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 present study was designed to investigate the characteristics of gintonin, one of components isolated from Korean Ginseng on secretion of catecholamines (CA) from the isolated perfused model of rat adrenal gland and to clarify its mechanism of action. Gintonin (1 to 30 µg/ml), perfused into an adrenal vein, markedly increased the CA secretion from the perfused rat adrenal medulla in a dose-dependent fashion. The gintonin-evoked CA secretion was greatly inhibited in the presence of chlorisondamine (1 µM, an autonomic ganglionic bloker), pirenzepine (2 µM, a muscarinic M₁ receptor antagonist), Ki14625 (10 µM, an LPA₁/₃ receptor antagonist), amiloride (1 mM, an inhibitor of Na⁺/Ca²⁺ exchanger), a nicardipine (1 µM, a voltage-dependent Ca²⁺ channel blocker), TMB-8 (1 µM, an intracellular Ca²⁺ antagonist), and perfusion of Ca²⁺-free Krebs solution with 5mM EGTA (a Ca²⁺chelater), while was not affected by sodium nitroprusside (100 µM, a nitrosovasodialtor). Interestingly, LPA (0.3~3 µM, an LPA receptor agonist) also dose-dependently enhanced the CA secretion from the adrenal medulla, but this facilitatory effect of LPA was greatly inhibited in the presence of Ki 14625 (10 µM). Moreover, acetylcholine (AC)-evoked CA secretion was greatly potentiated during the perfusion of gintonin (3 µg/ml). Taken together, these results demonstrate the first evidence that gintonin increases the CA secretion from the perfused rat adrenal medulla in a dose-dependent fashion. This facilitatory effect of gintonin seems to be associated with activation of LPA- and cholinergic-receptors, which are relevant to the cytoplasmic Ca²⁺ increase by stimulation of the Ca²⁺ influx as well as by the inhibition of Ca²⁺ uptake into the cytoplasmic Ca²⁺ stores, without the increased nitric oxide (NO). Based on these results, it is thought that gintonin, one of ginseng components, can elevate the CA secretion from adrenal medulla by regulating the Ca²⁺ mobilization for exocytosis, suggesting facilitation of cardiovascular system. Also, these findings show that gintonin might be at least one of ginseng-induced hypertensive components.
Acetylcholine ; Adrenal Glands ; Adrenal Medulla* ; Amiloride ; Animals ; Cardiovascular System ; Catecholamines ; Chlorisondamine ; Cytoplasm ; Egtazic Acid ; Exocytosis ; Ganglia, Autonomic ; Nicardipine ; Nitric Oxide ; Nitroprusside ; Panax ; Perfusion ; Pirenzepine ; Rats ; Veins

BACKGROUND: The authors evaluated the effect of intrathecal mixture of ginsenosides with neostigmine on formalin-induced nociception and made further clear the role of the spinal muscarinic (M) receptors on the activity of ginsenosides. METHODS: A catheter was located in the intrathecal space of male Sprague-Dawley rats. Pain was evoked by injection of formalin solution (5%, 50 microl) to the hindpaw. Isobolographic analysis was done to characterize drug interaction between ginsenosides and neostigmine. The antagonism of ginsenosides-mediated antinociception was determined with M1 receptor antagonist (pirenzepine), M2 receptor antagonist (methoctramine), M3 receptor antagonist (4-DAMP), M4 receptor antagonist (tropicamide). The expression of muscarinic receptor subtypes was examined with RT-PCR. RESULTS: Intrathecal ginsenosides and neostigmine produced an antinociceptive effect during phase 1 and phase 2 in the formalin test. Isobolographic analysis revealed an additive interaction between ginsenosides and neostigmine in both phases. Intrathecal pirenzepine, methoctramine, 4-DAMP, and tropicamide reversed the antinociception of ginsenosides in both phases. M1-M4 receptors mRNA detected in spinal cord of naive rats and the injection of formalin decreased the expression of M1 receptor mRNA, but it had no effect on the expression of other three muscarinic receptors mRNA. Intrathecal ginsenosides little affected the expression of all of muscarinic receptors mRNA in formalin-injected rats. CONCLUSIONS: Intrathecal ginsenosides additively interacted with neostigmine in the formalin test. Furthermore, M1-M4 receptors exist in the spinal cord, all of which contribute to the antinocieption of intrathecal ginsenosides.
Animals ; Catheters ; Diamines ; Drug Interactions ; Formaldehyde ; Ginsenosides ; Humans ; Male ; Neostigmine ; Nociception ; Pain Measurement ; Piperidines ; Pirenzepine ; Rats ; Rats, Sprague-Dawley ; Receptors, Muscarinic ; RNA, Messenger ; Spinal Cord ; Tropicamide

OBJECTIVE: To observe the inhibitive effect of pirenzepine on form deprivation myopia in guinea pigs and to explore the mechanism of Smad3 signaling pathway and connective tissue growth factor (CTGF) in the inhibition of myopia by pirenzepine. METHODS: Forty 1-week-old guinea pigs of either sex were randomly divided into 4 groups: a control group (Group I), a form deprivation group (Group II), a pirenzepine ophthalmic solution group (Group III), and a sodium chloride ophthalmic solution group (Group IV). Translucent blinders were used in the right eyes of Group II, III and IV. The left eyes were not given any treatment as the normal control group. Covered eyes of Group III and IV were given 3% pirenzepine ophthalmic solution and 0.1% azone ophthalmic solution respectively twice every day. Six weeks later, refraction and axial length were measured at the end of the experiment, and immunohistochemistry and Western blot were used to analyze the expression levels of Smad3 and CTGF in the sclera of all 4 groups. RESULTS: There was no significant difference between Group III and I in relative refraction and changes of axial length (P>0.05). The difference of Group II and IV compared with Group I was statistically significant (P<0.05). The number of Smad3 and CTGF positive cells in the sclera between Group III and I was not significantly different (P>0.05), while the difference in Group II, IV and I was significant (P<0.05). Western blot showed that the expression levels of Smad3 and CTGF in Group II and IV were much lower than those in Group I (P<0.05), but not evident in Group III and I (P>0.05). CONCLUSION Pirenzepine ophthalmic solution can inhibit the development of form deprivation myopia. Pirenzepine may affect Smad3 signaling pathway in the sclera by inhibiting the development of form deprivation myopia.
Animals ; Connective Tissue Growth Factor ; metabolism ; Guinea Pigs ; Humans ; Muscarinic Antagonists ; administration & dosage ; Myopia ; prevention & control ; Pirenzepine ; administration & dosage ; Random Allocation ; Sensory Deprivation ; Signal Transduction ; drug effects ; Smad3 Protein ; 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

Myopia is the most common refractive error throughout the world. Exact and relative etiologies of myopia have not been investigated in detail, although the high prevalence rate of myopia in school children has been well documented. Patients with myopia must endure the physical and financial burden of spectacles or contact lenses throughout their lives. The National Eye Institute estimated that the costs of refractive eye examinations amount to $1 billion annually, with another $1.5 billion spent on eyeglasses each year. The age of onset of myopia is frequently between 5 to 15 years. There has been a dramatic increase in the prevalence rates of myopia over the past decades in Korea and other parts of Asia. The prevalence rate was 8~15% in 1970s, 23% in 1980s, 38% in 1990s, and 46.2% in 2000s in Korean school children. The remarkable increase in Asian school children suggests that life style risk factors during the school periods may have a great impact on the development of school myopia and the overall population prevalence rate of myopia. Because the gene pool has not changed significantly over the past decades, the rapid increase of the prevalence rates of myopia has been attributed to increases in near-work activities and environmental factors. Atropine is effective in preventing myopia by a non-accommodative mechanism. Atropine is a broad-band muscarinic antagonist that binds to all five identified muscarinic receptors. Animal and clinical studies have shown that the M1-selective muscarinic antagonist, pirenzepine, is effective in reducing axial length enlargement and preventing myopia.
Age of Onset ; Animals ; Asia ; Asian Continental Ancestry Group ; Atropine ; Child ; Contact Lenses ; Eyeglasses ; Gene Pool ; Humans ; Korea ; Life Style ; Myopia* ; National Eye Institute (U.S.) ; Pirenzepine ; Prevalence ; Receptors, Muscarinic ; Refractive Errors ; Risk Factors

BACKGROUNDNonselective muscarinic receptor antagonist, atropine, was believed to inhibit myopic progression. The purpose of this study was to determine the efficacy, through topical administration, of the M1-selective muscarinic antagonist pirenzepine in preventing experimentally induced form-deprivation myopia in guinea pigs.

METHODSFifty-three guinea pigs, which underwent monocular deprivation with their eyelids sutured, were divided into 6 groups. Three groups were treated with 1%, 2% or 4% pirenzepine ophthalmic solutions; the fourth group with atropine; the fifth with saline and the last group left untreated. Ocular refraction, in vivo biometric measurements and wet eye weight were collected before and after the experiment. All the eyes were finally enucleated for histopathological examination to evaluate the possible toxic effects on ocular structures.

RESULTSAnimals untreated or treated with saline produced (-2.31+/-1.47) D and (-2.25+/-0.88) D of axial myopia respectively. Those treated with 1% pirenzepine ophthalmic solution produced relative myopia of (-1.63+/-0.48) D, and those under the treatment of 2% and 4% pirenzepine ophthalmic solution only developed a relative myopia of (-0.89+/-0.42) D and (-0.70+/-0.41) D (F=9.56, P<0.05). The significant reduction in myopia in 2% and 4% pirenzepine treated animals was caused by significantly less vitreous chamber elongation and axial elongation of the deprived eyes [2% group: (0.009+/-0.052) mm, 4% group: (0.006+/-0.078) mm] when compared with untreated, saline treated or 1% pirenzepine treated guinea pigs (0.057+/-0.056) mm, (0.064+/-0.053) mm and (0.033+/-0.035) mm, respectively]. Histological examinations revealed no obviously toxic effects on the eyes treated with pirenzepine.

CONCLUSIONTopical administration of the M1-selective muscarinic antagonist, pirenzepine, can prevent induced form-deprivation myopia in guinea pigs by inhibiting axial elongation without obvious damage to ocular tissues.

Animals ; Eye ; drug effects ; pathology ; Guinea Pigs ; Muscarinic Antagonists ; therapeutic use ; Myopia ; prevention & control ; Ophthalmic Solutions ; Organ Size ; drug effects ; Pirenzepine ; therapeutic use ; Refraction, Ocular ; drug effects

Cholinergic modulation of GABAergic spontaneous miniature inhibitory postsynaptic currents (mIPSCs) by the activation of muscarine receptors was investigated in mechanically dissociated rat nucleus basalis of the Meynert neurons using the conventional whole-cell patch recording configuration. Muscarine (10microM) reversibly and concentration-dependently decreased mIPSC frequency without affecting the current amplitude distribution. Muscarine action on GABAergic mIPSCs was completely blocked by 1microM methoctramine, a selective M2 receptor antagonist, but not by 1microM pirenzepine, a selective M1 receptor antagonist. NEM (10microM), a G-protein uncoupler, attenuated the inhibitory action of muscarine on GABAergic mIPSC frequency. Muscarine still could decrease GABAergic mIPSC frequency even in the Ca2+-free external solution. However, the inhibitory action of muscarine on GABAergic mIPSCs was completely occluded in the presence of forskolin. The results suggest that muscarine acts presynaptically and reduces the probability of spontaneous GABA release, and that such muscarine-induced inhibitory action seems to be mediated by G-protein-coupled M2 receptors, via the reduction of cAMP production. Accordingly, M2 receptor-mediated disinhibition of nBM neurons might play one of important roles in the regulation of cholinergic outputs from nBM neurons as well as the excitability of nBM neurons themselves.
Animals ; Cholinergic Neurons ; Colforsin ; gamma-Aminobutyric Acid ; GTP-Binding Proteins ; Inhibitory Postsynaptic Potentials ; Muscarine* ; Neurons* ; Pirenzepine ; Rats*

The present study was attempted to investigate the characteristics of epibatidine on secretion of catecholamines (CA) from the isolated perfused model of the rat adrenal gland, and to establish the mechanism of action. Epibatidine (3X10(-8) M) injected into an adrenal vein produced a great inhibition in secretory response of CA from the perfused rat adrenal gland. However, upon the repeated injection of epibatidine (3X10(-8) M) at 15 min-intervals, CA secretion was rapidly decreased after second injection of epibatidine. However, there was no statistical difference between CA secretory responses of both 1st and 2nd periods by the successive administration of epibatidine at 120 min-intervals. Tachyphylaxis to releasing effects of CA evoked by epibatidine was observed by the repeated administration. Therefore, in all subsequent experiments, epibatidine was not administered successively more than twice only 120 min-intervals. The epibatidine-induced CA secretion was markedly inhibited by the pretreatment with atropine, chlorisondamine, pirenzepine, nicardipine, TMB-8, and perfusion of Ca2+/-free Krebs solution containing EGTA, while was not affected by diphenhydramine. Moreover, the CA secretion evoked by ACh for 1st period (0apprx4 min) was greatly potentiated by the simultaneous perfusion of epibatidine (1.5X10(-8) M), but followed by time-dependently gradual reduction after 2nd period. The CA release evoked by high potassium (5.6+/-10(-8) M) for 1st period (0apprx4 min) was also enhanced by the simultaneous perfusion of epibatidine, but those after 2nd period were not affected. Taken together, these experimental data suggest that epibatidine causes catecholamine secretion in a calcium dependent fashion from the perfused rat adrenal gland through activation of neuronal cholinergic (nicotinic and muscarinic) receptors located in adrenomedullary chromaffin cells. It also seems that epibatidine-evoked catecholamine release is not relevant to stimulation of histaminergic receptors.
Adrenal Glands* ; Animals ; Atropine ; Calcium ; Catecholamines ; Chlorisondamine ; Chromaffin Cells ; Diphenhydramine ; Egtazic Acid ; Neurons ; Nicardipine ; Perfusion ; Pirenzepine ; Potassium ; Rats* ; Tachyphylaxis ; Veins

BACKGROUND: Although the efficacy of morphine in a neuropathic pain state is somewhat controversial, intrathecally administered morphine reversed the mechanical allodynia in a previous animal study. Using a behavioral test, we investigated the mechanism of the antiallodynic action of intrathecal morphine by administering opioids, alpha2 adrenergic and cholinergic receptor antagonists in a rat model of neuropathic pain induced by a spinal nerve ligation injury. METHODS: Male Sprague Dawley rats were prepared with a tight ligation of the left lumbar 5th and 6th spinal nerves and insertion of a chronic lumbar intrathecal catheter. Morphine 1 microgram was administered intrathecally to attenuate the mechanical allodynia. Naloxone 10 microgram, yohimbine 30 microgram, prazosin 30 microgram, atropine 10 microgram, pirenzepine 10 microgram, and methoctramine 10 microgram was administered intrathecally before and after the injection of morphine in order to investigate the reversal of an increased allodynic threshold by morphine. The allodynic thresholds for the left hindpaw withdrawal to von Frey hairs were assessed and converted to %MPE. RESULTS: A reduction of mechanical allodynia by intrathecal morphine was produced. Naloxone pretreatment, but not posttreatment, reversed the antiallodynic effect of intrathecal morphine (P < 0.01). All alpha2 adrenergic and cholinergic receptor antagonists used here did not reverse it. CONCLUSIONS: The results suggest that the reversal mechanism of mechanical allodynia by intrathecal morphine appears to be mediated mostly by the opioid receptor system, but not the alpha2 adrenergic and cholinergic receptor systems, at the spinal level in a rat model of a spinal nerve ligation injury.
Analgesics, Opioid ; Animals ; Atropine ; Catheters ; Cholinergic Antagonists ; Hair ; Humans ; Hyperalgesia ; Ligation* ; Male ; Models, Animal ; Morphine* ; Naloxone ; Neuralgia* ; Pirenzepine ; Prazosin ; Rats, Sprague-Dawley ; Receptors, Opioid ; Spinal Nerves* ; Yohimbine