Muscarinic receptors mediate cold stress-induced detrusor overactivity in type 2 diabetes mellitus rats.

OBJECTIVES: This study determined if muscarinic receptors could mediate the cold stress-induced detrusor overactivity induced in type 2 diabetes mellitus rats. METHODS: Ten-week-old female Goto-Kakizaki diabetic rats (n = 12) and Wister Kyoto non-diabetic rats (n = 12) were maintained on a high-fat diet for 4 weeks. Cystometric investigations of the unanesthetized rats were carried out at room temperature (27 ± 2°C) for 20 min. They were intravenously administered imidafenacin (0.3 mg/kg, n = 6) or vehicle (n = 6). After 5 min, the rats were transferred to a low temperature (4 ± 2°C) for 40 min where the cystometry was continued. The rats were then returned to room temperature for the final cystometric measurements. Afterwards, expressions of bladder muscarinic receptor M3 and M2 messenger ribonucleic acids and proteins were assessed by reverse transcription polymerase chain reaction and immunohistochemistry. RESULTS: In non-diabetic Wister Kyoto rats, imidafenacin did not reduce cold stress-induced detrusor overactivity. In diabetic Goto-Kakizaki rats, just after transfer to a low temperature, the cold stress-induced detrusor overactivity in imidafenacin-treated rats was reduced compared with vehicle-treated rats. Within the urinary bladders, the ratio of M3 to M2 receptor messenger ribonucleic acid in the diabetic Goto-Kakizaki rats was significantly higher than that of the non-diabetic Wister Kyoto rats. The proportion of muscarinic M3 receptor-positive area within the detrusor in diabetic Goto-Kakizaki rats was also significantly higher than that in non-diabetic Wister Kyoto rats. CONCLUSIONS: Imidafenacin partially inhibits cold stress-induced detrusor overactivity in diabetic Goto-Kakizaki rats. In this animal model, muscarinic M3 receptors partially mediate cold stress-induced detrusor overactivity.

Novel long-range inhibitory nNOS-expressing hippocampal cells.

The hippocampus, a brain region that is important for spatial navigation and episodic memory, benefits from a rich diversity of neuronal cell-types. Through the use of an intersectional genetic viral vector approach in mice, we report novel hippocampal neurons which we refer to as LINCs, as they are long-range inhibitory neuronal nitric oxide synthase (nNOS)-expressing cells. LINCs project to several extrahippocampal regions including the tenia tecta, diagonal band, and retromammillary nucleus, but also broadly target local CA1 cells. LINCs are thus both interneurons and projection neurons. LINCs display regular spiking non-pyramidal firing patterns, are primarily located in the stratum oriens or pyramidale, have sparsely spiny dendrites, and do not typically express somatostatin, VIP, or the muscarinic acetylcholine receptor M2. We further demonstrate that LINCs can strongly influence hippocampal function and oscillations, including interregional coherence. The identification and characterization of these novel cells advances our basic understanding of both hippocampal circuitry and neuronal diversity.

Evaluation of cholinergic functions in patients with Japanese encephalitis and Herpes simplex encephalitis.

Cognitive and memory impairment are related to cholinergic dysfunction and are important complications of viral encephalitis, In view of paucity of studies on cholinergic dysfunction in encephalitis, this study has been undertaken. We report acetyl choline esterase (AChE) and muscurinic 2 (M2) receptor levels in herpes simplex encephalitis (HSE) and Japanese encephalitis (JE) patients, and correlate these with cognitive functions and MRI findings. Patients with JE and HSE were evaluated for consciousness, neurological and MRI findings, plasma AChE and M2 receptor levels on admission and after one year. Twenty-nine patients with JE and 23 with HSE were included. Admission AChE levels in JE (48.32 ±â€¯5.36 nmol/min/ml) and HSE (41.92 ±â€¯5.12 nmol/min/ml) were significantly lower compared with controls (70.50 ±â€¯8.30 nmol/min/ml). M2 receptor levels were also low in JE (4.52 ±â€¯0.56 ng/ml) and HSE (4.35 ±â€¯0.57 ng/ml) compared with controls (7.95 ±â€¯0.41 ng/ml). In JE, AChE activity (r = 0.43, p = 0.02) and M2 receptor levels (r = 0.43, p = 0.02) correlated with caudate involvement, and AChE activity (r = 0.76, p = 0.03) with Mini Mental State Examination ( MMSE) score. In HSE, M2 receptor levels (r = 0.53, p = 0.03) correlated with MMSE. The levels of AChE and M2 receptors increased at one year compared to the baseline, which was greater in JE than in HSE. Both AChE and M2 receptors were reduced in JE and HSE and correlated with cognition at one year. Recovery of these biomarkers was more in JE than HSE.

Role of hyperglycaemia in the pathogenesis of hypotension observed in type-1 diabetic rats.

The role of hyperglycaemia in the pathogenesis of hypotension in diabetic disorders was investigated using the changes in cardiac M(2)-muscarinic receptor (M(2)-mAChR) gene expression in type-1-like diabetic rats and cultured cardiomyocytes. Blood pressure was markedly decreased in diabetic rats following the intravenous injection of streptozotocin (STZ) for 8 weeks. Also, the baroreflex sensitivity (Delta HR/Delta BP), as measured by the changes in heart rate (Delta HR) and mean blood pressure (Delta BP) 1 min after the intravenous injection of phenylephrine (10 microg/kg), was significantly increased. Arecaidine propargyl ester (APE), a M(2)-mAChR agonist produced a marked reduction in heart rate in these diabetic rats. Normalization of plasma glucose in diabetic rats using insulin (0.5 IU) or phlorizin (1 mg/kg) injection attenuated the blood pressure reduction and reversed the mRNA and protein levels of cardiac M(2)-mAChR. A high concentration of glucose (20 mmol/l) directly influenced the increase in gene expression of M(2)-mAChR in the H9c2 cardiac cell line. Hyperglycaemia induced an increase in cardiac M(2)-mAChR gene expression, suggesting a role in the pathogenesis of hypotension in diabetic disorders.

Hypoglossal premotor neurons of the intermediate medullary reticular region express cholinergic markers.

The inspiratory drive to hypoglossal (XII) motoneurons originates in the caudal medullary intermediate reticular (IRt) region. This drive is mainly glutamatergic, but little is known about the neurochemical features of IRt XII premotor neurons. Prompted by the evidence that XII motoneuronal activity is controlled by both muscarinic (M) and nicotinic cholinergic inputs and that the IRt region contains cells that express choline acetyltransferase (ChAT), a marker of cholinergic neurons, we investigated whether some IRt XII premotor neurons are cholinergic. In seven rats, we applied single-cell reverse transcription-polymerase chain reaction to acutely dissociated IRt neurons retrogradely labeled from the XII nucleus. We found that over half (21/37) of such neurons expressed mRNA for ChAT and one-third (13/37) also had M2 receptor mRNA. In contrast, among the IRt neurons not retrogradely labeled, only 4 of 29 expressed ChAT mRNA (P < 0.0008) and only 3 of 29 expressed M2 receptor mRNA (P < 0.04). The distributions of other cholinergic receptor mRNAs (M1, M3, M4, M5, and nicotinic alpha4-subunit) did not differ between IRt XII premotor neurons and unlabeled IRt neurons. In an additional three rats with retrograde tracers injected into the XII nucleus and ChAT immunohistochemistry, 5-11% of IRt XII premotor neurons located at, and caudal to, the area postrema were ChAT positive, and 27-48% of ChAT-positive caudal IRt neurons were retrogradely labeled from the XII nucleus. Thus the pre- and postsynaptic cholinergic effects previously described in XII motoneurons may originate, at least in part, in medullary IRt neurons.

Expression of muscarinic receptor subtypes in salivary glands of rats, sheep and man.

In rat parotid, submandibular and sublingual glands and in ovine parotid and in human labial glands, the expression of muscarinic receptor subtypes was examined by immunoblotting and immunohistochemistry. Functional correlates were searched for in rat salivary glands. In the rat submandibular and sublingual glandular tissues clear signals of muscarinic M1 and M5 receptors could be detected in the immunoblotting and vague bands for muscarinic M3 and, in particular for, M4 receptors. The rat parotid gland differed. In this gland, the signal was less obvious for the muscarinic M1 receptor, and further, muscarinic M4 receptors appeared more strongly marked than in the submandibular glands. The results from the immunohistochemistry could be interpreted as the muscarinic M4 receptors are located on nerve fibres, since the outer layer of lobuli were densely stained. Intraglandular vessels in the rat submandibular and parotid glands showed expression of M3 receptors. In contrast to the parotid gland, the submandibular vessels also expressed M1 and M2 receptors. Occasionally M5 receptors appeared in the arteries and veins also. The functional studies in the rat confirmed muscarinic M1 receptor mediated secretion in the submandibular gland. Since the M1 receptor blockade did not affect submandibular blood flow, indirect vascular effects could not in total explain the secretory inhibition. Also in the human labial glands, muscarinic M1, M3 and M5 receptors occurred. No or low amounts of muscarinic M2 and M4 receptors could be detected. In patients with Sjögren-like symptoms an up-regulation of M3, M4 and M5 receptors was apparent in the labial glands. In ovine parotid glands all receptors could be detected, but constantly with vague bands for muscarinic M2 receptors. In conclusion, muscarinic M1 receptors seem to be expressed in seromucous/mucous glands. A secretory effect by muscarinic M5 receptors is not to be excluded, since they were expressed in all the glands examined. However, other functions, such as promotion of inflammation, cell growth and proliferation are possible as well.

Decrease of muscarinic cholinergic receptors expression in placenta from rats exposed to methyl parathion.

Placental transfer of methyl parathion (MP), an organophosphate pesticide, could involve effects on cholinergic system. To analyze whether placental cholinergic system is altered by prenatal exposure to MP, expression of muscarinic cholinergic receptors (M1 and M2 subtypes; mAChR) was determined in pregnant rats exposed to MP at 0.0, 1.0, 1.5, and 2.0 mg/kg. An immunohistochemical analysis for M1 and M2 mAChR was performed, and the density of the mAChR signal was measured by image analysis. M1 and M2 mAChR were found in the trophoblast present in the labyrinth, with an 18% predominance of M2 over M1 in the non-exposed group. The expression of M1 and M2 mAChR in placentas exposed to MP showed a decrease when compared with the non-exposed group (P < 0.05); a dose-response effect was not detected. These results demonstrate that prenatal exposure to MP causes changes in the placental expression of mAChR M1 and M2, suggesting that related placental cholinergic functions could be affected.

Presence of a marked nonneuronal cholinergic system in human colon: study of normal colon and colon in ulcerative colitis.

BACKGROUND: The body has not only a neuronal but also a nonneuronal cholinergic system. Both systems are likely to be very important, particularly in inflammatory conditions. The patterns and importance of the nonneuronal cholinergic system in patients with ulcerative colitis (UC) are largely unknown. METHODS: The colons of UC and non-UC patients were examined for expression patterns of choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), and the muscarinic receptor of the M(2) subtype. RESULTS: ChAT and VAChT immunoreactions and mRNA reactions for ChAT were detected in epithelial and endocrine cells, in cells in the lamina propria, and in blood vessel walls. Furthermore, a marked M(2) immunoreaction was noted for epithelium, blood vessel walls, and smooth musculature. ChAT and VAChT immunoreactions were significantly higher in endocrine and epithelial cells, respectively, in non-UC mucosa than in UC mucosa. On the other hand, there was a tendency toward higher M(2) levels in epithelium of UC patients. CONCLUSIONS: There is a pronounced nonneuronal cholinergic system in the colon, which has previously been ignored when discussing cholinergic influences in UC. Furthermore, it is evident that certain changes in the nonneuronal cholinergic system occur in response to inflammation/derangement in UC. Cholinergic effects in the colon can be considered to be related not only to nerve-related effects but also to effects of acetylcholine from nonneuronal local cells. Thus, the recently discussed phenomenon of a "cholinergic antiinflammatory pathway" in the intestine may have a pronounced nonneuronal component.

The basal ganglia cholinergic neurochemistry of progressive supranuclear palsy and other neurodegenerative diseases.

BACKGROUND: Progressive supranuclear palsy (PSP) is a progressive neurodegenerative disorder involving motor and cognitive dysfunction. Currently, there is no effective treatment either for symptomatic relief or disease modification. This relates, in part, to a lack of knowledge of the underlying neurochemical abnormalities, including cholinergic receptor status in the basal ganglia. AIM: To measure muscarinic M2 and M4 receptors in the basal ganglia in PSP. METHODS: The muscarinic M2 (presynaptic) and M4 (postsynaptic) receptors in the striatum, pallidum and adjacent insular cortex were autoradiographically measured in pathologically confirmed cases of PSP (n = 18), and compared with cases of Lewy body dementias (LBDs; n = 45), Alzheimer's disease (AD; n = 39) and controls (n = 50). RESULTS: In cases of PSP, there was a reduction in M2 and M4 receptors in the posterior caudate and putamen compared to controls, but no significant changes in the pallidum. Cases with AD showed lower M2 receptors in the posterior striatum. Groups with LBD and AD showed higher M2 binding in the insular cortex compared with controls. CONCLUSIONS: The results suggest loss of posterior striatal cholinergic interneurones in PSP, and reduction in medium spiny projection neurones bearing M4 receptors. These results should be taken in the context of more widespread pathology in PSP, but may have implications for future trials of cholinergic treatments.

Decreased microRNA levels lead to deleterious increases in neuronal M2 muscarinic receptors in Spinal Muscular Atrophy models.

Spinal Muscular Atrophy (SMA) is caused by diminished Survival of Motor Neuron (SMN) protein, leading to neuromuscular junction (NMJ) dysfunction and spinal motor neuron (MN) loss. Here, we report that reduced SMN function impacts the action of a pertinent microRNA and its mRNA target in MNs. Loss of the C. elegans SMN ortholog, SMN-1, causes NMJ defects. We found that increased levels of the C. elegans Gemin3 ortholog, MEL-46, ameliorates these defects. Increased MEL-46 levels also restored perturbed microRNA (miR-2) function in smn-1(lf) animals. We determined that miR-2 regulates expression of the C. elegans M2 muscarinic receptor (m2R) ortholog, GAR-2. GAR-2 loss ameliorated smn-1(lf) and mel-46(lf) synaptic defects. In an SMA mouse model, m2R levels were increased and pharmacological inhibition of m2R rescued MN process defects. Collectively, these results suggest decreased SMN leads to defective microRNA function via MEL-46 misregulation, followed by increased m2R expression, and neuronal dysfunction in SMA.

Muscarinic M(2) acetylcholine receptor distribution in the guinea-pig gastrointestinal tract.

In the enteric nervous system, acetylcholine is the most common neurotransmitter to induce gastrointestinal smooth muscle contractions. Cholinergic signaling is mediated by muscarinic acetylcholine receptors on the surface of smooth muscle cells. Five different muscarinic receptor subtypes (M(1)-M(5)) have been identified and characterized, all of which belong to the superfamily of the G-protein-coupled receptor. The muscarinic M(2) acetylcholine receptor is the major muscarinic receptor subtype expressed by smooth muscle tissues in the gastrointestinal tract, where it is coexpressed with a smaller population of M(3) receptor. In this study, we examined the immunohistochemical distribution of the M(2) receptor using a specific antibody in the guinea-pig gastrointestinal tract. M(2) receptor-like immunoreactivity was mainly observed as associated with smooth muscle cells in the gastrointestinal tract. M(2) receptor-like immunoreactivity in smooth muscle cells was distributed throughout the cell membrane associated with caveolae. In the proximal colon, M(2) receptor-like immunoreactivity in the smooth muscle cells was weak. In the small intestine, interstitial cells of Cajal that possessed neurokinin 1 receptor-like immunoreactivity had intense M(2) receptor-like immunoreactivity. In the proximal colon, intramuscular and myenteric interstitial cells of Cajal exhibited M(2) receptor-like immunoreactivity. These findings indicate that, in the gastrointestinal musculature, M(2) receptors are distributed both in the smooth muscle cells and interstitial cells of Cajal, suggesting that the M(2) receptor elicits smooth muscle cell contraction and the interstitial cells of Cajal are the sites of innervation by enteric cholinergic neurons.

Immunohistochemical and histochemical findings favoring the occurrence of autocrine/paracrine as well as nerve-related cholinergic effects in chronic painful patellar tendon tendinosis.

The pathogenesis of the pain in patellar tendon tendinosis ("jumper's knee") is unclear. We have recently presented new information about the sensory nervous system in the human patellar tendon, but there is very little information regarding the possible occurrence of a cholinergic system in this tendon. In the present study, specimens of pain-free normal tendons and chronically painful tendinosis tendons were examined by different immunohistochemical and histochemical methods. Antibodies against the M(2) receptor, choline acetyltransferase (ChAT), and vesicular acetylcholine transporter (VAChT) were applied, and staining for demonstration of activity of acetylcholinesterase (AChE) was also utilized. It was found that immunoreactions for the M(2) receptor could be detected intracellularly in both blood vessel cells and tenocytes, especially in tendinosis specimens. Furthermore, in the tendinosis specimens, some tenocytes were seen to exhibit immunoreaction for ChAT and VAChT. AChE reactions were seen in fine nerve fibers associated with small blood vessels in both the normal control tendons and the tendinosis tendons. The observations suggest that there is both a nerve related and a local cholinergic system in the human patellar tendon. As ChAT and VAChT immunoreactions were detected in tenocytes of tendinosis tendons, these cells might be a source of local acetylcholine (Ach) production. As both tenocytes and blood vessel cells were found to exhibit immunoreactions for the M(2) receptor, it is likely that both of these tissue cells may be influenced by ACh. Thus, in conclusion, there appears to be an upregulation of the cholinergic system, and an occurrence of autocrine/paracrine effects in this system, in the tendinosis patellar tendon.

Localization of M2 muscarinic receptor protein in parvalbumin and calretinin containing cells of the adult rat entorhinal cortex using two complementary methods.

We investigated parvalbumin (PV) and calretinin (CR) containing interneurons in the rat entorhinal cortex. RNA amplification following single cell dissection of immunohistochemically labeled cells from layers II to VI revealed that PV cells, in contrast to CR cells, express the m2 muscarinic receptor (M2AchR) protein. Double immunostaining to confirm the results of RNA amplification indicated that the majority of PV cells contain M2AchR protein, whereas only a small proportion of CR cells do. In contrast, a large number of layer I CR cells, which are mostly Cajal-Retzius cells, were positive for M2AchR. RNA amplification following dissection of these cells also revealed that they contain the M2AchR protein. These findings emphasize that there are significant differences in the expression of different proteins, even among similar neuronal types in the same brain region. This highlights the importance of accurately collecting single cells, and knowledge of anatomical details in molecular biological studies.

Mitochondrial Ultrastructural Alterations and Declined M2 Receptor Density Were Involved in Cardiac Dysfunction in Rats after Long Term Treatment with Autoantibodies against M2 Muscarinic Receptor.

BACKGROUND: Previous studies showed that autoantibodies (M2-AA) against the second extracellular loop of M2 muscarinic receptor (M2AChR-el2) from dilated cardiomyopathy (DCM) serum could induce DCM-like morphological changes in mice hearts. However, the effects of M2-AA on the cardiac function during the process of DCM and the potential mechanisms are not fully known. The present study was designed to dynamically observe the cardiac function, mitochondrial changes, and M2 receptor binding characteristics in rats long-term stimulated with M2-AA in vivo. METHODS: M2-AA-positive model was established by actively immunizing healthy male Wistar rats with synthetic M2AChR-el2 peptide for 18 months. Meanwhile, vehicle group rats were administrated with physiological saline. The change of mitochondrial membrane potential (ΔΨm) was detected by radionuclide imaging. The ultrastructure of mitochondria was observed under electron microscopy. The M2 receptor binding characteristics were determined by radioactive ligand binding assay. RESULTS: After immunization for 12 months, compared with vehicle group, M2AChR-el2-immunized rats showed decreased myocardial contractility and cardiac diastolic function evidenced by declined maximal rate of rise of ventricular pressure and increased left ventricular end-diastolic pressure, respectively. Additionally, mitochondrial swelling and vacuolation were observed. At 18 months, M2AChR-el2-immunized rats manifested significant decreased cardiac systolic and diastolic function and pathological changes such as enlargement of right ventricular cavity and wall thinning; and the mitochondrial damage was aggravated. Furthermore, the M2 receptor maximum binding capacity (Bmax) of the M2AChR-el2-immunized rats significantly decreased, while the M2 receptor dissociation constant (Kd) was increased. CONCLUSIONS: Our study suggested that long-term stimulation with M2-AA leaded to the ventricular dilatation and gradual deterioration of cardiac dysfunction. Mitochondrial damage and the down-regulation of M2 receptor density and affinity may be involved in the process.

Gender comparison of muscarinic receptor expression and function in rat and human urinary bladder: differential regulation of M2 and M3 receptors?

Since symptoms of bladder dysfunction occur more frequently in women than in men and since muscarinic receptors are the physiologically most important system to mediate bladder contraction, we have compared the number, subtype distribution and function of muscarinic receptors in bladders from male and female rats. Muscarinic receptor function was also assessed in bladder strips from male and female human bladder. Male and female rats expressed a similar number of muscarinic receptors (144+/-5 vs. 140+/-6 fmol/mg protein in saturation radioligand binding). While competition binding curves for the moderately M(2)-selective methoctramine were not consistently better fitted by a two-site model, most competition curves for the M(3)-selective darifenacin were biphasic and yielded 29+/-10% and 31+/-7% high affinity sites (corresponding to M(3) receptors) in male and females, respectively. Immunoreactivity of alpha-subunits of the G-proteins G(q/11), G(i1/2), G(i3) and G(s) did not significantly differ between both genders. The muscarinic receptor agonist carbachol similarly stimulated inositol phosphate accumulation in bladder slices from male and female rats with calculated maximum responses of 69+/-17 and 77+/-18% over basal and pEC(50) values of 4.90+/-0.45 and 4.40+/-0.46, respectively. While darifenacin inhibited carbachol-stimulated inositol phosphate formation approximately 100-fold more potently than methoctramine, each antagonist was similarly potent in both genders. Carbachol concentration-dependently contracted bladder strips with a pEC(50) of 5.66+/-0.05 and 5.72+/-0.06 and maximum effects of 4.3+/-0.1 and 4.2+/-0.2 mN/mg wet weight in male and female rats, respectively. The contractile effect of carbachol was concentration-dependently antagonised by the non-selective atropine (1-30 nM), the M(1)-selective pirenzepine (1-30 M), the M(2)-selective methoctramine (1-10 microM) and the M(3)-selective darifenacin (10-100 nM), with the latter exhibiting a partly unsurmountable antagonism. The overall potency of all four antagonists suggested that contraction was mediated predominantly if not exclusively by M(3) receptors with no appreciable differences between both male and female rats. Similarly, the maximum effects (4.4+/-0.6 vs. 4.4+/-2.4 mN/mg) and pEC(50) (6.07+/-0.05 vs. 6.32+/-0.14) of carbachol did not differ between genders in bladder samples from 25 consecutive patients. We conclude that number und function of muscarinic receptors and the relative roles of their M(2) and M(3) subtypes do not differ between urinary bladders of male and female rats; at least with regard to overall muscarinic responsiveness this situation appears to be similar in humans.

[The acute effects of dimethoate on the muscarinic-receptors of rat brains and the relationship between muscarinic-receptors and cholinesterase].

OBJECTIVE: To study the acute effects of dimethoate on the muscarinic-receptors(M1, M2) in the brain of rats. METHODS: 24 Sprague-Dawley rats were divided into 4 groups randomly. They were administered subcutaneously with 0, 25, 50, 100 mg/kg dimethoate, respectively. Brains were removed after 48 hours of administration. Radioligand binding assay was used to determine the density and affinity of M1 and M2 receptors. RESULTS: Rats in the treated group showed low density of M1 and M2 receptors compared with the control rats. The brain M1 receptor density of the rats in the highest dosage group was significantly lower than that in the control group while brain M2 receptors density had a decrease trend with increasing dosage, but the difference showed no significance. However, there were no differences of the affinity of both M1 and M2 among different treated groups. Correlation analysis showed there is positive relationship between cholinesterase activity and density of M1 receptors(r = 0.583, P < 0.01). CONCLUSION: M1 and M2 receptors density decreased with the increasing dosage of dimethoate. It is suggested that the alleviating of cholinergic symptoms may be due to the decrease of M1 and M2 receptors in rat brain.

Expression and function of muscarinic subtype receptors in bladder interstitial cells of cajal in rats.

PURPOSE: To locate the muscarinic (M) M2 and M3 receptors in bladder interstitial cells of Cajal (ICCs) and to determine the effects of M2 and M3 agonists on bladder ICCs. MATERIALS AND METHODS: A total of 30 adult male Sprague-Dawley rats weighing 225-250 g were used in this study. Double-labeled fluorescence of muscarinic receptors and c-kit was performed for co-localization. To evaluate the effect of muscarinic agents on the excitation of bladder ICCs, we analyzed the inward current of bladder ICCs using the whole-cell patch clamp. The effect of muscarinic agents on the carbachol-induced inward currents was evaluated with the whole-cell patch clamp. RESULTS: M2 and M3 receptors were confirmed in the stroma ICCs in rats' bladders with double-labeled immunofluorescence. Spontaneous action potential was observed in freshly isolated bladder ICCs. The carbachol-induced inward Ca2+ current in ICCs can be blocked by atropine. The M2 receptor antagonist methoctramine (1 µM) showed a weak inhibitory capability on the inward Ca2+ current [from 74.8 ± 9.6 to 63.3 ± 13.8 Pascal (pA), n = 12, P = .03]. While the M3 receptor antagonist 4-diphenyl-acetoxy-N-methyl-piperidine methiodide (4-DAMP) (1 µM) significantly inhibited the inward Ca2+ current (from 78.4 ± 11.2 to 17.3 ± 7.9 pA, n = 12, P < .001). CONCLUSION: Bladder ICCs express M2 and M3 cholinergic receptors. Most muscarinic cholinergic receptor antagonists, especially the M3 antagonists, can effectively inhibit the carbamylcholine- induced inward current of bladder ICCs.

Muscarinic acetylcholine receptors in the mouse esophagus: focus on intraganglionic laminar endings (IGLEs).

BACKGROUND: IGLEs represent the only low-threshold vagal mechanosensory terminals in the tunica muscularis of the esophagus. Previously, close relationships of vesicular glutamate transporter 2 (VGLUT2) immunopositive IGLEs and cholinergic varicosities suggestive for direct contacts were described in almost all mouse esophageal myenteric ganglia. Possible cholinergic influence on IGLEs requires specific acetylcholine receptors. In particular, the occurrence and location of neuronal muscarinic acetylcholine receptors (mAChR) in the esophagus were not yet characterized. METHODS: This study aimed at specifying relationships of VGLUT2 immunopositive IGLEs and vesicular acetylcholine transporter (VAChT)-immunopositive varicosities using pre-embedding electron microscopy and the location of mAChR1-3 (M1-3) within esophagus and nodose ganglia using multilabel immunofluorescence and retrograde tracing. KEY RESULTS: Electron microscopy confirmed synaptic contacts between cholinergic varicosities and IGLEs. M1- and M2-immunoreactivities (-iry; -iries) were colocalized with VGLUT2-iry in subpopulations of IGLEs. Retrograde Fast Blue tracing from the esophagus showed nodose ganglion neurons colocalizing tracer and M2-iry. M1-3-iries were detected in about 80% of myenteric ganglia and in about 67% of myenteric neurons. M1- and M2-iry were present in many fibers and varicosities within myenteric ganglia. Presynaptic M2-iry was detected in all, presynaptic M3-iry in one-fifth of motor endplates of striated esophageal muscles. M1-iry could not be detected in motor endplates of the esophagus, but in sternomastoid muscle. CONCLUSIONS & INFERENCES: Acetylcholine probably released from varicosities of both extrinsic and intrinsic origin may influence a subpopulation of esophageal IGLEs via M2 and M1-receptors.

Effect of aging on airway remodeling and muscarinic receptors in a murine acute asthma model.

BACKGROUND AND OBJECTIVES: The influence of aging on the development of asthma has not been studied thoroughly. The aim of this study was to investigate age-related airway responses involving lung histology and expression of muscarinic receptors in a murine model of acute asthma. METHODS: Female BALB/c mice at the ages of 6 weeks and 6, 9, and 12 months were sensitized and challenged with ovalbumin (OVA) for 1 month (n = 8-12 per group). We analyzed inflammatory cells and T-helper (Th)2 cytokines in bronchoalveolar lavage (BAL) fluid and parameters of airway remodeling and expression of muscarinic receptors in lung tissue. RESULTS: Among the OVA groups, total cell and eosinophil numbers in BAL fluid were significantly higher in the older (6-, 9-, and 12-month-old) mice than in the young (6-week-old) mice. Interleukin (IL) 4 (IL-4) concentration increased, but IL-5 and IL-13 concentrations showed a decreased tendency, with age. IL-17 concentration tended to increase with age, which did not reach statistical significance. Periodic acid-Schiff (PAS) staining area, peribronchial collagen deposition, and area of α-smooth muscle staining were significantly higher in the 6-month older OVA group than in the young OVA group. The expression of the M3 and M2 muscarinic receptors tended to increase and decrease, respectively, with age. CONCLUSION: The aged mice showed an active and unique pattern not only on airway inflammation, but also on airway remodeling and expression of the muscarinic receptors during the development of acute asthma compared with the young mice. These findings suggest that the aging process affects the pathogenesis of acute asthma and age-specific approach might be more appropriate for better asthma control in a clinical practice.