dissociation constants of edrophonium for M2 and M3 receptors were 1.02 and 1.07 and 21 and 34 microM, respectively. Edrophonium also changed dissociation constant values of [3H]NMS without affecting its maximum binding capacities. CONCLUSION: Edrophonium binds to muscarinic M2 and M3 receptors nonselectively, and acts as a competitive antagonist.     

Atropine and glycopyrronium show similar binding patterns to M2 (cardiac) and M3 (submandibular gland) muscarinic receptor subtypes in the rat

Atropine and glycopyrronium are frequently used for premedicationto reduce oral and respiratory secretions and prevent bradycardia.Glycopyrronium is said to have similar antisialagogue effects,but is less likely to cause significant tachycardia than atropine.Different antimuscarinic receptor selectivity patterns couldexplain the differences. The aim of this investigation was todetermine the possible selectivity of glyco pyrronium for M₂and M₃ muscarinic receptor subtypes. Muscarinic receptor subtypesin Wistar rat ventricle and submandibular gland homogenateswere characterized with [³H]-N-methylscopolamine ([³H]-NMS)by ligand binding studies. Inhibition of [³H]-NMS binding bynon-labelled compounds showed the following order: in rat ventricle:glycopyrronium > atropine >> otenzepad > hexahydrosiladiphenidol(HHSiD) >> pirenzepine; in rat submandibular gland: glycopyrronium> atropine >> HHSiD >> pirenzepine > otenzepad.These were similar to the expected order of frequency of M₂and M₃ subtypes, respectively. Glycopyrronium showed similarlyhigh affinities for both M₂ (k_i = 1.889 (SEM 0.049) nmol litre^–1and M (K_i = 1.686 (0.184) nmol litre^–1 subtypes. Glycopyrroniumbound to a homogeneous population of binding sites in both tissuesand showed no selectivity for M₂ or M₃ muscarinic receptor subtypes.(Br. J. Anaesth. 1995; 74: 549–552)     

A mechanism for rapacuronium-induced bronchospasm: M2 muscarinic receptor antagonism

BACKGROUND: A safe and effective ultra-short-acting nondepolarizing neuromuscular blocking agent is required to block nicotinic receptors to facilitate intubation. Rapacuronium, which sought to fulfill these criteria, was withdrawn from clinical use due to a high incidence of bronchospasm resulting in death. Understanding the mechanism by which rapacuronium induces fatal bronchospasm is imperative so that newly synthesized neuromuscular blocking agents that share this mechanism will not be introduced clinically. Selective inhibition of M2 muscarinic receptors by muscle relaxants during periods of parasympathetic nerve stimulation (e.g., intubation) can result in the massive release of acetylcholine to act on unopposed M3 muscarinic receptors in airway smooth muscle, thereby facilitating bronchoconstriction. METHODS: Competitive radioligand binding determined the binding affinities of rapacuronium, vecuronium, cisatracurium, methoctramine (selective M2 antagonist), and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; selective M3 antagonist) for M2 and M3 muscarinic receptors. RESULTS: Rapacuronium competitively displaced 3H-QNB from the M2 muscarinic receptors but not from the M3 muscarinic receptors within clinically relevant concentrations. Fifty percent inhibitory concentrations (mean +/- SE) for rapacuronium were as follows: M2 muscarinic receptor, 5.10 +/- 1.5 microm (n = 6); M3 muscarinic receptor, 77.9 +/- 11 microm (n = 8). Cisatracurium and vecuronium competitively displaced 3H-QNB from both M2 and M3 muscarinic receptors but had affinities at greater than clinically achieved concentrations for these relaxants. CONCLUSIONS: Rapacuronium in clinically significant doses has a higher affinity for M2 muscarinic receptors as compared with M3 muscarinic receptors. A potential mechanism by which rapacuronium may potentiate bronchoconstriction is by blockade of M2 muscarinic receptors on prejunctional parasympathetic nerves, leading to increased release of acetylcholine and thereby resulting in M3 muscarinic receptor-mediated airway smooth muscle constriction.     

Rapacuronium augments acetylcholine-induced bronchoconstriction via positive allosteric interactions at the M3 muscarinic receptor

BACKGROUND: Neuromuscular blocking agents' detrimental airway effects may occur as a result of interactions with muscarinic receptors, allergic reactions, or histamine release. Rapacuronium, a nondepolarizing muscle relaxant, was withdrawn from clinical use because of its association with fatal bronchospasm. Despite its withdrawal from clinical use, it is imperative that the mechanism by which bronchospasm occurred is understood so that new muscle relaxants introduced to clinical practice do not share these same detrimental airway effects. METHODS: Airway smooth muscle force was measured in guinea pig tracheal rings in organ baths exposed to muscle relaxants with or without subthreshold concentrations of acetylcholine. Antagonism of muscarinic, histamine, neurokinin, leukotriene receptors, or blockade of L-type calcium channels or depletion of nonadrenergic, noncholinergic neurotransmitters was performed. Muscle relaxants' potentiation of acetylcholine-stimulated inositol phosphate synthesis and allosteric interactions on the kinetics of atropine-induced [3H]N-methylscopolamine dissociation were measured in cells expressing recombinant human M3 muscarinic receptors. RESULTS: Rapacuronium, within clinically achieved concentrations, contracted tracheal rings in the presence but not in the absence of subthreshold concentrations of acetylcholine. This effect was prevented or reversed only by atropine. The allosteric action of rapacuronium was demonstrated by the slowing of atropine-induced dissociation of [3H]N-methylscopolamine, and positive cooperativity was demonstrated by potentiation of acetylcholine-induced inositol phosphate synthesis. CONCLUSION: Many muscle relaxants have allosteric properties at muscarinic receptors; however, positive cooperativity at the M3 muscarinic receptor within clinically relevant concentrations is unique to rapacuronium. These findings establish novel parameters that should be considered in the evaluation of airway safety of any newly synthesized neuromuscular blocking agents considered for clinical practice.     

Effects of the M3 receptor selective muscarinic antagonist darifenacin on bladder afferent activity of the rat pelvic nerve

OBJECTIVE: Previous studies have revealed that intravesical and systemic administration of oxybutinin suppress pelvic afferent nerves. This study evaluates the efficacy of a selective M3 antimuscarinic, darifenacin, on bladder afferent activity. METHODS: Sixteen single bladder afferent fibers were isolated in nine female Sprague-Dawley rats. On the basis of their conduction velocities, they were grouped as Adelta or C fibers. The effect of repeat bladder filling was studied on the mechanosensitive properties of these units. The M3 receptor selective muscarinic antagonist darifenacin (0.1mg/kg) was administered intravenously. Unitary afferent activity was again analyzed 30, 60, 90, and 120 min after the drug administration. RESULTS: Seven units corresponded to criteria for Adelta fibers, nine for C fibers. Repeat bladder filling did not change nerve activity in Adelta or C fibers. When nerve activity was expressed as a percent of control activity, afferent sensitivity changed after darifenacin in Adelta fibers: 86+/-27%, 30 min (p>0.05), 69 +/- 32%, 60 min (p<0.05), 56 +/- 36%, 90 min (p<0.05), and 61+/- 49%, 120 min (p>0.05), and in C fibers: 70+/-39%, 30 min (p<0.05), 57 +/- 49%, 60 min (p<0.05), 45 +/- 42%, 90 min (p<0.01), and 47 +/- 43%, 120 min (p<0.01). CONCLUSIONS: In this study we show that darifenacin reduces bladder afferent activity in both Adelta and C fibers. The decrease in afferent spikes in C fibers may be more pronounced than that in Adelta fibers. These results may explain that the efficacy of darifenacin in overactive bladder symptoms is partly due to bladder afferent desensitization.     

Localization of M2 and M3 muscarinic receptors in human bladder disorders and their clinical correlations

PURPOSE: We studied the cellular localization of muscarinic receptor subtypes 2 and 3 in the human bladder and related any changes in overactive and painful bladder syndromes to measures of clinical dysfunction. MATERIALS AND METHODS: Bladder specimens obtained from patients with painful bladder syndrome (11), idiopathic detrusor overactivity (12) and from controls with asymptomatic microscopic hematuria (16) were immunostained using specific antibodies to muscarinic receptor subtypes 2 and 3, and to vimentin, which is a marker for myofibroblasts. Immunostaining results were quantified with computerized image analysis and correlated with clinical dysfunction using frequency and urgency scores. RESULTS: Muscarinic receptor subtype 2 and 3 immunoreactivity was observed in the urothelium, nerve fibers and detrusor layers. In addition, strong myofibroblast-like cell staining, similar to vimentin, was present in the suburothelial region and detrusor muscle. A significant increase in suburothelial myofibroblast-like muscarinic receptor subtype 2 immunoreactivity was seen in patients with painful bladder syndrome (p = 0.0062) and idiopathic detrusor overactivity (p = 0.0002), and in muscarinic receptor subtype 3 immunoreactivity in those with idiopathic detrusor overactivity (p = 0.0122) with a trend in painful bladder syndrome. Muscarinic receptor subtype 2 and 3 immunoreactivity significantly correlated with the urgency score (p = 0.0002 and 0.0206, respectively) and muscarinic receptor subtype 2 immunoreactivity correlated with the frequency score (p = 0.0029). No significant difference was seen in urothelial and detrusor muscarinic receptor subtypes 2 and 3 or vimentin immunostaining. CONCLUSIONS: To our knowledge this is the first study to show the cellular localization of muscarinic receptor subtypes 2 and 3 in the human bladder. The increase in muscarinic receptor subtypes 2 and 3 immunostaining in myofibroblast-like cells in clinical bladder syndromes and its correlation with clinical scores suggests a potential role in pathophysiological mechanisms and the therapeutic effect of anti-muscarinic agents.     

糖尿病大鼠精囊组织中神经生长因子和胆碱能M3受体的变化

目的:观察糖尿病大鼠精囊组织中神经生长因子(NGF)和胆碱能毒蕈碱M3受体含量的变化,以探讨糖尿病自主神经病变对精囊的影响,为糖尿病不育的临床防治提供理论依据。方法:成年雄性普通级SD大鼠15只,随机分为正常对照组5只和糖尿病模型组10只。链脲佐菌素(STZ)制备糖尿病模型成功后饲养8周,取精囊组织进行苏木精-伊红(HE)染色和免疫组化染色。结果:与正常对照组相比,糖尿病模型组HE染色可见大鼠精囊平滑肌细胞减少,腺上皮胞质变薄,结构紊乱。免疫组织化学染色显示,糖尿病模型组NGF阳性强度明显增强,而M3受体则明显减弱,糖尿病模型组与正常对照两组间NGF和M3平均累积光密度(IA)[0.018 7±0.002 4 vs 0.010 0±0.001 5和0.020 9±0.008 5 vs 0.041 2±0.011 7]的差异均具有统计学意义(P均<0.01)。结论:NGF和M3受体在糖尿病鼠精囊中表达改变,提示糖尿病可致精囊自主神经病变。     

Metoclopramide causes airway smooth muscle relaxation through inhibition of muscarinic M3 receptor in the rat trachea

Although metoclopramide, often used as an antiemetic, is reported to have an anticholinesterase action, the effect on airway smooth muscle remains unclear. We investigated the effect of metoclopramide on the contraction, phosphatidylinositol response, and binding affinity of muscarinic M(3) receptors in rat trachea preparations. Male Wistar rats were anesthetized and their tracheas excised and chopped into 3-mm-wide rings, 1-mm-wide slices, or frozen 10- microm-thick sections. Contraction was induced with 0.55 microM carbachol (CCh) and, 30 min later, metoclopramide (10 microM to 1 mM) was added. The slices were incubated with (3)[H]myo-inositol, 0.55 microM CCh, and metoclopramide, and the formation of (3)[H] inositol monophosphate was measured. A radioligand binding study was conducted to examine the effects of metoclopramide using [(3)H] 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP), a muscarinic M(3) receptor antagonist, in sections of the trachea. Metoclopramide concentration dependently attenuated CCh-induced contraction and inositol monophosphate accumulation, and also attenuated the binding affinity of 4-DAMP to muscarinic M(3) receptors. The 50% inhibitory concentration of metoclopramide against the binding affinity of 4-DAMP to muscarinic M(3) receptors of rat trachea was 24 micro M. These findings suggest that the attenuation by metoclopramide of CCh-induced contraction and phosphatidylinositol response may be mediated through the muscarinic M(3) receptors. IMPLICATIONS: We investigated the effect of metoclopramide on the contraction, phosphatidylinositol response, and binding affinity of muscarinic M(3) receptors in rat trachea preparations. Our findings suggest that the attenuation by metoclopramide of carbachol-induced contraction and phosphatidylinositol response may be mediated through the muscarinic M(3) receptors.     

Rapacuronium preferentially antagonizes the function of M2 versus M3 muscarinic receptors in guinea pig airway smooth muscle

BACKGROUND: Rapacuronium, a nondepolarizing muscle relaxant that was proposed as a replacement for succinylcholine for rapid intubation, was withdrawn from clinical use as a result of fatal bronchospasm, but the mechanism of this effect is not known. Preferential antagonism of presynaptic M2 muscarinic receptors versus postsynpatic M3 muscarinic receptors can facilitate bronchoconstriction. The authors questioned whether rapacuronium preferentially antagonized M2 versus M3 muscarinic receptors in intact airway. METHODS: Guinea pig tracheal rings were suspended in organ baths and muscle relaxants' antagonism of prejunctional M2 muscarinic autoreceptors was evaluated by augmentation of muscle contraction elicited by electrical field stimulation. Muscle relaxants' antagonism of postjunctional M3 muscarinic receptors was assessed by attenuation of muscle contraction elicited by acetylcholine. RESULTS: Rapacuronium displayed a 50-fold higher affinity for antagonism of the M2 versus M3 muscarinic receptor. Moreover, its affinity for the M2 but not the M3 receptor was within concentrations achieved clinically. In addition, rapacuronium caused an increase in baseline tone of airway smooth muscle that was antagonized by atropine but not by previous depletion of nonadrenergic noncholinergic neurotransmitters or by inhibitors of histamine receptors, tachykinin receptors, leukotriene receptors, or calcium channels. CONCLUSION: These findings are consistent with the hypothesis that rapacuronium may precipitate bronchoconstriction by selective antagonism of the M2 muscarinic receptor on parasympathetic nerves, enhancing acetylcholine release to act upon unopposed M3 muscarinic receptors on airway muscle. An additional mechanism of rapacuronium-induced bronchoconstriction is suggested by increases in baseline muscle tension.     

The role of M2 muscarinic receptor subtypes in mediating contraction of the pig bladder base after cyclic adenosine monophosphate elevation and/or selective M3 inactivation.

PURPOSE: In the bladder body M2 muscarinic receptors predominate but a smaller population of M3 receptors mediates direct detrusor contraction. M2 receptors have an indirect role by inhibiting cyclic adenosine monophosphate mediated relaxation in the bladder body. We investigated whether a similar mechanism also exists in the bladder base. METHODS: Experiments were performed on pig detrusor muscle. In receptor binding studies using l-quinuclidinyl [phenyl-4-(3)H] benzilate ([(3)H]QNB) (NEN Life Science Products, Inc., Boston, Massachusetts) displacement experiments were performed with subtype selective antagonists to determine the M2-to-M3 receptor ratio. In functional studies the affinity of these antagonists against carbachol induced contractions was calculated in normal tissues and in tissues after cyclic adenosine monophosphate elevation by pre-contraction with KCl and relaxation with isoprenaline, and/or selective M3 inactivation by incubation with 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) mustard (Sigma Chemical Co., St. Louis, Missouri) in the presence of methoctramine (Sigma Chemical Co.) to protect M2 receptors. RESULTS: In saturation binding studies receptor density was 130.5 of fmol./mg. protein and the dissociation constant for [(3)H]QNB was 0.27 nM. Displacement of [(3)H]QNB by the M3 selective antagonist 4-DAMP and the M2 antagonist methoctramine indicated an M2-to-M3 ratio of about 3:1. In functional studies 4-DAMP and methoctramine caused competitive antagonism of responses with affinity values of 9.5 and 6.3 in normal tissues, and 9.3 and 6.1, respectively, in cyclic adenosine monophosphate elevated tissues, suggesting the involvement of M3 receptors only. In tissues in which M3 receptors were inactivated and cyclic adenosine monophosphate levels were elevated the affinity of 4-DAMP was significantly reduced to 8.5 but that of methoctramine was significantly increased to 6.5, suggesting the involvement of M2 receptors. CONCLUSIONS: The M3 subtype appears to mediate contraction of the normal and cyclic adenosine monophosphate elevated tissues of the bladder base. Involvement of M2 receptors was only noted after selective M3 inactivation and cyclic adenosine monophosphate elevation.