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*

Stimulation of muscarinic receptors by carbachol (CCh) in the circular smooth muscle of the guinea pig gastric antrum causes muscle contraction. In the present study, muscarinic receptor subtype controlling the muscle contraction in response to CCh was studied using putative muscarinic receptor antagonists. Isometric force of the isolated circular muscle strips was measured in an organ bath. CCh contracted the muscle in a dose-dependent way, and each of the three muscarinic receptor antagonists, 4-diphenylacetoxy-N-methylpeperdine methiodide (4-DAMP), methoctramine and pirenzepine shifted the concentration-response curves to the right without significantly reducing the maximum force. The affinities of the muscarinic antagonists (pA2 values) obtained from Schild plot analysis were 10.15, 7.05 and 6.84 for 4-DAMP, methoctramine and pirenzepine, respectively. These results suggest that the M3-subtype mainly mediate the muscle contraction in response to CCh in guinea pig gastric antrum.
Animals ; Baths ; Carbachol ; Guinea Pigs* ; Guinea* ; Muscarinic Antagonists ; Muscle Contraction* ; Muscle, Smooth ; Pirenzepine ; Pyloric Antrum* ; Receptors, Muscarinic*

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: 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

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

Effects of a M1 receptor antagonist, pirenzepine, a M2 receptor antagonist, AF-DX116, and a nicotinic receptor antagonist, mecamylamine on the pressor responses to preganglionic sympathetic nerve stimulation(PNS) and McN-A-343 and DMPP in spinal(pithed) rabbits were investigated in order to elucidate a functional role of M1, M2 and nicotinic receptors in ganglionic transmission. Pirenzepine and AF-DX116 selectively inhibited the McN-A-343-induced pressor reponse in chlorisondamine-treated rabbit and the BCh-induced bradycardia, respectively. Electrical stimulations of preganglionic sympathetic outflow at T8 level produced increases in blood pressure. Pirenzepine(3 microgram/kg) significantly inhibited the PNS-induced pressor response and the degree of inhibition was not changed by increasing the doses to 100 microgram/kg. AF-DX116(100 microgram/kg) had no effect on the PNS-induced pressor response. Mecamylamine inhibited the PNS-induced pressor response in a dose-dependent manner. The inhibitory action of mecamylamine was significantly augmented by combined-treatment with pirenzepine(30 microgram/kg) but AF-DX116(100 microgram/kg) did not affect the inhibitory action of mecamylamine. McN-A-343 and DMPP elicited pressor response in the spinal rabbit. Pirenzepine and AF-DX116 dose-dependently inhibited the McN-A-343-induced pressor response but they did not affect DMPP-induced pressor response. Mecamylamine inhibited both pressor responses induced by Mc-N-343- and DMPP. These results suggest that not only nicotinic receptors but also M1 receptors play a facilitatory role in ganglionic transmission but M2 receptors do not contribute the transmission in spinal(pithed) rabbits.
(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride ; Blood Pressure ; Bradycardia ; Dimethylphenylpiperazinium Iodide ; Electric Stimulation ; Ganglia, Sympathetic* ; Ganglion Cysts ; Mecamylamine ; Pirenzepine ; Rabbits ; Receptors, Nicotinic*

BACKGROUND: Peripheral nerve injury may produce a syndrome consisting of spontaneous pain, allodynia and hyperpathia. In previous study, we examined the antiallodynic action produced by intrathecal (i.t.) cholinesterase inhibitors (ChEi) in a neuropathic pain rat model and the reversal of antiallodynic state by i.t. atropine, muscarinic antagonist, but not by nicotinic antagonist mecamylamine. The purpose of this study was to determine the selective antagonistic action of four subtypes of muscarinic receptor on antiallodynic state by i.t. ChEi in a rat model of neuropathic pain. METHODS: Sprague Dawley rats were prepared with tight ligation of left L5/L6 spinal nerves with 6-0 black silk and chronic lumbar intrathecal catheters. After obtaining the baseline hindpaw withdrawal scores, edrophonium (100 microgram) or neostigmine (10 microgram) was administered intrathecally. Tactile allodynia was measured using von Frey filaments and allodynic threshold was calculated by the up-down method. Allodynic changes were tested at 15, 30, 45, 60, 90, 120 and 180 minutes. To examine the reversal of antiallodynia and to compare the antagonizing action of antiallodynic state produced by i.t. administration of ChEi, non-selective muscarinic receptor antagonists atropine (10 microgram), M1 antagonist pirenzepine (3 microgram), M2 antagonist methoctramine (3 microgram), M3 antagonist 4-DAMP (3 microgram) and M4 antagonist tropicamide (3 microgram) were injected intrathecally respectively 5 minutes prior to the injection of edrophonium or neostigmine. RESULTS: Antiallodynia produced by i.t. edrophonium was reversed by pretreatment with i.t. methoctramine, 4-DAMP, tropicamide and pirenzepine (P<0.05). On the contrary, antiallodynic state made by i.t. neostigmine was not antagonized by methoctramine, 4-DAMP and tropicamide. M1 antagonist pirenzepine had a moderate, statistically significant (P<0.05) effect on reversal of increased allodynic threshold while atropine showed a complete antagonism. CONCLUSION: These experiments suggest that antialllodynic action of cholinesterase inhibitors is likely due to mediation of spinal muscarinic system and M1 receptor subtype is more likely involved in this mechanism.
Animals ; Atropine ; Catheters ; Cholinesterase Inhibitors ; Edrophonium ; Hyperalgesia ; Ligation ; Mecamylamine ; Models, Animal* ; Negotiating ; Neostigmine ; Neuralgia* ; Peripheral Nerve Injuries ; Pirenzepine ; Rats* ; Rats, Sprague-Dawley ; Receptors, Muscarinic* ; Silk ; Spinal Nerves ; Tropicamide

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

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

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