OBJECTIVE: To observe the expression of Muscarinic receptor M1, M3, M5 subunits in rat flocculus following left unilateral labyrinthectomy (UL). METHOD: The RT-PCR was used to observe the expression of Muscarinic receptor M1, M3, M5 subunits post-unilateral labyrinthectomy and investigate its effect on vestibular compensation. RESULT: Muscarinic receptor M1, M3, M5 subunits were induced decrease in both side flocculus after unilateral labyrinthectomy. The expression was the least in the 1 d flocculus of following UL. The expression is rising from the 3-7 d flocculus of following UL. No difference was observed in the 7 d and sham operation flocculus following UL. No difference was observed in the ipsilateral and contralateral flocculus at any group. CONCLUSION Muscarinic receptor M1, M3, M5 subunits were induced decrease in the flocculus after unilateral labyrinthectomy. But the significance of the change of Muscarinic receptor M1, M3, M5 subunits in the vestibular compensation is still unknown.
Animals ; Cerebellum ; metabolism ; Functional Laterality ; Gene Expression ; Male ; Postoperative Period ; Rats ; Receptor, Muscarinic M1 ; metabolism ; Receptor, Muscarinic M3 ; metabolism ; Receptor, Muscarinic M5 ; metabolism ; Vestibule, Labyrinth ; metabolism ; surgery

OBJECTIVE[corrected] To characterize the insulinotropic action of hippocampal cholinergic neurostimulating peptide (HCNP) and analyze the role of type 3 muscarinic receptor (M(3)R) pathway in the action of HCNP.

METHODSINS-1 cells were incubated in routine RPMI 1640 medium (control group), RPMI 1640 supplemented with 50 pg/ml synthetic HCNP (HCNP group), or HCNP-containing medium with the addition of PMA 18 h prior to insulin release assay. The insulin levels in the medium was measured using radioimmunoassay following stimulation with different concentrations of glucose. Real-time quantitative PCR was used for detecting the gene expression of HCNP-pp, choline acetyltransferase (ChAT) and M(3)R in HCNP group and control group.

RESULTSAfter stimulation with different concentrations of glucose (5.6 and 16.7 mmol/L), HCNP group showed significantly higher insulin levels than the control and HCNP+ PMA groups. Compared with those in the control group, the mRNA levels of HCNP-pp, ChAT, and M(3)R were all lowered in HCNP group.

CONCLUSIONHCNP can promote insulin release in INS-1 cells by increasing ChAT activity and activating M(3)R, and this effect is inhibited by PMA.

Animals ; Cell Line ; Insulin ; secretion ; Neuropeptides ; pharmacology ; Rats ; Receptor, Muscarinic M3 ; metabolism

OBJECTIVE: To investigate the expression of M3 subtype of muscarinic receptors (M3R) during the incised wound healing of the skin in mice and the characteristics of its time-dependent. METHODS: The change of M3R in skin incised wound was detected by immunohistochemical staining and Western blot. RESULTS: M3R-positive cells were detected in epidermis, hair follicle, sebaceous glands, sweat glands, dermomuscular layer in normal mouse skin. Expression of M3R was mainly detectable in polymorphonuclear cells (PMNs) in the wound specimens aged from 6h to 12h after injury. Afterwards, the M3R-positive cells were mostly mononuclear cells (MNCs) and fibroblastic cells (FBCs) at 1 d to 3d post-injury, whereas the M3R-positive cells were mostly FBCs aged from 5 d to 14d. Morphometrically, the ratio of the M3R-positive cells increased aged from 6h to 12h after injury, with a peak at 12h. The ratios kept a high relatively level aged from 1 d to 5 d, but significantly that lowered as compared with aged 12h after injury. The ratio reached the peak at 7 d again after injury, and then decreased gradually. The M3R protein also revealed a time-dependent tendency with double peaks at 12h and 7 d after injury as detected by Western blotting. CONCLUSION M3R is time-dependently expression in PMNs, MNCs and FBCs suggesting that it may play roles during the skin incised wound healing, and M3R may be used as a marker for wound age determination.
Animals ; Blotting, Western ; Fibroblasts/metabolism* ; Immunohistochemistry ; Male ; Mice ; Monocytes/metabolism* ; Neutrophils/metabolism* ; Receptor, Muscarinic M3/metabolism* ; Skin/metabolism* ; Time Factors ; Wound Healing ; Wounds and Injuries/metabolism*

OBJECTIVETo investigate the effect of diabetic autonomic neuropathy on the seminal vesicle and search for the theoretical evidence for the prevention and treatment of diabetic infertility by observing changes in the contents of the nerve growth factor (NGF) and muscarinic M3 receptor in the seminal vesicle of diabetic rats.

METHODSDiabetic models were established in 10 of the 15 male adult SD rats by intraperitoneal injection of streptozotocin (STZ), and the other 5 were included in a normal control group. Eight weeks after modeling, seminal vesicles were collected from the rats for HE and immunohistochemical staining.

RESULTSCompared with the normal controls, the diabetic models showed a decreased number of smooth muscle cells, thinner cytoplasm of glandular epithelial cells and disordered structure in the seminal vesicle. The intensity of NGF-positive staining was significantly enhanced, but that of M3 markedly reduced in the diabetic group. There were statistically significant differences in the mean integrated optical density (IA) of muscarinic M3 receptors and NGF between the control and diabetic groups (0.0187 +/- 0.0024 vs 0.0100 +/- 0.0015 and 0.0209 +/- 0.0085 vs 0.0412 +/- 0.0117, P<0.01).

CONCLUSIONThe changes in the expressions of NGF and M3 receptors in the seminal vesicle of diabetic rats suggest that diabetes mellitus may induce autonomic neuropathy of the seminal vesicle.

Animals ; Diabetes Mellitus, Experimental ; metabolism ; Male ; Nerve Growth Factor ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, Muscarinic M3 ; metabolism ; Seminal Vesicles ; metabolism

Our previous studies document the expression of adrenoceptors and purinoceptors in the rat prostate neuroendocrine cells (RPNECs). However, a direct investigation of the receptors for acetylcholine (ACh) is still lacking in the prostate neuroendocrine cells. RPNECs were freshly isolated from the ventral lobes of rat prostate by using collagenase. Effects of ACh and various muscarinic antagonists on the intracellular Ca2+ concentration ([Ca2+]c ) were investigated by using the fura-2 spectrofluorimetry. Single-cell RT-PCR analysis was applied to identify the transcripts for the muscarinic receptor subtypes. ACh (5 micrometer) induced a sharp transient increase in the [Ca2+]c of RPNECs, which was independent of the extracellular Ca2+. In the same RPNECs, high KCl (60 mM), phenylephrine (5micrometer), UTP (P2Y1/2 agonist, 50, micrometer), and alpha, beta-meATP (P2X1/3 agonist, 0.5micrometer) also increased the [Ca2+]c. The ACh-induced [Ca2+]c change (delta[Ca2+]c ) was blocked by atropine or by para-fluorohexahydrosiladifenidol (M3 antagonist, 0.3micrometer), but not by telenzepine (M1 antagonist, 1 micrometer) and himbacine (M2 and M4 antagonist, 1 mircoM). The single-cell RT-PCR demonstrated the selective expression of mRNAs for M3 in RPNECs. In summary, RPNECs express M3 muscarinic receptors that are linked to the release of Ca2+ from intracellular stores. The Ca2+ signals of RPNECs might mediate the parasympathetic regulation of prostate gland.
Acetylcholine/pharmacology ; Animals ; Calcium/*metabolism ; Calcium Signaling ; Male ; Neurosecretory Systems/*metabolism ; Prostate/*metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, Muscarinic M3/*physiology ; Research Support, Non-U.S. Gov't

The relationship between M3 cholinergic receptor agonist (carbachol) hyperstimulation-induced pancreatic acinar cellular injury and trypsinogen activation or NF-kappaB activation in rats was studied in vitro. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, the active protease inhibitor (pefabloc), and NF-kappaB inhibitor (PDTC) in vitro. Intracellular trypsin activity was measured by using a fluorogenic substrate. The cellular injury was evaluated by measuring the leakage of LDH from pancreatic acinar cells. The results showed that as compared with control group, 10(-3) mol/L carbachol induced a significant increase of the intracellular trypsin activity and the leakage of LDH from pancreatic acinar cells. Pretreatment with 2 mmol/L pefabloc could significantly decrease the activity of trypsin and the leakage of LDH from pancreatic acinar cells (P < 0.01) following the treatment with a high concentration of carbachol (10(-3) mol/L) in vitro. The addition of 10(-2) mol/L PDTC didn't result in a significant decrease in the activity of trypsin and the leakage of LDH from pancreatic acinar cells treated with a high concentration of carbachol (10(-3) mol/L) in vitro (P > 0.05). It was concluded that intracellular trypsinogen activation is likely involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro. NF-kappaB activation may not be involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro.
Carbachol/*pharmacology ; Cholinergic Agonists/pharmacology ; NF-kappa B/*metabolism ; Pancreas/metabolism ; Pancreas/*pathology ; Rats, Wistar ; Receptor, Muscarinic M3/agonists ; Trypsinogen/*metabolism

AIMTo optimize the method of investigating structural integration between proteins and study the integration between arrhythmia related proteins in molecular level.

METHODSImmunostaining the normal ventricular myocytes was used to observe the distribution of connexin 43 and muscarinic acetylcholine receptor (mAChR). The five mAChR subtypes were precipitated using immunoprecipitation. Then, SDS-PAGE and Western blotting with the anti-connexin 43 antibody were performed to observe whether they were structurally integrated. Further, different concentrations of detergent were used to observe whether this relationship could be broken.

RESULTSThe five subtypes of mAChR existed in the cardiac myocyte of the rat, and all the five mAChR subtypes combined with connexin 43. In the normal rat ventricular myocyte membrane, connexin 43 and M3 receptor are co-located. When adding certain concentration of detergent to the membrane protein, the integration between M3 receptor and connexin 43 was broken, and the phosphorylated form of connexin 43 integrated with M3 receptor.

CONCLUSIONThe results indicated that the structural integration between mAChR and phosphorylation of connexin 43 existed in rat ventricular myocardium, and this integration could be broken by certain concentration of detergent.

Animals ; Cell Membrane ; metabolism ; Connexin 43 ; metabolism ; Heart Ventricles ; Immunoprecipitation ; Male ; Microscopy, Confocal ; Myocytes, Cardiac ; metabolism ; Phosphorylation ; drug effects ; Rats ; Rats, Wistar ; Receptor, Muscarinic M3 ; metabolism ; Receptors, Muscarinic ; metabolism ; Sodium Dodecyl Sulfate ; pharmacology

Animals ; Arecoline ; pharmacology ; Cholinergic Agonists ; pharmacology ; Female ; Guinea Pigs ; In Vitro Techniques ; Male ; Muscle Contraction ; drug effects ; Muscle, Smooth ; physiology ; Pyloric Antrum ; physiology ; Receptor, Muscarinic M3 ; metabolism

AIMTo observe the effect of activation of M3 receptor on H2O2 induced apoptosis in cultured rat myocytes and to investigate its possible mechanisms.

METHODSIsolated neonatal cardiomyocytes were cultured. Morphologic changes were observed by microscopy. The apoptosis in cardiomyocyte was detected by terminal deoxynucleotide transferase directed d-UTP nick and end labeling (TUNEL) assay. The expression of apoptosis-related protein in Bcl-2 and Fas was measured by immunohistochemistry assay. [Ca2+]i in single cardiomyocyte loaded with Fluo 3-AM was measured by confocal microscope.

RESULTSH2O2-mediated myocyte apoptosis was attenuated by M3 receptor agonist choline (10 mmol x L(-1)). Pretreatment of cardiac myocytes with choline also increased Bcl-2, decreased Fas expression, and inhibited the increase in FI value of [Ca2+]i in H2O2-stimulated cardiac myocytes. However, blockade of M3 receptor by 4DAMP (10 nmol x L(-1)) completely inhibited the effects of choline on H2O2-stimulated cardiac myocytes.

CONCLUSIONActivation of M3 receptor showed protective effect on H2O2-induced apoptosis in cultured rat myocytes and this effect might be related to modulating the expression of some genes including Bcl-2 and Fas as well as the downregulation of [Ca2+]i.

Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Calcium ; metabolism ; Cells, Cultured ; Choline ; pharmacology ; Hydrogen Peroxide ; antagonists & inhibitors ; Myocytes, Cardiac ; cytology ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Wistar ; Receptor, Muscarinic M3 ; agonists ; fas Receptor ; metabolism

AIMAcetylcholine(ACh) is a neurotransmitter and a potent vasodilator in many vascular beds. ACh hyperpolarizes the smooth muscle cells(SMCs) of arteries including the cochlear spiral modiolar artery(SMA) via an endothelium-dependent mechanism, but the biochemical and biophysical basis of the hyperpolarization and vasodilation remain unclear and controversial.

METHODSUsing intracellular recording techniques and an in vitro preparation of the SMA, the ionic mechanism of the hyperpolarization and a possible role of nitric oxide(NO) were investigated.

RESULTSWith 5 mmol/L K(+) in the bathing solution and a minimum longitudinal tension, ACh (0.1-10 micromol/L) induced a robust hyperpolarization in low RP cells but caused a depolarization in the high RP cells. The ACh hyperpolarization was fast in onset and offset and the amplitude was concentration-dependent(22 and 30 mV by 1 micromol/L and 10 micromol/L ACh, respectively, n = 7 ). ACh also hyperpolarized the cells that initially had a high resting potential (RP) but were pre-depolarized by Ba(2+) (50-100 micromol/L). The onset time courses of the hyperpolarization were often slower in these cases than those without the presence of Ba(2+) . The ACh-induced hyperpolarization was blocked by atropine (0.1- 1 micromol/L, n = 6) or DAMP (50 -100 nmol/L, n = 6, a selective M3 antagonist) and also by BAPTA-AM (10 micromol/L, n = 7, a membrane-permeable Ca(2+)-chelator), or charybdotoxin plus apamin (50-100 nmol/L, n= 4, Ca(2+) -activated K(+) -channel blockers), but not by Nomega-nitro-L-arginine methyl ester (L-NAME, 300 micromol/L, n = 8, an inhibitor of NO-synthase), glipizide (10 micromol/L, n = 4, ATP-sensitive K(+) -channel blocker) and indomethacin (10 micromol/L, n = 4, cyclo-oxygenase inhibitor).

CONCLUSIONIt is concluded that ACh-induced hyperpolarization in the arterial SMCs is primarily due to an activation of calcium-activated potassium channels via M3 receptors of endothelial cell and is independent of NO-release in the spiral modiolar artery.

Acetylcholine ; physiology ; Animals ; Arteries ; Cell Polarity ; physiology ; Cochlea ; blood supply ; physiology ; Guinea Pigs ; Membrane Potentials ; physiology ; Muscle, Smooth, Vascular ; metabolism ; physiology ; Nitric Oxide ; metabolism ; Potassium Channels, Calcium-Activated ; metabolism ; Receptor, Muscarinic M3 ; metabolism