The autonomic nervous system contributes to prostate cancer proliferation and metastasis. However, the exact molecular mechanism remains unclear. In this study, muscarinic acetylcholine receptor M1 (CHRM1) expression was measured via immunohistochemical analysis in human prostate cancer tissue array slides. PC-3, LNCaP, and A549 cells were treated with pirenzepine or carbachol, and the cell migration and invasion abilities were evaluated. Western blotting and quantitative real-time PCR were performed to measure GLI family zinc finger 1 (GLI1), patched 1 (PTCH1), and sonic hedgehog (SHH) expression levels. High expression of CHRM1 was found in early-stage human prostate cancer tissues. In addition, the selective CHRM1 antagonist pirenzepine inhibited PC-3, LNCaP, and A549 cell migration and invasion, but the agonist carbachol promoted the migration and invasion of these three cell lines. Muscarinic signaling can be relayed by hedgehog signaling. These data show that CHRM1 is involved in the regulation of prostate cancer migration and invasion through the hedgehog signaling pathway.
Carbachol/pharmacology* ; Cell Movement/genetics* ; Cell Proliferation ; Hedgehog Proteins/genetics* ; Humans ; Male ; Muscarinic Agonists/pharmacology* ; Muscarinic Antagonists/pharmacology* ; Patched-1 Receptor/genetics* ; Pirenzepine/pharmacology* ; Prostatic Neoplasms/pathology* ; Receptor, Muscarinic M1/genetics* ; Zinc Finger Protein GLI1/genetics*

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

We isolated mouse embryonic cardiomyocytes derived from timed-pregnant females at different periods and used patch-clamp technique to investigate the muscarinic cholinergic modulation of pacemaker current I(f) in different developmental stages. In early development stage (EDS), muscarinic agonist carbachol (CCh) significantly decreased the magnitude of the pacemaker current I(f) but had no effect in late development stage (LDS). Forskolin (a direct adenylate cyclase activator) and IBMX (a non-selective phosphodiesterase inhibitor) increased I(f) in both EDS and LDS cells. Interestingly, although both forskolin and IBMX increased basal I(f), their effects on CCh-inhibited I(f) were different. Forskolin did not reverse the inhibitory action of CCh until intermediate development stage (IDS). In contrast, IBMX reversed the inhibitory action of CCh on I(f) in EDS but not in IDS. It is suggested that a decrease in intracellular cAMP is a possible mechanism for CCh to modulate I(f). During the EDS and IDS CCh controls the cytoplasmic cAMP level by different pathways: In EDS, CCh modulates I(f) possibly by activating PDE which accelerates the breakdown of cAMP, but in IDS possibly by inhibiting adenylate cyclase (AC) which then reduces the synthesis of cAMP.
Animals ; Carbachol ; pharmacology ; Colforsin ; metabolism ; pharmacology ; Female ; Heart ; embryology ; physiology ; Mice ; Muscarinic Agonists ; pharmacology ; Myocytes, Cardiac ; drug effects ; physiology ; Pacemaker, Artificial ; Phosphodiesterase Inhibitors ; metabolism ; pharmacology ; Pregnancy ; Receptors, Muscarinic ; metabolism

Synaptotagmin is a Ca2+ sensing protein, which triggers a fusion of synaptic vesicles in neuronal transmission. Little is known regarding the expression of Ca2+ - dependent synaptotagmin isoforms and their contribution to the release of secretory vesicles in mouse and rat parotid acinar cells. We investigated a type of Ca2+ - dependent synaptotagmin and Ca2+ signaling in both rat and mouse parotid acinar cells using RT-PCR, microfluorometry, and amylase assay. Mouse parotid acinar cells exhibited much more sensitive amylase release in response to muscarinic stimulation than did rat parotid acinar cells. However, transient [Ca2+]i increases and Ca2+ influx in response to muscarinic stimulation in both cells were identical, suggesting that the expression or activity of the Ca2+ sensing proteins is different. Seven Ca2+ - dependent synaptotagmins, from 1 to 7, were expressed in the mouse parotid acinar cells. However, in the rat parotid acinar cells, only synaptotagmins 1, 3, 4 and 7 were expressed. These results indicate that the expression of Ca2+ - dependent synaptotagmins may contribute to the release of secretory vesicles in parotid acinar cells.
Synaptotagmins/*metabolism ; Signal Transduction ; Rats ; Protein Isoforms/metabolism ; Parotid Gland/cytology/*metabolism ; Muscarinic Agonists/pharmacology ; Mice ; Exocytosis/drug effects/physiology ; Carbachol/pharmacology ; Calcium/metabolism/*physiology ; Animals ; Amylases/secretion

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

BACKGROUNDCa(2+) in the central nervous system plays important roles in brain physiology, including neuronal survival and regeneration in rats with injured facial motoneurons. The present research was to study the modulations of intracellular free Ca(2+) concentrations by cholinergic receptors in rat facial nucleus, and the mechanisms of the modulations.

METHODSThe fluorescence intensity of facial nucleus in Fluo-3 AM loaded acute brainstem slices was detected by applying intracellular free Ca(2+) measurement technique via confocal laser scanning microscope. The changes of fluorescence intensity of facial nucleus indicate the average changes of intracellular free Ca(2+) levels of the neurons.

RESULTSAcetylcholine was effective at increasing the fluorescence intensity of facial nucleus. Muscarine chloride induced a marked increase of fluorescence intensity in a concentration dependent fashion. The enhancement of fluorescence intensity by muscarine chloride was significantly reduced by thapsigargin (depletor of intracellular Ca(2+) store; P < 0.01), rather than Ca(2+) free artifical cerebrospinal fluid or EGTA (free Ca(2+) chelator; P > 0.05). And the increase of fluorescence intensity was also significantly inhibited by pirenzepine (M(1) subtype selective antagonist; P < 0.01) and 4-DAMP (M(3) subtype selective antagonist; P < 0.01). In addition, fluorescence intensity was markedly increased by nicotine. The enhancement of fluorescence intensity by nicotine was significantly reduced by EGTA, nifedipine (L-type voltage-gated Ca(2+) channel blocker), dihydro-beta-erythroidine (alpha4beta2 subtype selective antagonist), and in Ca(2+) free artificial cerebrospinal fluid (P < 0.01), but not in the presence of mibefradil (M-type voltage-gated Ca(2+) channel blocker) or thapsigargin (P > 0.05).

CONCLUSIONSThe data provide the evidence that muscarinic receptors may induce the increase of intracellular free Ca(2+) levels through the Ca(2+) release of intracellular Ca(2+) stores, in a manner related to M(1) and M(3) subtypes of muscarinic receptors in rat facial nucleus. Nicotine may increase intracellular free Ca(2+) concentrations via the influx of extracellular Ca(2+)+ mainly across L-type voltage-gated Ca(2+) channels, in a manner related to the alpha4beta2 subtype of nicotinic receptors.

Acetylcholine ; pharmacology ; Aniline Compounds ; administration & dosage ; Animals ; Brain Stem ; cytology ; drug effects ; metabolism ; Calcium ; metabolism ; Diamines ; pharmacology ; Facial Nerve ; cytology ; Female ; Fluorescent Dyes ; administration & dosage ; In Vitro Techniques ; Male ; Microscopy, Confocal ; Motor Neurons ; drug effects ; metabolism ; Muscarinic Agonists ; pharmacology ; Nicotine ; pharmacology ; Nicotinic Agonists ; pharmacology ; Piperidines ; pharmacology ; Pirenzepine ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, Cholinergic ; metabolism ; Receptors, Muscarinic ; metabolism ; Receptors, Nicotinic ; metabolism ; Tropicamide ; pharmacology ; Xanthenes ; administration & dosage

Muscarinic receptors play key roles in the control of gastrointestinal smooth muscle activity. However, specific physiological functions of each subtype remain to be determined. In this study, the nonselective cation channel activated by carbachol (ICCh) was examined in circular smooth muscle cells of the guinea pig gastric antrum using patch-clamp technique. 4-DAMP inhibited ICCh dose- dependently with IC50 of 1.1 +/- 0.1 nM (n = 6). GTPgS- induced current, however, was not inhibited by 10 nM 4-DAMP. ICCh was not recorded in pertussis- toxin (PTX)-pretreated smooth muscle cells of gastric antrum. ICCh values in response to 10 mM CCh at a holding potential of 60 mV were -330 32 pA (n=4) and -15 +/- 3 pA (n = 6) in the control and PTX-treated cells, respectively (P<0.01). Sensitivities to nanomolar 4-DAMP and PTX suggest the possible involvement of m4 subtype. Using sequence information obtained from cloned guinea pig muscarinic receptor genes, it is possible to amplify the cDNAs encoding m1-m5 from guinea pig brain tissue. Single cell RT-PCR experiments showed that all five subtypes of muscarinic receptor were present in circular smooth muscle cells of the guinea pig gastric antrum. Together with our previous results showing that Go protein is important for activation of ACh-activated NSC channels, our results suggest that ICCh might be activated by acetylcholine through m4 subtype as well as m2 and m3 subtypes in guinea-pig stomach.
Animals ; Base Sequence ; Carbachol/pharmacology ; Cations ; Cholinergic Agonists/pharmacology ; Dose-Response Relationship, Drug ; Drug Interactions ; Guinea Pigs ; Ion Channels/drug effects/metabolism/physiology ; Muscarinic Antagonists/pharmacology ; Muscle Contraction/drug effects ; Muscle, Smooth/drug effects/*metabolism ; Piperidines/pharmacology ; Receptors, Muscarinic/chemistry/classification/*metabolism ; Stomach/drug effects/*metabolism

Animals ; Delayed Rectifier Potassium Channels ; physiology ; Humans ; Membrane Potentials ; drug effects ; Myocardial Ischemia ; pathology ; physiopathology ; Pilocarpine ; pharmacology ; Piperidines ; pharmacology ; Receptor, Muscarinic M3 ; agonists ; antagonists & inhibitors ; physiology ; Signal Transduction ; drug effects

Corticosteroids are used in the treatment of many diseases; however, they also induce various side effects. Dexamethasone is one of the most potent corticosteroids, and it has been reported to induce the side effect of impaired salivary gland function. This study aimed to evaluate the effects of dexamethasone on mouse submandibular gland function to gain insight into the mechanism of dexamethasone-induced salivary hypofunction. The muscarinic agonist carbachol (CCh) induced salivary secretion and was not affected by short-term dexamethasone treatment but was decreased following long-term dexamethasone administration. The expression levels of the membrane proteins Na-K-2Cl cotransporter, transmembrane member 16A, and aquaporin 5 were comparable between the control and long-term dexamethasone treatment groups. The CCh-induced increase in calcium concentration was significantly lower in the presence of extracellular Ca in the long-term dexamethasone treatment group compared to that in the control group. Furthermore, CCh-induced salivation in the absence of extracellular Ca and Ca ionophore A23187-induced salivation was comparable between the control and long-term dexamethasone treatment groups. Moreover, salivation induced by the Ca-ATPase inhibitor thapsigargin was diminished in the long-term dexamethasone treatment group. In summary, these results demonstrate that short-term dexamethasone treatment did not impair salivary gland function, whereas long-term dexamethasone treatment diminished store-operated Ca entry, resulting in hyposalivation in mouse submandibular glands.
Acinar Cells ; drug effects ; metabolism ; Animals ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; Carbachol ; pharmacology ; Dexamethasone ; therapeutic use ; Mice ; Muscarinic Agonists ; pharmacology ; Saliva ; metabolism ; Salivation ; drug effects ; Submandibular Gland ; drug effects ; metabolism

Whether M3 cholinergic receptor signal transduction pathway is involved in regulation of the activation of NF-kappaB and the expression of chemokine MOB-1, MCP-lgenes in pancreatic acinar cells was investigated. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, atropine and PDTC in vitro. The MOB-1 and MCP-1 mRNA expression was detected by using RT-PCR. The activation of NF-kappaB was monitored by using electrophoretic mobility shift assay. The results showed that as compared with control group, M3 cholinergic receptor agonist (10(-3) mol/L, 10(-4) mol/L carbachol) could induce a concentration-dependent and time-dependent increase in the expression of MOB-1, MCP-1 mRNA in pancreatic acinar cells. After treatment with 10(-3) mol/L carbachol for 2 h, the expression of MOB-1, MCP-1 mRNA was strongest. The activity of NF-kappaB in pancreatic acinar cells was significantly increased (P<0.01) after treated with M3 cholinergic receptor agonist (10(-3) mol/L carbachol) in vitro for 30 min. Either M3 cholinergic receptor antagonist (10(-5) mol/L atropine) or NF-kappaB inhibitor (10(-2) mol/L PDTC) could obviously inhibit the activation of NF-kappaB and the chemokine MOB-1, MCP-1 mRNA expression induced by carbachol (P<0.05). This inhibitory effect was significantly increased by atropine plus PDTC (P<0.01). The results of these studies indicated that M3 cholinergic receptor signal transduction pathway was likely involved in regulation of the expression of chemokine MOB-1 and MCP-lgenes in pancreatic acinar cells in vitro through the activation of NF-kappaB.
Adaptor Proteins, Signal Transducing ; Carbachol ; pharmacology ; Carrier Proteins ; biosynthesis ; genetics ; Chemokine CCL2 ; biosynthesis ; genetics ; Chemokines ; biosynthesis ; genetics ; Humans ; NF-kappa B ; biosynthesis ; genetics ; Pancreas, Exocrine ; metabolism ; Pancreatitis ; etiology ; RNA, Messenger ; biosynthesis ; genetics ; Receptor, Muscarinic M3 ; agonists ; physiology ; Signal Transduction