Expression Of M3 Receptors Research Articles

Parasympathetic regulation of heart rate in rats after 5/6 nephrectomy is impaired despite functionally intact cardiac vagal innervation

Chronic renal failure is frequently associated with a high risk of sudden cardiac death due to dysfunction of the autonomic nervous system. The pathogenic mechanisms underlying the parasympathetic cardiac dysautonomia are not fully elucidated yet. Chronic renal failure was induced in rats by 5/6 nephrectomy. Blood pressure, resting heart rate and plasma levels of creatinine, urea and asymmetric dimethylarginine (ADMA) were measured. To characterize the parasympathetic innervation of the heart, chronotropic responses to atropine, metipranolol and to vagal stimulation in the absence or presence of ADMA were investigated in vivo. In vitro, chronotropic and inotropic effects of carbachol and ADMA and mRNA expression of muscarinic M2 receptors, high affinity choline transporter (CHT1), vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT) were assessed in the isolated cardiac tissues. In 5/6 nephrectomy rats, the resting heart rate was significantly higher and the parasympathetic tone, measured as the effect of atropine after administration of metipranolol was significantly lower than in control animals. Plasma ADMA levels were significantly elevated in the uraemic rats and significantly inversely correlated with the effect of atropine on the heart rate. No differences were revealed in the plasma norepinephrine concentrations, negative chronotropic responses to stimulation of the vagus nerves, chronotropic and inotropic responses to carbachol and the relative expression of M2 receptors, CHT1, VAChT and ChAT. The data suggest that cardioacceleration in chronic renal failure is caused by a diminished cardiac parasympathetic tone in the presence of a functionally intact intrinsic cardiac cholinergic signalling system.

Protein Kinase C Regulates the Expression of M1 Receptors and BDNF in Rat Retinal Cells

Protein kinase C (PKC) plays a key role in cellular events including proliferation, survival and differentiation. Our previous study showed the effect of phorbol 12-myristate 13-acetate (PMA), a PKC activator, inducing a decrease in retinal cells proliferation. This effect was mediated by muscarinic type 1 receptors (M1) activation and brain derived neurotrophic factor (BDNF) treatment also induced a decrease in cell proliferation. Based on these results we analyzed the expression of either M1 receptors or BDNF following PMA treatment of retinal cell cultures. Our data demonstrated that PMA induced a decrease in both protein expressions after 48 h in culture. However, after 45 min, PMA induced a transient increase in BDNF expression and a decrease in M1 receptors expression. Analyzing the expression of M1 receptors and BDNF during the postnatal development in vivo, we observed a decrease in both proteins. Taken together our results suggest the involvement of PKC in the control of M1 expression in retinal cells.

Muscarinic receptor expression increases following exposure to intravesical pressures of ≤40 cm-H2O: a possible mechanism for pressure-induced cell proliferation

We studied the effect of increased hydrostatic pressure on muscarinic (M) receptor expression in isolated human bladder smooth muscle cells (HBSMCs) and in an in vivo model of acute bladder outlet obstruction (BOO). We evaluated additionally whether acetylcholine functions as BSMCs mitogen. HBSMCs were exposed to sustained levels of increased hydrostatic pressure. Expression of the M2 and M3 receptors was measured by Western blot analysis. Acute BOO was maintained in five male pigs for 24 h; six normal animals served as controls. Expression of the M2 and M3 in tissues was evaluated in a similar fashion to the isolated cells. HBSMCs were subsequently exposed to 100 microM acetylcholine in the presence or absence of hydrostatic pressure. DNA and protein synthesis were measured using a 3H-tymidine and leucine incorporation assay, respectively. Mean (range) detrusor pressure in acute BOO and controls was 19 (1-36.1) and 3.1 (1-16) cm-H2O, respectively. M2 and M3 receptor expression in HBSMCs increased in a time-dependent and pressure-dependent manner. Both M2 and M3 expressions in BOO model were markedly upregulated when compared to controls. Compared to controls, thymidine and leucine incorporation in HBSMCs demonstrated a significant increase following exposure to 100 microM acetylcholine and/or 40 cm-H2O. M2 and M3 receptors are upregulated in a time-dependent and pressure-dependent fashion after as little as a 24 h exposure to increased hydrostatic pressure. Acetylcholine has a more profound proliferative effect when cells are simultaneously exposed to increased levels of hydrostatic pressure.

Compensatory recovery of vagal control of hemodynamics after unilateral vagotomy

This study investigated whether each part of the heart is evenly innervated by the left or right vagus and observed the mechanism of compensatory recovery after unilateral cervical vagotomy. HR, BP, LVSP and +/-dp/dt max all decreased one week after left vagotomy, whereas only BP and -dp/dt max decreased one week after right vagotomy. Western blot analyses revealed that the expression of M(2) receptors in the left atrium and left ventricle was upregulated after subacute (1 week) left/right vagotomy. However, significantly more cholinesterase-positive nerves in LV and RV were seen one week after unilateral vagotomy compared to the sham-operated group. In addition, baroreflex sensitivity was increased after subacute right vagotomy. The decreasing effects of ACh (0.5 microg/kg) on LVSP and +/-dp/dt max (but not on HR and BP) were facilitated by subacute unilateral vagotomy. Our present experiments indicate that 1) the working myocardium is innervated bilaterally by the vagus, 2) ventricular contractility is influenced more by denervation of the left than the right vagus and 3) up-regulation of M(2) muscarinic receptors in the left heart, increase of cholinergic nerves, and high baroreflex sensitivity could be involved in the mechanism of compensatory hemodynamic recovery via contralateral vagus overactivity, thereby amplifying contralateral vagal activity and decreasing cardiac contractility.

Muscarinic receptor expression and receptor-mediated detrusor contraction: comparison of juvenile and adult porcine tissue

Urinary bladder function is known to mature during fetal and postnatal development, including changes in neurotransmitter regulation of detrusor contraction. However, only few experimental data are available about muscarinic receptor antagonist function in the urinary bladder from young animals. In the present study, we compare the muscarinic receptor-mediated contractions in juvenile and adult porcine detrusor and the effects of antimuscarinic compounds. Urinary bladders from young (8-12 weeks; 12- to 35-kg body weight) and mature pigs (>40 weeks; >100 kg) were compared. Muscarinic receptor expression was assessed by real time polymerase chain reaction and radioligand binding. Muscle contraction was measured with a force transducer; L-type Ca2+ currents (ICa,L) of isolated detrusor myocytes were recorded with standard voltage clamp technique. Juvenile and adult detrusor expressed similar quantities of the messenger RNA of M2 and M3 receptors. The number of [3H]QNB-binding sites and their affinity for the radioligand were also similar between juvenile and adult detrusor. In contrast, maximum contractile responses to the muscarinic receptor agonist carbachol were slightly larger in juvenile than adult bladders. On the other hand, carbachol was slightly less potent in juvenile than in adult tissue. The M3 antagonist DAU 5884 and the spasmolytic drug propiverine inhibited contractile responses with comparable efficacies and potencies in juvenile and adult tissue. ICa,L was somewhat smaller in juvenile than in adult cells. Taken together, these data suggest that expression and function of M2 and M3 receptors are similar in the detrusor of juvenile and mature pigs. Therefore, similar responses to antimuscarinic compounds could be expected in young and adult patients.

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.

Neue Verfahren der optimalen Patientenselektion für die anticholinerge Therapie

M3-specific inhibitors are currently preferred for anticholinergic therapy of OAB. However, not all of the patients profit from this regimen. This might reflect a heterogeneity of the patient group. The aim of this work is to define subgroups of patients with specific alterations of receptor expression and to profile the receptor expression individually. These receptor profiles might be used for the development of evidence-based "tailored" therapies. Detrusor probes from bladder carcinoma patients (BCa, n=9 F, n=7 male) and interstitial cystitis patients (IC, n=9 female) were examined using confocal immunofluorescence and PCR. M2, M3, P2X1-3, and H1-3 mRNAs were demonstrated in detrusor tissue. As revealed by immunofluorescence, the M2 receptor expression was significantly higher in female compared to male BCa tissues. In addition, the M2 receptor was further upregulated in IC vs BCa in female detrusor. IC patients showed specific alterations of their receptor profile. Individual receptor profiles might be used to optimize medicinal therapies.

Molecular characterization of M2 and M3 muscarinic receptor expression in bladder from women with refractory idiopathic detrusor overactivity

To examine the expression of muscarinic M2 and M3 receptors in human bladder detrusor and mucosa, from controls and patients with idiopathic detrusor overactivity (IDO), as antimuscarinic agents are the primary pharmacological treatment for IDO. Biopsies from the bladder body were collected at cystoscopy from 20 women with urodynamically confirmed refractory IDO (age range 25-86 years); biopsies were also collected from 30 asymptomatic female controls (age range 32-87 years). Samples were collected into RNA extraction medium and dissected into mucosa (urothelium plus lamina propria) and detrusor. RNA was extracted and the expression of M2 and M3 receptor mRNA determined by quantitative competitive reverse transcription-polymerase chain reaction. Results were normalized to beta-actin expression in the same sample. Expression of M3 receptor mRNA, in mucosa of IDO patients (median 0.057 pg M3/100 ng total RNA; interquartile range 0.03-0.13, 12 samples), was four times (P = 0.039, Mann-Whitney) lower than from the control (median 0.22 pg M3/100 ng total RNA; 0.13-0.51, 11 samples). The expression of muscarinic M3 receptor mRNA was higher (14-35 times) in detrusor (control median 3.17; 26 samples) than in mucosa and did not change in IDO (median 2.03; 14 samples). M2 expression was not significantly different with region or with IDO. These data show that M3 muscarinic receptor mRNA expression was significantly less in mucosa from IDO patients than from age-matched controls. The role of mucosal M3 receptors is unknown at present and elucidation of this role might provide a greater understanding of the aetiology of IDO.

Changes in muscarinic cholinergic receptor expression in human peripheral blood lymphocytes in allergic rhinitis patients

Background Parasympathetic nerves provide the dominant autonomic innervation of the upper and lower airways. They release acetylcholine that, activating post-junctional muscarinic receptors, causes bronchoconstriction, mucous secretion and vasodilation. Dysfunction of the upper and lower airways frequently coexist, and they appear to share key elements of pathogenesis. Objective The present study has assessed the expression and pattern of cholinergic muscarinic receptor subtypes in peripheral blood lymphocytes harvested from allergic rhinitis patients with different degree of bronchial hyperresponsiveness detected by methacholine challenge test. Methods Radioligand binding assay for determining the density of muscarinic cholinergic receptor subtypes; immunoblot analysis for assessing the characteristic of muscarinic cholinergic receptor subtype protein and immunocytochemical techniques for investigating the cellular localization of receptors. Results An increased expression of M2 and M5 receptor proteins was observed in peripheral blood lymphocytes of allergic rhinitis patients in comparison with healthy control individuals. M3 receptor subtype decreased in allergic rhinitis patients with normal or mild responses to methacholine. A trend versus a return to normal value was found in moderate and severe responders. No changes of the M4 receptor subtype were found. Conclusions and clinical implications Increase in M2 receptor expression correlated with disease severity and bronchial hyperreactivity. Changes in muscarinic cholinergic receptor expression in allergic rhinitis underline a role of cholinergic system of immune cells in allergic airway disease. Capsule summary Studies addressed to rhinitis and asthma have identified many similarities. Our results indicate that changes in peripheral blood lymphocyte muscarinic receptor expression may reflect the cholinergic involvement into allergic airway diseases.

Expression of muscarinic acetylcholine receptor-1 in human retinal pigment epithelium

The aim of this study was to identify the presence of muscarinic acetylcholine receptors-I (M1 receptor) in human retinal pigment epithelium (RPE) in order to determine the role of M1 receptor in the maintenance of function of RPE and its role in the occurrence and development of myopia. The 3rd-5th passages of RPE cells established in our laboratory were used in the present study. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect mRNA expression of M1 receptor in cultured RPE. Immunocytochemistry was used to detect M1 receptor protein in the RPE cells. Cultured RPE demonstrated mRNA expression of M1 receptor in RT-PCR. Protein of M1 receptor was presented in the RPE under immunocytochemistry. This study demonstrated the presence of M1 receptor in human RPE at both mRNA and protein levels. M1 receptor plays an important role in the maintenance of function of RPE. Injection of M1 receptor antagonist into the vitreous can delay the occurrence and inhibit the development of myopia, which is possibly related to the inhibition of RPE cells function.

A Role for ADP-ribosylation Factor 6 in the Processing of G-protein-coupled Receptors

After agonist-induced internalization, the vasopressin V2 receptor (V2R) does not recycle to the plasma membrane. The ADP-ribosylation factor (ARF) proteins initiate vesicular intracellular traffic by promoting the recruitment of adaptor proteins; thus, we sought to determine whether ARF6 could promote V2R recycling. Neither the agonist-induced internalization nor the recycling of the V2R was regulated by ARF6, but a constitutively active mutant of ARF6 reduced cell-surface V2Rs 10-fold in the absence of agonist treatment. Visualization of the ARF6 mutant-expressing cells revealed a vacuolar-staining pattern of the V2R instead of the normal plasma membrane expression. Analysis of V2R maturation revealed that reduced cell-surface expression was due to the diminished ability of the newly synthesized receptor to migrate from the endoplasmic reticulum to the Golgi network. The same mechanism affected processing of the V1aR and acetylcholine M2 receptors. Therefore, ARF6 controls the exit of the V2 and other receptors from the endoplasmic reticulum in addition to its established role in the trafficking of plasma-membrane-derived vesicles.

The Effects of Five Alkaloids from Bulbus Fritillariae on the Concentration of cAMP in HEK Cells Transfected with Muscarinic M2 Receptor Plasmid

The aim of this study was to investigate the effects of five alkaloids, namely verticine, verticinone, imperialine, imperialine-3beta-D-glucoside, and puqietinone, purified from Bulbus Fritillariae and used as an antitussive drug in traditional Chinese medicine, on their antimuscarinic M(2) function and the cAMP level of HEK cells transfected with muscarinic M(2) receptor plasmid. By transfecting the HEK cells with the method of calcium phosphate co-precipitation and screening with G418, the cells stably expressing M(2) receptor were identified. The expression of M(2) receptor in HEK cells was confirmed by both RT-PCR and western blot. The cAMP level in the treated cells was analyzed with RIA method ((125)I-cAMP KIT). And the results suggested that the five alkaloids could significantly elevate the cAMP concentration in the HEK cells transfected with muscarinic M(2) receptor plasmid (p < 0.01).

Muscarinic cholinergic receptor subtypes in cerebral cortex of Fisher 344 rats: A light microscope autoradiography study of age-related changes

The density and localization of muscarinic cholinergic M1–M5 receptor subtypes was investigated in frontal and occipital cortex of male Fisher 344 rats aged 6 months (young-adult), 15 months (mature) and 22 months (senescent) by combined kinetic and equilibrium binding and light microscope autoradiography. In 6-month-old rats, the rank order density of muscarinic cholinergic receptor subtypes was M1 > M2 > M4 > M3 > M5 both in frontal and occipital cortex. A not homogeneous distribution of different receptor subtypes throughout cerebrocortical layers of frontal or occipital cortex was found. In frontal cortex silver grains corresponding to the M1 and M2 receptor subtypes were decreased in 15- and 22-month-old groups. The M3 receptor density was remarkably and moderately decreased in layers II/III and V, respectively, of rats aged 15 and 22 months. A reduced M4 receptor density was observed in layer I and to a lesser extent in layer V of mature and senescent rats, whereas no age-related changes of M5 receptor were found. In occipital cortex a diminution of M1 receptor was observed in layers II/III and V of mature and senescent rats. The M2 receptor expression decreased in layer I of 15- and 22-month-old senescent rats, whereas M3–M5 receptors were unchanged with exception of a slight decrease of the M4 receptor in layer IV and of M5 receptor in layers II/III. These findings indicate a different sensitivity to aging of muscarinic receptor subtypes located in various cerebrocortical layers. This may account for the difficulty in obtaining relevant results in manipulating cholinoceptors to counter age-related impairment of cholinergic system.

Expression of muscarinic acetylcholine receptor-1 in human scleral fibroblasts

The aim of this study was to identify the presence of muscarinic acetylcholine receptors-1 (mAChRs-1) in human sclera in order to determine whether the sclera is a potential site of action for mAChR antagonists. Cell lines of human scleral fibroblasts were cultured in Dulbecco Modified Eagle's Medium. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was used to detect mRNA expression of M1 receptors in the fibroblasts. Immunocytochemistry was used to detect proteins of mAChRs in the cell lines. Immunohistochemical study was used to further detect the presence of M1 receptor in the frozen scleral sections. The cultured fibroblasts demonstrated mRNA expression of M1 receptor in RT-PCR. Protein of the M1 was present in the fibroblasts by examination of immunocytochemistry and immunohistochemistry staining. This study demonstrated the presence of M1 receptor in human scleral fibroblasts at both mRNA and protein levels. This finding indicates that the sclera is a potential site of action for the currently used mAChR antagonists in prevention of human myopia.

Muscarinic receptors, leukotriene B4 production and neutrophilic inflammation in COPD patients

Acetylcholine (ACh) plays an important role in smooth muscle contraction and in the development of airway narrowing; preliminary evidences led us to hypothesize that ACh might also play a role in the development of airways inflammation in chronic obstructive pulmonary disease (COPD). We evaluated the concentrations of leukotriene B4 (LTB4) in induced sputum, and the expression of Ach M1, M2, and M3 receptors in sputum cells (SC) obtained from 16 patients with COPD, 11 smokers, and 14 control subjects. The SC were also treated with ACh and the production of LTB4 assessed in the presence or absence of a muscarinic antagonist (oxitropium). In blood monocytes, we evaluated LTB4 release and activation of the extracellular signal-regulated kinases (ERK) pathway after treatment with Ach. The LTB4 concentrations were higher in COPD than in controls (P < 0.01) and correlated with the number of neutrophil (P < 0.01). The M3 receptors expression was increased in COPD subjects when compared to smokers and control (P < 0.05 and 0.0001, respectively), while M2 expression resulted decreased (P < 0.05 and 0.01). The ACh-induced LTB(4) production was observed in peripheral blood monocytes, and was sensitive to ERK inhibition. Similarly, ACh significantly increased neutrophil chemotactic activity and LTB4 released from SC of COPD patients only, and these effects were blocked by pretreatment with the inhibitor of ERK pathway PD98059. The results obtained show that muscarinic receptors may be involved in airway inflammation in COPD subjects through ACh-induced, ERK1/2-dependent LTB4 release. Muscarinic antagonism may contribute to reduce neutrophil infiltration and activation in COPD.

Reducing airways inflammation to prevent exacerbations in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is amajor cause of handicap, mortality and health careexpenses (1). Altered quality of life (QOL) is a hallmarkof the disease, and relates mainly to dyspnoea andexacerbations: both QOL at steady-state and its rate ofdecline over time correlate to the frequency of acuterespiratory events (2, 3). In parallel, hospitalizations forexacerbations in the most severe patients are importantdeterminants of health care costs (4). Thus, preventingexacerbations is a major goal of care for COPD patients,both from an individual and collective point of view.Inflammation and COPD exacerbationsIn most cases, COPD occurs in smokers and ex-smokers,in whom airflow limitation is the consequence of variousdegrees of emphysema, chronic bronchiolitis and mucusplugging (5). These components of the disease have beenlinked to inflammatory and remodelling processes in theairways and lung parenchyma, involved inflammatorycells being mainly neutrophils, B and T lymphocytes andmacrophages (5, 6). Airways inflammation is moreintense during exacerbations, as shown by increases inthe levels of (i) expression of neutrophil elastase, CXCL-5, CXCL-8 and CXCR1/2 in biopsies (7), (ii) exhaledLTB4 and 8-isoprostane in breath condensates (8), (iii)interleukin (IL)-8 and tumour necrosis factor (TNF)-a insputum (9).Infections are a leading cause of exacerbations andfurther amplify airway inflammation (10). However,about one half of exacerbations occur in the absence ofany detectable infective pathogen, which may not preventinflammation from increasing (11). Besides, higher levelsof inflammatory markers in the airways of patients atsteady-state are also associated with more frequentexacerbations (12). Studies of the East London COPDcohort even suggest the existence of a particular subgroupof COPD patients called frequent exacerbators, whoconsistently have more than 2–3 exacerbations per year(13). In these patients, QOL is more altered, forcedexpiratory volume at 1 s (FEV

Cholinergic agonist-induced pepsinogen secretion from murine gastric chief cells is mediated by M1and M3muscarinic receptors

Muscarinic cholinergic mechanisms play a key role in stimulating gastric pepsinogen secretion. Studies using antagonists suggested that the M3 receptor subtype (M3R) plays a prominent role in mediating pepsinogen secretion, but in situ hybridization indicated expression of M1 receptor (M1R) in rat chief cells. We used mice that were deficient in either the M1 (M1R-/-) or M3 (M3R-/-) receptor or that lacked both receptors (M(1/3)R-/-) to determine the role of M1R and M3R in mediating cholinergic agonist-induced pepsinogen secretion. Pepsinogen secretion from murine gastric glands was determined by adapting methods used for rabbit and rat stomach. In wild-type (WT) mice, maximal concentrations of carbachol and CCK caused a 3.0- and 2.5-fold increase in pepsinogen secretion, respectively. Maximal carbachol-induced secretion from M1R-/- mouse gastric glands was decreased by 25%. In contrast, there was only a slight decrease in carbachol potency and no change in efficacy when comparing M3R-/- with WT glands. To explore the possibility that both M1R and M3R are involved in carbachol-mediated pepsinogen secretion, we examined secretion from glands prepared from M(1/3)R-/- double-knockout mice. Strikingly, carbachol-induced pepsinogen secretion was nearly abolished in glands from M(1/3)R-/- mice, whereas CCK-induced secretion was not altered. In situ hybridization for murine M1R and M3R mRNA in gastric mucosa from WT mice revealed abundant signals for both receptor subtypes in the cytoplasm of chief cells. These data clearly indicate that, in gastric chief cells, a mixture of M1 and M3 receptors mediates cholinergic stimulation of pepsinogen secretion and that no other muscarinic receptor subtypes are involved in this activity. The development of a murine secretory model facilitates use of transgenic mice to investigate the regulation of pepsinogen secretion.

Regional differences in cholinergic regulation of potassium current in feline esophageal circular smooth muscle

Potassium channels are important contributors to membrane excitability in smooth muscles. There are regional differences in resting membrane potential and K(+)-channel density along the length of the feline circular smooth muscle esophagus. The aim of this study was to assess responses of K(+)-channel currents to cholinergic (ACh) stimulation along the length of the feline circular smooth muscle esophageal body. Perforated patch-clamp technique assessed K(+)-channel responses to ACh stimulation in isolated smooth muscle cells from the circular muscle layer of the esophageal body at 2 (distal)- and 4-cm (proximal) sites above the lower esophageal sphincter. Western immunoblots assessed ion channel and receptor expression. ACh stimulation produced a transient increase in outward current followed by inhibition of spontaneous transient outward currents. These ACh-induced currents were abolished by blockers of large-conductance Ca(2+)-dependent K(+) channels (BK(Ca)). Distal cells demonstrated a greater peak current density in outward current than cells from the proximal region and a longer-lasting outward current increase. These responses were abolished by atropine and the specific M(3) receptor antagonist 4-DAMP but not the M(1) receptor antagonist pirenzipine or the M(2) receptor antagonist methoctramine. BK(Ca) expression along the smooth muscle esophagus was similar, but M(3) receptor expression was greater in the distal region. Therefore, ACh can differentially activate a potassium channel (BK(Ca)) current along the smooth muscle esophagus. This activation probably occurs through release of intracellular calcium via an M(3) pathway and has the potential to modulate the timing and amplitude of peristaltic contraction along the esophagus.

Bile-pancreatic juice exclusion increases cholinergic M3 and CCK-A receptor expression and interleukin-6 production in ligation-induced acute pancreatitis

Background Using an original model, the Donor Rat Model, we showed that bile-pancreatic juice (BPJ) exclusion from gut exacerbates ligation-induced acute pancreatitis in rats. We also showed that muscarinic cholinergic M3 and CCK-A receptor expression is induced following duct ligation. Increased receptor number potentially could exacerbate cytokine production. We hypothesize that BPJ exclusion is responsible for M3 and CCK-A receptor induction and increased interleukin-6 (IL-6) production. Methods M3 and CCK-A receptor expression and IL-6 production were compared in rat pancreata 1 to 3 hours after duct ligation with or without BPJ replacement. Results Our studies showed that BPJ replacement attenuates duct ligation–induced increases in M3 and CCK-A receptor expression and IL-6 production. Conclusions In this model, BPJ exclusion from gut induces M3 and CCK-A receptor expression and increases IL-6 production. In this experimental corollary of gallstone pancreatitis, BPJ exclusion from gut may play a key role in the mechanism of disease pathogenesis.

G protein-mediated dysfunction of excitation-contraction coupling in ileal inflammation

Inflammation impairs the circular muscle contractile response to muscarinic (M) receptor activation. The aim of this study was to investigate whether the expression of muscarinic receptors, their binding affinity, and the expression and activation of receptor-coupled G proteins contribute to the suppression of contractility in inflammation. The studies were performed on freshly dissociated single smooth muscle cells from normal and inflamed canine ileum. Northern blotting indicated the presence of only M(2) and M(3) receptors on canine ileal circular muscle cells. Inflammation did not alter the mRNA or protein expression of M(2) and M(3) receptors. The maximal binding and K(d) values also did not differ between normal and inflamed cells. However, the contractile response to ACh in M(3) receptor-protected cells was suppressed, whereas that in M(2) receptor-protected cells was enhanced. Further experiments indicated that the expression and binding activity of G alpha(q/11) protein, which couples to M(3) receptors, were downregulated, whereas those of G alpha(i3), which couples to M(2) receptors, were upregulated in inflamed cells. We concluded that inflammation depresses M(3) receptor function, but it enhances M(2) receptor function in ileum. These effects are mediated by the differentially altered expression and binding activity of their respective coupled G alpha(q/11) and G alpha(i3) proteins.