Endogenous noradrenaline release from guinea-pig isolated trachea is inhibited by activation of M2 receptors.

Overflow of endogenous noradrenaline (NA) from guinea-pig isolated tracheae was evoked by electrical field stimulation (3 Hz, 540 pulses). The muscarinic receptor agonist oxotremorine inhibited the evoked overflow of NA in a concentration-dependent manner (EC50 84 nM). Methoctramine, pirenzepine and p-fluoro-hexahydrosiladiphenidol (each 1 microM) shifted the concentration-response curves of oxotremorine to the right with apparent pA2 values of 7.60, 6.74 and 6.18, respectively. It is concluded that sympathetic nerve terminals in the guinea-pig trachea are endowed with inhibitory muscarinic M2 receptors.     

星形胶质细胞毒蕈碱样乙酰胆碱受体M2、M4亚型基因克隆及序列分析

目的：克隆胶质细胞M_2、M_4受体亚型基因序列，并比较胶质细胞M_2、M_4受体亚型基因序列和蛋白质序列与神经元细胞M_2、M_4受体基因序列和蛋白质序列间的差异。方法：根据神经元细胞M_2、M_4受体基因序列设计出针对M_2、M_4受体基因序列全长的特异性探针，采用RT-PCR方法扩增胶质细胞M_2、M_4受体亚型基因序列，并对其进行克隆测序。结果：通过RT-PCR方法扩增胶质细胞M_2、M_4受体亚型基因序列，与神经元细胞M_2、M_4受体比较，M_2受体差异碱基17个，发生氨基酸改变的有8个；M_4受体差异碱基3个，发生氨基酸改变的有2个。结论：胶质细胞M_2、M_4受体与神经细胞M_2、M_4受体亚型在基因序列和氨基酸序列上具有明显差异。     

Determination of muscarinic agonist potencies at M1 and M2 muscarinic receptors in a modified pithed rat preparation

Summary The agonistic potencies of (±)muscarine, (±)cis - 2 - methyl - 5 - [(dimethylamino)methyl] - 1,3 -oxathiolane methiodide (cis-oxathiolane) and its two enantiomers were determined at muscarinic M₁ and M₂ receptors in the pithed rat. In non-pretreated animals, i.v. administration of these agents produced bradycardic effects mediated by cardiac M₂ receptors followed by increases in heart rate mediated by M1 receptors in sympathetic ganglia. As these responses have been shown to partly overlap, “true” M₁ and M₂ potencies were determined after selective blockade of M₁ and M₂ receptors by pirenzepine and methoctramine, respectively. A similar rank order of agonist potencies was obtained at M₁ and M₂ receptors: (+)cis-oxathiolane > (±)cis-oxathiolane > (±)muscarine > (-)cis-oxathiolane. At both receptor subtypes, (+)cis-oxathiolane was considerably more potent (ca. 30-fold) than its corresponding (−) enantiomer indicating that the agonist binding sites of the two receptor subtypes may have similar stereochemical properties. While (±)muscarine showed similar potencies at M₁ and M₂ receptors, racemic cis-oxathiolane and its two enantiomers showed a slight selectivity (3–7 fold) for M₁ receptors indicating the potential usefulness of these compounds in the development of selective M₁ receptor agonists. Send offprint requests to F. Cantalamessa 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.     

Increased function of inhibitory neuronal M2 muscarinic receptors in diabetic rat lungs

1. The function of inhibitory neuronal M₂ muscarinic receptors in diabetic rat lungs was investigated.
2. Neuronal M₂ muscarinic receptors inhibit acetylcholine release from parasympathetic nerves. Thus, stimulation of neuronal M₂ muscarinic receptors with muscarinic agonists, such as pilocarpine, inhibits acetylcholine release and vagally induced bronchoconstriction. In contrast, blockade of neuronal M₂ muscarinic receptors with selective M₂ muscarinic antagonists, such as AF-DX 116, potentiates acetylcholine release and vagally induced bronchoconstriction.
3. Rats were made diabetic by streptozotocin (65 mg kg⁻¹, i.v.). After 7–14 days the rats were anaesthetized with urethane (1.5 g kg⁻¹, i.p.), tracheostomized, vagotomized, ventilated and paralysed with suxamethonium (30 mg kg⁻¹, i.v.). Some 7 day diabetic rats were treated with low doses of long acting (NPH) insulin (2 units day⁻¹, s.c.) for 7 days before experimentation. This dose of insulin was not sufficient to restore normoglycaemia in diabetic rats. Thus, insulin-treated diabetic rats remained hyperglycaemic.
4. Distal electrical stimulation (5–70 Hz, 6 s, 40 V, 0.4 ms) of the vagi caused bronchoconstriction, measured as an increase in inflation pressure and bradycardia. In diabetic rats, vagally induced bronchoconstriction was significantly depressed vs controls. In contrast, bronchoconstriction caused by i.v. acetylcholine was similar in diabetic and control animals.
5. The function of neuronal M₂ muscarinic receptors was tested with the muscarinic agonist pilocarpine (0.001–100.0 μg kg⁻¹, i.v.) and the antagonist AF-DX 116 (0.01–3.0 mg kg⁻¹, i.v.). Pilocarpine inhibited vagally-induced bronchoconstriction (30 Hz, 20–40 V, 0.4 ms at 6 s) and AF-DX 116 potentiated vagally-induced bronchoconstriction (20 Hz, 20–40 V, 0.4 ms at 6 s) to a significantly greater degree in diabetic rats compared to controls.
6. Both frequency-dependent vagally-induced bronchoconstriction and M₂ muscarinic receptor function could be restored to nearly control values in diabetic rats treated with low doses of insulin.
7. Displacement of [³H]QNB (1 nM) with the agonist carbachol (10.0 nM–10.0 mM) from diabetic cardiac M₂ muscarinic receptors revealed a half log increase in agonist binding affinity at both the high and low affinity binding sites vs controls. In contrast, M₂ receptors from insulin-treated diabetic rat hearts showed no significant difference in binding affinity vs controls.
8. These data show that neuronal M₂ muscarinic receptors in the lungs have increased function in diabetic rats, suggesting that insulin modulates M₂ muscarinic receptor function.

     

Evidence of paired M2 muscarinic receptors

Binding assays involving various antagonists, including N-[3H] methylscopolamine, [3H]quinuclidinyl benzilate, AFDX-116, pirenzepine, and propylbenzilylcholine mustard, disclosed only a single population of M2 muscarinic receptors in membranes from the rat "brainstem" (medulla, pons, and colliculi). However, competition curves between N-[3H]methylscopolamine and various agonists, including oxotremorine, cis-dioxolane, and acetylethylcholine mustard, showed approximately equal numbers of guanine nucleotide-sensitive high affinity (H) sites and guanine nucleotide-insensitive low affinity (L) sites. This 50% H phenomenon persisted in different buffers, at different temperatures, after the number of receptors was halved (and, thus, the remaining receptor to guanine nucleotide-binding protein ratio was doubled), after membrane solubilization with digitonin, and when rabbit cardiac membranes were used instead of rat brainstem membranes. Preferential occupation of H sites with acetylethylcholine mustard, and of L sites with quinuclidinyl benzilate or either mustard, yielded residual free receptor populations showing predominantly L and H sites, respectively. Low concentrations of [3H]-oxotremorine-M labeled only H sites, and the Bmax for these sites was 49% of the Bmax found with [3H]quinuclidinyl benzilate plus guanine nucleotide. These and other results are most consistent with the idea that H and L receptor sites exist on separate but dimeric receptor molecules and with the hypothesis that only the H receptors cycle between high and low affinity, depending upon interactions between this receptor molecule and a guanine nucleotide-binding protein.     

Pharmacological differentiation of presynaptic M1 muscarinic receptors modulating acetylcholine release from postsynaptic muscarinic receptors in guinea-pig ileum

1. Effects of three muscarinic antagonists on electrically evoked ACh release and contractile response were investigated in longitudinal muscle strips of guinea-pig ileum suspended in an organ-bath and superfused with Krebs solution. ACh release was determined by a specific radioimmunoassay. 2. Telenzepine, a selective M1 muscarinic antagonist, increased the ACh release at a concentration of 100-fold less than that inhibiting the contractile response (10 vs 1000 nM). 3. AF-DX 116, a cardioselective M2 muscarinic antagonist, inhibited the contractile response at 10 microM, but did not affect the ACh release at this concentration. 4. (-)N-Methylscopolamine (NMS) did not affect the ACh release, but inhibited the contractile response at all concentrations tested (1-1000 nM), indicating (-)NMS can be used as an ileal specific postsynaptic muscarinic antagonist. 5. These data demonstrate that presynaptic muscarinic receptors modulating ACh release are distinct from postsynaptic ones involved in the contractile response and can be classified as M1 subtype.     

Evidence for prejunctional M2 muscarinic receptors in pulmonary cholinergic nerves in the rat.

1. The effects of muscarinic antagonists considered to be selective for M1 receptors (pirenzepine) and for M2 receptors (gallamine and methoctramine) were used to investigate the existence of prejunctional muscarinic receptors on cholinergic nerves in the rat lung. The tracheal tube preparation was used in vitro, and contraction of the trachealis muscle was induced by electrical field stimulation (EFS) and by application of an exogenous muscarinic agonist (pilocarpine), and measured as an increase in intraluminal pressure in the tube. 2. The muscarinic antagonists, gallamine and methoctramine, enhanced the contractions induced by nerve stimulation, while contractions elicited by exogenous application of pilocarpine were inhibited by the antagonists. 3. In contrast, pirenzepine blocked contractions induced by both EFS and pilocarpine in a dose-dependent manner (EC50 0.1 microM) due to blockade of the postjunctional muscarinic receptors on airway smooth muscle. Potentiation of the response to EFS was never seen with this antagonist. 4. The muscarinic agonist, pilocarpine, caused a slow maintained increase in tone of the tracheal tube and at the same time reduced the contractions induced by EFS. This inhibitory effect was blocked by gallamine and methoctramine. 5. The results suggest that prejunctional inhibitory muscarinic receptors may be localised on the parasympathetic cholinergic nerve terminals innervating tracheal smooth muscle in the rat. This confirms previous findings obtained by measuring transmitter release in this species. The present results suggest that these receptors are of the M2 subtype. Blockade of these autoreceptors with gallamine or methoctramine would increase the output of acetylcholine (ACh) and thereby enhance the nerve-induced contraction of tracheal smooth muscle.     

2型糖尿病大鼠膀胱M_3受体改变的实验研究

目的研究病程3周的2型糖尿病大鼠膀胱M3受体含量及其基因转录水平的变化情况,探讨糖尿病性膀胱早期发病机制中逼尿肌M3受体的改变情况。方法2 d龄雌性Wistar大鼠随机分成2型糖尿病组和正常对照组,链脲佐菌素按90 mg/kg体重腹腔注射并结合高糖高脂饮食诱导2型糖尿病大鼠模型。于糖尿病病程3周时进行下列实验:反转录-聚合酶链反应(RT-PCR)方法检测M3受体mRNA的含量;Western blotting方法检测膀胱M3受体蛋白的含量。结果RT-PCR方法检测结果显示:糖尿病组大鼠膀胱M3受体mRNA的数量显著高于正常对照组[(52.0±5.7)%vs(35.6±11.7)%,P<0.05]。Western blotting方法检测结果显示:糠尿病组大鼠膀胱M3受体含量显著高于正常对照组[(30.9±2.1)%vs(23.4±4.7)%,P<0.01]。结论本研究证实了糖尿病性膀胱早期M3受体的生物合成上调这一改变,从而解释了糖尿病早期膀胱逼尿肌收缩力增加这一现象。这可能是早期糖尿病性膀胱病变的一个发病机制。     

M1 and M3 muscarinic receptors are involved in the release of urinary bladder-derived relaxant factor

We have investigated the muscarinic receptor subtype(s) mediating the release of urinary bladder-derived relaxant factor that is demonstrated by a coaxial bioassay system. Acetylcholine-induced relaxation of a precontracted anococcygeus muscle mounted within the bladder was considered as an evidence for the release of this factor. M₁-muscarinic agonist McN-A-343 and the cholinesterase inhibitor physostigmine also elicited relaxation responses in the coaxial bioassay besides acetylcholine. Acetylcholine-induced relaxation was antagonized by the subtype-selective muscarinic antagonists (pK_B): M₃-antagonist darifenacin (9.36 ± 0.11), M₃/M₁-antagonist 4-DAMP (9.30 ± 0.11), M₁-antagonist telenzepine (8.56 ± 0.21), M₄-antagonist tropicamide (6.63 ± 0.17) and M₂-antagonist AF-DX 116 (6.01 ± 0.21). The pK_B values of these antagonists have suggested that stimulation of M₁- and M₃-muscarinic receptors in the bladder wall mediates the release of urinary bladder-derived relaxant factor. In addition, McN-A-343, by activating the facilitatory M₁ receptors and physostigmine by inhibiting the acetylcholinesterase may induce the release of this factor through endogenous acetylcholine in the coaxial bioassay system.     

Selective blockade of muscarinic M2 receptors in vivo by the new antagonist tripitramine

The antimuscarinic effects of tripitramine (1, 1, 24--tris [[5, 11-dihydro-6-oxo-6H-pyrido [2, 3-b][1, 4]-benzodiazepin-11-yl)(carbonyl] methyl]-8, 17-dimethyl-1, 8, 17, 24-tetraazatetracosane tetraoxalate), a member of a series of polymethylene tetraamines with in vitro cardioselectivity, were assessed in two in vivo preparations: anaesthetized and pithed rats. The well-known M₂ selective antagonist methoctramine was used in a comparative study. Tripitramine (0.0202 mol/kg i.v.) proved to be a potent antagonist at cardiac M₂ receptors that mediate the decrease in heart rate in the pithed rat; the same dose of this antagonist in the anaesthetized rat did not significantly affect the depressor action of methacholine mediated by vascular M₃ receptors. In the pithed rat, this dose did not affect the ganglionic M₁ receptor-mediated tachycardia and pressor response to muscarme or McN-A-343. These in vivo data are consistent with the in vitro findings and confirm that tripitramine is a more potent and selective muscarinic M₂ receptor antagonist than methoctramine.     

Putative M2 muscarinic receptors of rat heart have high affinity for organophosphorus anticholinesterases

The M2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-[3H]cis-methyldioxolane ([3H]CD), which has been used to label a high affinity population of M2 receptors. A single population of sites (KD 2.74 nM; Bmax of 82 fmol/mg protein) was detected and [3H]CD binding was sensitive to the M2 antagonist himbacine but much less so to pirenzepine, the M1 antagonist. These cardiac receptors had different sensitivities to NiCl2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with [3H]CD and considered to be of the M2 subtype. Up to 70% of the [3H]CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. [3H]CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K0.5 values of 0.8, 2, 20, and 50 nM, respectively. It was also inhibited by echothiophate and paraoxon with K0.5 values of 100 and 300 nM, respectively. The apparent competitive nature of inhibition of [3H]CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting less than 50% of 5 nM [3H]CD binding by 1 microM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting [3H]CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration. Nevertheless, the high affinities that cardiac muscarinic receptors have for these toxicants point to their extra vulnerability. It is suggested that the success of iv administration of the muscarinic receptor inhibitor atropine in initial therapy of poisoning by OP anticholinesterases may be related in part to the extra sensitivity of M2 receptors to certain OPs.     

Calcium channels involved in the inhibition of acetylcholine release by presynaptic muscarinic receptors in rat striatum.

1. The mechanism of the inhibitory action of presynaptic muscarinic receptors on the release of acetylcholine from striatal cholinergic neurons is not known. We investigated how the electrically stimulated release of [3H]-acetylcholine from superfused rat striatal slices and its inhibition by carbachol are affected by specific inhibitors of voltage-operated calcium channels of the L-type (nifedipine), N-type (omega-conotoxin GVIA) and P/Q-type (omega-agatoxin IVA). 2. The evoked release of [3H]-acetylcholine was not diminished by nifedipine but was lowered by omega-conotoxin GVIA and by omega-agatoxin IVA, indicating that both the N- and the P/Q-type (but not the L-type) channels are involved in the release. The N-type channels were responsible for approximately two thirds of the release. The release was >97% blocked when both omega-toxins acted together. 3. The inhibition of [3H]-acetylcholine release by carbachol was not substantially affected by the blockade of the L- or P/Q-type channels. It was diminished but not eliminated by the blockade of the N-type channels. 4. In experiments on slices in which cholinesterases had been inhibited by paraoxon, inhibition of [3H]-acetylcholine release by endogenous acetylcholine accumulating in the tissue could be demonstrated by the enhancement of the release after the addition of atropine. The inhibition was higher in slices with functional N-type than with functional P/Q-type channels. 5.We conclude that both the N- and the P/Q-type calcium channels contribute to the stimulation-evoked release of acetylcholine in rat striatum, that the quantitative contribution of the N-type channels is higher, and that the inhibitory muscarinic receptors are more closely coupled with the N-type than with the P/Q-type calcium channels.     

Transducing system operated by adenosine A(2A) receptors to facilitate acetylcholine release in the rat hippocampus

In spite of the increasing evidence concerning its neurotoxicity, young human individuals are often involved in the recreational use of amphetamine-type stimulants such as 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy"). A study aimed to investigate short- and long-term consequences of a repeated and intermittent MDMA administration (0, 5 or 10 mg/kg i.p., 3 days treatment history) was conducted in mice. Mice were injected at different phases in development, namely at early (28 days old), middle (38 days old) or late (52 days old) adolescence. When assessed for nociceptive response, a dose-dependent analgesia was found in middle and late adolescent mice. Carryover consequences of previous MDMA treatment were then investigated at adulthood (80 days old). In a social interaction test, levels of environment exploration and social behaviour resulted markedly increased in drug-free state as a function of drug exposure during development, whereas others behaviours were reduced. MDMA challenge (5-mg/kg dose) produced the expected hyperactivity, as well as a marked increment of hypothalamic serotonin (5-hydroxyhyptamine, 5-HT) levels. Mice treated chronically with MDMA during middle and late adolescence were associated with important reductions of the indoleamine. As a whole, these results indicate a differential long-term vulnerability to behavioural and neurotoxicant effects of MDMA as a function of the developmental stage of exposure.     

Methoctramine selectively blocks cardiac muscarinic M2 receptors in vivo

Summary The antimuscarinic effects of methoctramine (N, N- bis[6-[(2-methoxybenzyl)amino]hexyl]-1,8-octanediamine tetrahydrochloride), a polymethylene tetraamine endowed with high cardioselectivity in vitro, were assessed in two in vivo preparations. Methoctramine (300 g/kg i.v.) strongly inhibited the methacholine- and muscarine-induced bradycardia in the anaesthetized and pithed rat, respectively. The same dose of methoctramine did not significantly affect the depressor action of methacholine in the anaesthetized rat mediated by vascular M₂ receptors. Furthermore, even high doses of methoctramine (up to 1 mg/kg i. v.) did not reduce the ganglionic M₁ receptor-mediated tachycardia and pressor response to muscarine or McN-A-343 in the pithed rat. These data suggest that methoctramine while showing high affinity for cardiac M₂ receptors has rather low affinity for ganglionic M₁ and vascular M₂ receptors. This in vivo study thus provides further evidence to support the view that methoctramine is a potent and highly selective antagonist of cardiac M₂ receptors. Send offprint requests to: G. Lambrecht at the above address     

In vivo modulation of histamine release by autoreceptors and muscarinic acetylcholine receptors in the rat anterior hypothalamus

The modulation of histamine release by histamine and muscarinic acetylcholine receptors was investigated by using the push-pull technique. The anterior hypothalamic area of the conscious, freely moving rat was superfused through the push-pull cannula with CSF or with CSF containing drugs and the release of endogenous histamine was determined in the superfusate.Hypothalamic superfusion with tetrodotoxin (10 mol/1) led to a pronounced and sustained decrease in the histamine release rate. Superfusion with compound 48/80 (100 mg/1) was ineffective. Hypothalamic superfusion with the H₃ agonist (R)--methylhistamine inhibited, while superfusion with the H₃ antagonist thioperamide enhanced the release of histamine. The release of histamine was inhibited on hypothalamic superfusion with the muscarinic receptor agonists carbachol or oxotremorine. Histamine release was enhanced by atropine, and this release-enhancing effect was abolished by oxotremorine. The selective M₁ antagonist pirenzepine (100 mol/I) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 10 ol/1), which blocks M₁ and M₃ receptors, also enhanced the release rate of histamine. On the other hand, 50 and 100 moI/I methoctramine (M₂ receptor antagonist) 10 and 100 moI/l p-fluoro-hexahydro-siladifenidol (p-F-HHSiD, a M₃ receptor antagonist) were ineffective.It is concluded that histamine released in the hypothalamus originates predominantly from neurons. The release of histamine is modulated by H₃ autoreceptors. The histamine release is also modulated by cholinergic neurons which modify histamine release from histaminergic neurons by stimulating M₁ muscarinic acetylcholine heteroreceptors probably located on histaminergic neurons.Supported by the Fonds zur Förderung der wissenschaftlichen Forschung Correspondence to: H. Prast at the above address     

星形胶质细胞毒蕈碱样乙酰胆碱受体M1、M3、M5亚型基因克隆及序列分析

目的克隆胶质细胞M1、M3、M5受体亚型基因序列,比较胶质细胞和神经细胞M1、M3、M5受体亚型基因序列、蛋白质序列的差异。方法根据神经细胞M1、M3、M5受体基因序列全长设计特异性探针,采用RT-PCR方法扩增胶质细胞M1、M3、M5受体亚型基因序列,并对其进行克隆测序。结果测序得到胶质细胞M1、M3、M5受体亚型基因序列,与神经细胞比较,M1受体差异碱基4个,发生氨基酸改变的有1个;M3受体差异碱基有8个,发生氨基酸改变的位点有4个;M5受体差异碱基1个,发生氨基酸改变的有1个。结论胶质细胞与神经细胞M1、M3、M5受体亚型在基因和蛋白序列上具有明显差异。     

Postjunctional modulation by muscarinic M2 receptors of responses to electrical field stimulation of rat detrusor muscle preparations

1. The aim of the present study was to examine the modulator influence of muscarinic M(2) receptors on responses of rat urinary bladder detrusor muscle evoked by endogenous stimuli, i.e. by stimulation of the bladder innervation. 2. Responses were evoked by electrical field stimulation (EFS; 2-20 Hz, 0.8 ms, 60 V) of isolated strip preparations mounted in organ baths. The tension of the muscle strips was recorded digitally. EFS was performed by applying stimulation with either a short duration (5 s) or a longer duration (to reach peak response; approximately 20 s). 3. Effects of muscarinic receptor antagonists (muscarinic M(1)/M(3) receptor selective: 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP); muscarinic M(2) receptor selective: methoctramine), a beta-adrenergic antagonist (propranolol) and an adenosine receptor antagonist (8-p-sulfophenyltheophylline) were assessed on contractile activity and on poststimulatory relaxations. 4. Low concentrations of methoctramine (10(-8) m) reduced or tended to reduce the EFS-induced contraction, e.g. at 2 Hz by 12% while methoctramine at 10(-7) m had no significant effect. In addition, in the presence of 4-DAMP (10(-9) m), which tended to inhibit contractions at all frequencies (2-20 Hz; -17 to -25%), methoctramine at 10(-8) and 10(-7) m induced a further reduction of the contractile responses (-5 to -10%; 2-20 Hz). 5. The beta-adrenergic receptor antagonist propranolol (10(-6) m) and the adenosine receptor antagonist 8-p-sulfophenyltheophylline (10(-6) m) both increased contractile responses by 9-21% (2-10 Hz, long duration; P < 0.05-0.001) as a consequence of antagonizing relaxatory stimuli. Neither antagonist affected the contractile responses to EFS with the short duration stimulation. Poststimulatory relaxations were reduced by 30-60% (P < 0.05) by propranolol and by 40-60% (P < 0.001) by 8-p-sulfophenyltheophylline, but for 8-p-sulfophenyltheophylline only after stimulation with the short duration. 6. In the presence of methoctramine (10(-7) m), the 8-p-sulfophenyltheophylline-induced increases of the contractile response to long duration EFS were significantly enhanced at 10 Hz (+12 +/- 4%; P < 0.05), whereas no such enhancement of the propranolol inhibitory effect occurred in the presence of methoctramine. However, poststimulatory beta-adrenoceptor-evoked relaxations after short duration EFS were increased by about 35% in the presence of methoctramine, but not those after long duration. 7. Thus, muscarinic M(2) receptor activation inhibits adenosine receptor- and beta-adrenoceptor-evoked relaxations of the rat detrusor muscle. The inhibition occurs via a transient postjunctional mechanism that mainly affects responses with a short latency.     

Differential agonist-induced regulation of human M2 and M3 muscarinic receptors

We have compared the regulation of M(2) and M(3) muscarinic receptors heterologously expressed in HEK-293 cells upon long-term exposure towards the agonist carbachol. Carbachol time- and concentration-dependently reduced M(2) receptor density with a maximum reduction of about 60%. Treatment with 1mM carbachol for 24hr was accompanied by desensitisation of carbachol-induced Ca(2+) elevations (maximum response reduced by 70%) but not by alterations in the expression of various G-protein alpha-subunits. Consistently, heterologous desensitisation of Ca(2+) elevations by the purinergic receptor agonist ATP or by sphingosine-1-phosphate was not detected. In contrast, carbachol time- and concentration-dependently up-regulated M(3) receptors with maximum increases to about 350% of control values. The up-regulation was fully blocked by cycloheximide indicating that it was dependent on protein synthesis. Concomitant with the up-regulation of the M(3) receptor was a reduction in the expression of the alpha-subunit of G(q/11). The net effect of these two opposite regulatory mechanisms was a lack of alteration of carbachol-stimulated Ca(2+) elevation. However, the reduction of G(q/11) was accompanied by a heterologous desensitisation of Ca(2+) elevations by ATP and sphingosine-1-phosphate. Levels of M(2) and M(3) receptor mRNA as assessed by real-time PCR were not significantly altered by carbachol exposure for either receptor, suggesting that alterations of mRNA stability did not contribute to the observed changes in receptor number. We conclude that M(2) and M(3) receptor expression within the same cell undergoes differential agonist-induced regulation being accompanied by distinct regulation of G-protein expression leading to differential effects on signal transduction by other receptor systems.