Acquired Idiopathic Generalized Anhidrosis (AIGA) and Its Complications: Implications for AIGA as an Autoimmune Disease.

Acquired idiopathic generalized anhidrosis (AIGA) is a rare disorder in which systemic anhidrosis/hypohidrosis occurs without causative dermatological, metabolic or neurological disorder. Most cases of AIGA have been reported in Asia, especially in Japan, but there have been only a few reports in Europe and the United States. Severe AIGA may result in heatstroke and can reduce quality of life due to restriction of exercise and outdoor works. AIGA is often accompanied by cholinergic urticaria (CholU), and it is thought that AIGA and CholU with anhidrosis/hypohidrosis belong to the same spectrum of the disease. However, the pathophysiology of AIGA has not yet been clarified. Decreased expression of cholinergic receptor M3 on the epithelial cells of eccrine sweat glands is often accompanied by T cell infiltration around eccrine apparatus, suggesting an immunological mechanism of disordered perspiration. AIGA is occasionally associated with various complications indicative of autoimmune disorders. The association of autoimmune complications further suggests that AIGA is an autoimmune disorder. Studies on complications may lead to a better understanding of the pathophysiology of AIGA.

Highly specific detection of muscarinic M3 receptor, G protein interaction and intracellular trafficking in human detrusor using Proximity Ligation Assay (PLA).

PURPOSE: Muscarinic acetylcholine receptors (mAChRs) regulate a number of important physiological functions. Alteration of mAChR expression or function has been associated in the etiology of several pathologies including functional bladder disorders (e.g bladder pain syndrome/interstitial cystitis - BPS/IC). In a previous study we found specific mAChR expression patterns associated with BPS/IC, while correlation between protein and gene expression was lacking. Posttranslational regulatory mechanisms, e.g. altered intracellular receptor trafficking, could explain those differences. In addition, alternative G protein (GP) coupling could add to the pathophysiology via modulation of muscarinic signaling. In our proof-of-principle study, we addressed these questions in situ. We established PLA in combination with confocal laserscanning microscopy (CLSM) and 3D object reconstruction for highly specific detection and analysis of muscarinic 3 receptors (M3), G protein (GP) coupling and intracellular trafficking in human detrusor samples. MATERIAL AND METHODS: Paraffin sections of formalin-fixed bladder tissue (FFPE) of BPS/IC patients receiving transurethral biopsy were examined by Cy3-PLA for M3 expression, coupling of M3 to GPs (Gαq/11, Gαs, Gαi) and interaction of M3 with endocytic regulator proteins. Membranes were labeled with wheat germ agglutinin-Alexa Fluor®488, nuclei were stained with DAPI. Object density and co-localization were analyzed in 3D-reconstruction of high resolution confocal z-stacks. RESULTS: Confocal image stack processing resulted in well demarcated objects. Calculated receptor densities correlated significantly with existing confocal expression data, while significantly improved specificity of M3 detection by PLA was verified using bladder tissue samples from transgenic mice. 50-60% of the M3 receptor complexes were plasma membrane associated in human bladder detrusor. Application of PLA for M3 and GPs allowed visualization of M3-GP interactions and revealed individual GP-subtype coupling patterns. Detection of M3 interactions with endocytic trafficking proteins by PLA resulted in object sizes correlating with well-documented vesicle sizes of the endocytosis pathway. CONCLUSION: PLA enabled highly specific detection of M3 receptor expression, demonstration of M3/GP differential coupling and intracellular M3 trafficking in human detrusor smooth muscle cells. This new approach minimized background fluorescence and antibody cross-reactions resulting from single antibody application, and enhanced specificity due to the use of two primary antibodies. Use of subcellular markers allowed visualization of subcellular receptor location. PLA/CLSM allows analyses of muscarinic "receptor - G protein - promiscuity" and intracellular trafficking even in bladder paraffin sections and may give new insights into the etiology and pathology of BPS/IC.

Role of flotillins in the endocytosis of GPCR in salivary gland epithelial cells.

Endocytosis has numerous functions in cellular homeostasis. Defects in the endocytic pathway of receptors may lead to dysfunction of salivary gland secretion. Therefore, elucidating the complex mechanisms of endocytosis may facilitate solutions for disease treatment and prevention. The muscarinic type 3 receptor (M3R), a G-protein-coupled receptor (GPCR) located in the plasma membrane, is involved in numerous physiological activities such as smooth muscle contraction and saliva secretion. M3R enters cells through clathrin-mediated endocytosis (CME), while flotillins (flot-1 and -2), highly conserved proteins residing in lipid-raft microdomains, make use of clathrin-independent endocytosis (CIE) for their internalization. Since these two proteins use two discrete pathways for endocytic entry, the association of flotillins with CME is poorly understood. We examined whether flotillins play a role in CME of M3R using immunoblotting, immunocytochemistry, confocal immunofluorescence microscopy, co-immunoprecipitation, and RNA interference techniques in secretory epithelial cells. Upon stimulation with a cholinergic agonist, M3R, flot-1, and flot-2 each internalized from the plasma membrane into intracellular sites. The addition of chlorpromazine and cytochalasin D, well-known inhibitors of CME, inhibited internalization of M3R via CME. Filipin III and methyl-ß-cyclodextrin (mßCD) acting as lipid raft inhibitors disrupted internalization of flot-1/2 via CIE. Interestingly, filipin III and mßCD slightly reduced expression level of M3R whereas chlorpromazine and cytochalasin D did not affect internalization of the flotillin isoforms. M3R and flot-1/2 colocalized and interacted with each other as they entered the cytosol during limited periods of incubation. Moreover, knockdown of flot-1 or -2 by flotillin-specific siRNA prevented internalization and reduced the endocytic efficiency of M3R. Our results suggest that flot-1 and -2 are partially involved in CME of M3R by facilitating its internalization.

The F-BAR Protein NOSTRIN Dictates the Localization of the Muscarinic M3 Receptor and Regulates Cardiovascular Function.

RATIONALE: Endothelial dysfunction is an early event in cardiovascular disease and characterized by reduced production of nitric oxide (NO). The F-BAR protein NO synthase traffic inducer (NOSTRIN) is an interaction partner of endothelial NO synthase and modulates its subcellular localization, but the role of NOSTRIN in pathophysiology in vivo is unclear. OBJECTIVE: We analyzed the consequences of deleting the NOSTRIN gene in endothelial cells on NO production and cardiovascular function in vivo using NOSTRIN knockout mice. METHODS AND RESULTS: The levels of NO and cGMP were significantly reduced in mice with endothelial cell-specific deletion of the NOSTRIN gene resulting in diastolic heart dysfunction. In addition, systemic blood pressure was increased, and myograph measurements indicated an impaired acetylcholine-induced relaxation of isolated aortic rings and resistance arteries. We found that the muscarinic acetylcholine receptor subtype M3 (M3R) interacted directly with NOSTRIN, and the latter was necessary for correct localization of the M3R at the plasma membrane in murine aorta. In the absence of NOSTRIN, the acetylcholine-induced increase in intracellular Ca(2+) in primary endothelial cells was abolished. Moreover, the activating phosphorylation and Golgi translocation of endothelial NO synthase in response to the M3R agonist carbachol were diminished. CONCLUSIONS: NOSTRIN is crucial for the localization and function of the M3R and NO production. The loss of NOSTRIN in mice leads to endothelial dysfunction, increased blood pressure, and diastolic heart failure.

Reinnervated nerves contribute to the secretion function and regeneration of denervated submandibular glands in rabbits.

This study aimed to investigate the contribution of redistributed nerves in the secretory function and regeneration of a denervated submandibular gland (SMG). The postganglionic parasympathetic and sympathetic denervated SMGs of rabbits were wrapped in polyester or acellular dermal matrices to block nerve regeneration either partially or completely. Submandibular glands were removed 4, 8, 16, and 24 wk after the operation and examined histologically. Furthermore, the aquaporin-5 (AQP5), muscarinic-3 (M3), and ß1-adrenergic receptors were evaluated by immunofluorescence and western blot analysis. After denervation, salivary flow was decreased and acinar cells were atrophic, and the expression levels of the M3, ß1-adrenergic, and AQP5 receptors were decreased. However, both impaired secretion function and atrophic parenchyma were gradually ameliorated with the growing redistribution of parasympathetic and sympathetic nerves. Apoptosis was markedly inhibited and expression of the M3, ß1-adrenergic, and AQP5 receptors was increased after reinnervation. In contrast, SMGs without reinnervated nerves maintained hyposecretion and atrophic parenchyma. In conclusion, reinnervated nerves in a rabbit's denervated SMG played an important role in the secretion function and regeneration of SMGs via up-regulation of the expression of neurotransmitter receptors and AQP5.

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 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.

Hypersensitive mAChRs are involved in the epiphora of transplanted glands.

Autologous transplantation of the submandibular gland is an effective treatment for severe dry eye syndrome. However, more than 40% of patients experience epiphora 3 to 6 months after transplantation. The underlying mechanism of epiphora remains to be elucidated. To investigate the potential roles of muscarinic acetylcholine receptors (mAChRs) in the induction of epiphora in transplanted glands, we assessed and found elevated mRNA and protein expression of M1- and M3-mAChR in transplanted glands from epiphora patients. The content of inositol 1, 4, 5-trisphosphate was also elevated. Moreover, carbachol (5 and 10 µM) induced greater increase of [Ca(2+)]i in isolated epiphora submandibular cells than in controls. Although aquaporin-5 (AQP5) content and distribution in the apical and lateral plasma of epiphora glands did not change, AQP5 content was reduced in lipid microdomains (lipid rafts and caveolae) but increased in non-lipid microdomains compared with controls. Carbachol (10 µM) increased the ratio of non-lipid microdomain to total AQP5 in the cultured control submandibular gland tissue. Taken together, these results indicated that hypersensitive mAChRs might be involved in the epiphora of transplanted submandibular glands by modulating AQP5 trafficking.

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.

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.

Monoclonal antibody against α-actinin 4 from human umbilical vein endothelial cells inhibits endothelium-dependent vasorelaxation.

BACKGROUND: This study was attempted to identify new molecules expressed on the plasma membrane of human umbilical vein endothelial cells (HUVECs) using monoclonal antibody-based proteomics technology and to determine the effect of the identified antibody on vascular reactivity. METHODS: Twenty-two antibodies were developed from rats inoculated with HUVECs, and their effects were determined by observing vascular reactivity. RESULTS: Among the 22 antibodies, the C-7 antibody significantly inhibited endothelium-dependent vasorelaxation in response to acetylcholine (ACh) but not to histamine. Moreover, the C-7 antibody did not affect norepinephrine-induced contraction in either the endothelium-intact or -denuded aorta. A proteomics study involving immunoprecipitation of the C-7 antibody with biotinylated HUVECs showed that this antibody binds to plasma membrane proteins corresponding to immunoglobulin heavy chain (VHDJ region), chaperonin-containing T-complex polypeptide 1 and α-actinin 4. The muscarinic M3 ACh receptor and α-actinin 4 were colocalized on the plasma membrane of HUVECs, and the colocalization was found to increase in response to ACh and was inhibited by pretreatment with the C-7 antibody. CONCLUSIONS: These results demonstrate that monoclonal C-7 antibody exerts an inhibitory effect on endothelium-dependent vasorelaxation induced by ACh and that this response may at least partially result from the inhibition of α-actinin 4.

Alterations in the non-neuronal acetylcholine synthesis and release machinery in esophageal epithelium.

AIMS: A non-neuronal cholinergic system has been described in epithelial cells including that of the urinary bladder (urothelium) and the upper gastrointestinal tract (esophagus). Epithelial dysfunction has been implicated in the pathophysiology of persistent pain conditions such as painful bladder syndrome as well as functional heartburn. For example, alterations in the ability to synthesize and release acetylcholine may contribute to changes in epithelial sensory and barrier function associated with a number of functional genitourinary and intestinal disorders. MAIN METHODS: We examined using immunoblot, acetylcholine (ACh)-synthesis and release components in cat esophageal mucosa and whether elements of these components are altered in a naturally occurring model of chronic idiopathic cystitis termed feline interstitial cystitis (FIC). KEY FINDINGS: We identified proteins involved in ACh synthesis and release (high affinity choline transporter, CHT1; ACh synthesizing enzyme choline acetyltransferase ChAT and carnitine acetyltransferase CarAT; vesicular ACh transporter VAChT and the organic cation transporter isoforms 1-3 or OCT-1-3) in cat esophageal mucosa. Significant alterations in CHT, ChAT, VAChT and OCT-1 were detected in the esophageal mucosa from FIC cats. Changes in the vesicular nucleotide transporter (VNUT) and the junctional protein pan-cadherin were also noted. SIGNIFICANCE: Taken together, these findings suggest that changes in the non-neuronal cholinergic system may contribute to alterations in cell-cell contacts and possibly communication with underlying cells that may contribute to changes in sensory function and visceral hyperalgesia in functional esophageal pain.

Tiotropium increases cytosolic muscarinic M3 receptors and acetylated H3 histone proteins in induced sputum cells of COPD patients.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is characterized by irreversible progressive airflow limitation related to tobacco smoking. This limitation is caused by chronic inflammation of the airways and lung parenchyma and is associated with increased activity of parasympathetic system. The most effective bronchodilators in COPD are muscarinic receptor antagonists (MRA), which reverse, at least in part, compromised respiratory function. MRA also contribute to control inflammatory processes via interactions with inflammatory signaling molecules. The use of the long-acting cholinolytic bronchodilatator - tiotropium, with high affinity to M3 receptors, is suggested as a first line maintenance treatment in COPD patients. MATERIAL AND METHODS: In this study we assessed M3 receptor protein expression in induced sputum of 27 stable COPD patients before and after therapy consisting of 18 µg once daily tiotropium for 12 weeks. Lung function tests including spirometry, lung volumes, and DLCO were performed before and after therapy in all COPD patients. The patients were subjected to the sputum induction procedures before and after therapy. Sputum cells were isolated, sample-specific cell profiles were characterized, and the cells were processed to isolate pure cytosolic fractions. Cytosolic M3 protein and HDAC2 levels and nuclear acetylated histone H3 (AcH3) expression was quantified using specific antibodies against human proteins and Western blot with enhanced luminescence detection. RESULTS: Therapy significantly increased the mean forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) volume (P<0.05). The mean expression of M3 protein was higher by 37% after therapy (P<0.05), HDAC2 expression was not altered, while AcH3 level was increased by about 90% (P<0.01), compared with the corresponding data before therapy. HDAC2 expression before therapy was positively correlated with AcH3 expression (r = 0.74), while after therapy no correlation was detected. FEV1, FCV, and cytosolic M3 protein expression did not correlate with other biochemical parameters tested. CONCLUSIONS: Twelve weeks of tiotropium therapy in COPD patients improves clinical indices of lung function and involves alterations in sputum cell chromatin acetylation and also increased cholinergic M3 receptor internalization.

Leucine supplementation augments insulin secretion in pancreatic islets of malnourished mice.

OBJECTIVES: We investigated the influence of leucine supplementation on insulin secretion and on some proteins related to insulin secretion in malnourished mice. METHODS: Swiss mice (aged 21 days) received isocaloric normo-17% (NP) or 6% low-protein (LP) diet for 120 days. Half of the NP and LP mice received 1.5% leucine in the drinking water during the last 30 days (NPL and LPL, respectively). RESULTS: The LP mice were hypoinsulinemic compared with the NP group, whereas LPL mice exhibited increased insulinemia in the fed state versus LP mice. The LP mouse islets were less responsive to 22.2 mM glucose, 100 microM carbachol (Cch), and 10 mM leucine than the NP group. However, LPL islets were more responsive to all these conditions compared with the LP group. The muscarinic type 3 receptor, (M3R) Cabeta2, and PKC-alpha protein contents were reduced in LP compared with NP islets but significantly higher in LPL than LP islets. The p-AKT/AKT ratio was higher in LPL compared with LP islets. CONCLUSIONS: Leucine supplementation increases insulin secretion in response to glucose and leucine and to agents that potentiate secretion, such as Cch, in malnourished mice. The enhanced levels of M3R, Cabeta2, and PKC-alpha proteins, as well as of the p-AKT/AKT ratio, may play a role in this process.

Histological determination of the areas enriched in cholinergic terminals and M2 and M3 muscarinic receptors in the mouse central auditory system.

Cholinergic projections to auditory system are vital for coupling arousal with sound processing. Systematic search with in situ hybridization and immunohistochemistry indicated that the ventral nucleus of the medial geniculate body and the nucleus of the brachium of the inferior colliculus constituted cholinergic synaptic sites in the brainstem auditory system, containing a significant number of cholinergic axon terminals and m2 receptor-expressing cell bodies.

[Characterization of muscarinic receptors in undifferentiated thyroid cells in Fisher rats]. / Caracterización de receptores muscarínicos en células indiferenciadas tiroideas de ratas Fisher.

INTRODUCTION: The parasympathetic autonomous nervous system exerts control over thyroid function by activation of the muscarinic receptors in follicular cells. Various pharmacological and molecular subtypes of muscarinic receptors (M(1), M(2), M(3), M(4), M(5)) have been identified in central nervous system and peripheral tissues. Controversy surrounds receptor characterization in thyroid cells. MATERIALS AND METHODS: Undifferentiated Fisher rat thyroid epithelial cells (FRT) were cultured. Association and dissociation kinetics assays and antagonist competition studies of the binding of (3)H-N-methylscopolamine ((3)H-NMS) to muscarinic receptors were performed to demonstrate the presence of muscarinic receptors. RESULTS: Specific muscarinic receptors in the plasma membrane of FRT cells were observed with an equilibrium dissociation constant (K(d)) of 0.44 nmol. The order of affinities obtained fitting the data to one binding site model in competition experiments with the muscarinic receptor antagonist was: dicyclomine > hexahydrosiladifenidol (HHSD) = 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) > pirenzepine > himbacine = 11-[[2-[(diethylamino)methyl]- 1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido (414)benzodiazepine (AF-DX 116). CONCLUSIONS: The results obtained indicate the existence of specific (3)H-NMS muscarinic binding sites located in the plasma membrane of FRT cells. The results obtained in competition experiments suggest that the receptors present in FRT cells belong to the M(3) subtype.

Suitability of muscarinic acetylcholine receptor antibodies for immunohistochemistry evaluated on tissue sections of receptor gene-deficient mice.

Acetylcholine (ACh) is a major regulator of visceral function exerting pharmacologically relevant effects upon smooth muscle tone and epithelial function via five types of muscarinic receptors (M1R-M5R). In this paper, we assessed the specificity of muscarinic receptor (MR) antibodies in immunohistochemical labelling on tissue sections by analysing specimens from wild-type and respective gene-deficient mice. Of 24 antibodies evaluated in this study, 16 were tested at 18 different conditions each, and eight of them in 21 different protocols, resulting in a total number of 456 antibody/protocol combinations. Each of them was tested at four antibody dilutions at minimum, so that finally, at least 1,824 conditions were evaluated. For each of them, dorsal root ganglia, urinary bladder and cross-sections through all thoracic viscera were investigated. In all cases where the antigen was available, at least one incubation condition was identified in which only select cell types were immunolabelled in the positive control but remained unlabelled in the pre-absorption control. With two exceptions (M2R antibodies), however, all antibodies produced identical immunohistochemical labelling patterns in tissues taken from corresponding gene-deficient mice even when the pre-absorption control in wild-type mice suggested specificity. Hence, the present data demonstrate the unpleasant fact that reliable immunohistochemical localisation of MR subtypes with antibodies is the exception rather than the rule. Immunohistochemical detection of MR subtype localisation in tissue sections of peripheral organs is limited to the M2R subtype utilising the most commonly used methodological approaches.

Innervation and receptor profiles of the human apocrine (epitrichial) sweat gland: routes for intervention in bromhidrosis.

BACKGROUND: Human apocrine (epitrichial) sweat glands secrete in response to local or systemic administration of catecholamines and cholinergic agonists. As the process of secretion in human apocrine glands is not fully understood and no literature detailing the expression of adrenergic, cholinergic and purinergic receptors is available, there is a need to know the receptor types. Such data could provide new approaches for the treatment of axillary bromhidrosis. OBJECTIVES: To investigate the localization of nerve fibres, adrenergic, cholinergic and purinergic receptors in human axillary apocrine sweat glands by immunohistochemistry. METHODS: Human axillary apocrine sweat glands were investigated by serial sectioning of paraffin wax-embedded skin samples from volunteers. Sections were examined by light microscopy and immunohistochemistry, using antibodies against neurofilament, alpha- and beta-adrenoceptors, P2Y(1), P2Y(2) and P2Y(4) purinoceptors, and M(3) cholinoceptors. RESULTS: Neurofilaments were found near the eccrine but not the apocrine gland. Apocrine glands demonstrated the presence of beta-2 and beta-3 adrenoceptors in the secretory coil of the gland, but not alpha-1, beta-1 or M(3) receptors. Glandular purinergic staining (P2Y(1), P2Y(2) and P2Y(4)) was found in what looked like myoepithelial cells, while P2Y(1) and P2Y(2) staining was found on apical membranes and diffusely throughout secretory cells. Eccrine gland staining acted as internal positive controls. CONCLUSIONS: No nerve fibres were found near the apocrine gland, suggesting that any catecholamine influence is through humoral effects and that glands could be influenced by beta-adrenoceptor subtypes and purinoceptors. Blockage of both these types of receptors offers a route to controlling apocrine secretion from axillary glands and reducing the opportunity for the development of bromhidrosis.

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.