• Archives of Pharmacal Research
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• 제28권8호
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• pp.930-935
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• 2005

We have investigated the effects of relatively high concentration of carbachol (CCh), an agonist of muscarinic acetylcholine receptor (mAChR), on cardiac automaticity in mouse heart. Action potentials from automatically beating right atria of mice were measured with conventional microelectrodes. When atria were treated with $100{\mu}M$ CCh, atrial beating was immediately arrested and diastolic membrane potential (DMP) was depolarized. After exposure of the atria to CCh for $\~4 min$, action potentials were regenerated. The regenerated action potentials had lower frequency and shorter duration when compared with the control. When atria were pre-exposed to pirenzepine $(1{\mu}M)$, an $M_1$ mAChR antagonist, there was complete inhibition of CCh-induced depolarization of DMP and regeneration of action potentials. Pre-exposure to AFDX-116 (11 ({2-[(diethylamino)-methyl]-1-piperidyl}acetyl)-5, 11-dihydro-6H-pyridol[2,3-b][1,4] benzodiazepine-6-one base, $1{\mu}M$), an $M_2$ mAChR antagonist, failed to block CCh-induced arrest of the beating. However, prolonged exposure to CCh elicited gradual depolarization of DMP and slight acceleration in beating rate. Our data indicate that high concentration of CCh depolarizes membrane potential and recovers right atrial automaticity via $M_1$ mAChR, providing functional evidence for the role of $M_1$ mAChR in the atrial myocytes.

• Archives of Pharmacal Research
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• 제21권4호
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• pp.423-428
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• 1998

The ligand binding signals to a wide variety of seven transmembrane cell surface receptors are transduced into intracellular signals through heterotrimeric G-proteins. Recently, there have been reports which show diverse coupling patterns of ligand-activated receptors to the members of Gq family $\alpha$ subunits. In order to shed some light on these complex signal processing networks, interactions between G$\alpha$q family of G protein and neurokinin-2 receptor as well as muscarinic M$_{1}$ receptor, which are considered to be new thearpeutic targets in asthma, were studied. Using washed membranes from Cos-7 cells co-transfected with different G.alpha.q and receptor cDNAs, the receptors were stimulated with various concentrations of carbachol and neurokinin A and the agonist-dependent release of [$^3H$]inositol phosphates through phospholipase C beta-1 activation was measured. Differential coupling of Gaq family of G-protein to muscarinic M$_{1}$ receptor and neurokinin-2 receptor was observed. The neurokinin-2 receptor shows a ligand-mediated response in membranes co-transfected with G$\alpha$q, G$\alpha$11 and G$\alpha$14 but not G$\alpha$16 and the ability of the muscarinic $M_1$ receptor to activate phospholipase C through G$\alpha$/11 but not G$\alpha$14 and G$\alpha$16 was demonstrated. Clearly G$\alpha$/11 can couple $\M_1$ and neurokinin-2 receptor to activate phospholipase C. But, there are differences in the relative coupling of the G$\alpha$14 and G$\alpha$16 subunits to these receptors.

• 약학회지
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• 제37권4호
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• pp.397-407
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• 1993

The muscarinic antagonist l-[benzilic-4,4'-$^3H$]quinuclidinyl benzilate([$^3H$]QNB) bound to a single class of muscarinic receptor with high affinity in guinea pig ileal membranes. The $K_{D}$ and B$_{max}$ values for [$^3H$]QNB calculated from analysis of saturation isotherms were 54 pM and 156fmol/mg, respectively. H$_{1}$-blockers inhibited [$^3H$]QNB binding to ileal membranes with $K_{i}$ values ranged from 0.008 $\mu{M}$ to 1.6 $\mu{M}$. The pseudo-Hill coefficients of H$_{1}$-blockers for inhibition of [$^3H$]QNB binding to the ileal membranes were close to unit. The $K_{i}$ values for H$_{1}$-blockers were similar to the $K_{M}$ values calculated by Schild plot of functional data obtained from inhibition of the carbachol-induced contraction in guinea-pig ileum, suggesting that binding of H$_{1}$-blockers vs [$^3H$]QNB in ileal membranes represents an interaction with a receptor of physiological relevance. The $K_{H}$ values of H$_{1}$-blockers for H$_{1}$-receptor estimated from inhibition of the histamine-induced contraction were the range of 0.15 nM to 56.5 nM. The $K_{M}$/K$_{H}$ ratio of H$_{1}$-blockers varied over a wide range of 3 to 2300. Thus, the antihistaminic potencies of H$_{1}$-blockers do not correlate with their antimuscarinic potencies, which suggest that antihistamines have different antimuscarinic potencies in therapeutic blood levels causing similar antiallergic effect. Among 13 traditional antihistaminics examined in this study, drug having the highest and the lowest $K_{M}$/K$_{H}$ ratio is triprolidine and diphenidol, respectively. The present results demonstrate that the antimuscarinic property of antihistamines is not necessary for their antiallergic effect, and data on the affinity of antihistamines for muscarinic and H$_{1}$-receptors can be an important parameter in the selection and evaluation of these drugs.

• The Korean Journal of Physiology and Pharmacology
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• 제24권6호
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• pp.517-527
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• 2020

Layer 2/3 pyramidal neurons (L2/3 PyNs) of the cortex extend their basal dendrites near the soma and as apical dendritic tufts in layer 1, which mainly receive feedforward and feedback inputs, respectively. It is suggested that neuromodulators such as serotonin and acetylcholine may regulate the information flow between brain structures depending on the brain state. However, little is known about the dendritic compartment-specific induction of synaptic transmission in single PyNs. Here, we studied layer-specific serotonergic and cholinergic induction of long-term synaptic plasticity in L2/3 PyNs of the agranular insular cortex, a lateral component of the orbitofrontal cortex. Using FM1-43 dye unloading, we verified that local electrical stimulation to layers 1 (L1) and 3 (L3) activated axon terminals mostly located in L1 and perisomatic area (L2/3). Independent and AMPA receptor-mediated excitatory postsynaptic potential was evoked by local electrical stimulation of either L1 or L3. Application of serotonin (5-HT, 10 μM) induced activity-dependent longterm depression (LTD) in L2/3 but not in L1 inputs. LTD induced by 5-HT was blocked by the 5-HT₂ receptor antagonist ketanserin, an NMDA receptor antagonist and by intracellular Ca²⁺ chelation. The 5-HT₂ receptor agonist α-me-5-HT mimicked the LTD induced by 5-HT. However, the application of carbachol induced muscarinic receptor-dependent LTD in both inputs. The differential layer-specific induction of LTD by neuromodulators might play an important role in information processing mechanism of the prefrontal cortex.

• 대한한방부인과학회지
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• 제18권1호
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• pp.64-80
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• 2005

The pharmacological effects of medicinal remedies traditionally used in Asian countries for improving the blood circulation were examined on isolated rat thoracic aorta strips in organ baths. Each experimental medicine was consecutively extracted under reflux with water. Of 17 plants, Curcuma longa (CL) having the strongest acute relaxant activity in endothelium-intact arteries, Mucunae caulis (MC), Cirsium pendulum (CP), Rumex longiflius (RL), Paeonia suffruticosa (PS), Curcuma zedoaria (CZ), Scirpus maritimus (SM), Siphonostegia chinensis (SC), Leonurs sibiricus (LS) and Typha orientalis (TO) were showing dose-dependent relaxant activity. Long-term relaxant effects were showed in Curcuma aromatia (CA), MC, CP, RL, PS, Potulacae grandiflorae (PG), CZ, Panax notoginseng (PN), Achyranthes japonica (AJ), CL, SC, Lycoppus lucidus (LL) and Corydalis turtschaninovii (CT). In endothelium-injury test using carbachol, CL, SC, MC, RL and PS which are having the acute vasorelaxing activity and CA and CT which are not showing vasorelaxaing activity were damaged to endothelium. As a result of this study, the possibility that a part of medicinal remedy may contribute to the beneficial effects in blood circulation was proposed, but inter-individual variation has been observed. Also, further studies on the vasorelaxant effects of these remedies are still required.

• International Journal of Oral Biology
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• 제46권3호
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• pp.134-139
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• 2021

Under physiological conditions, calcium (Ca²⁺) regulates essential functions of polarized secretory cells by the stimulation of specific Ca²⁺ signaling mechanisms, such as increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) via the store-operated Ca²⁺ entry (SOCE) and the receptor-operated Ca²⁺ entry (ROCE). Homer proteins are scaffold proteins that interact with G protein-coupled receptors, inositol 1,4,5-triphosphate (IP₃) receptors, Orai1-stromal interaction molecule 1, and transient receptor potential canonical (TRPC) channels. However, their role in the Ca²⁺ signaling in exocrine cells remains unknown. In this study, we investigated the role of Homer2 in the Ca²⁺ signaling and regulatory channels to mediate SOCE and ROCE in pancreatic acinar cells. Deletion of Homer2 (Homer2^-/-) markedly increased the expression of TRPC3, TRPC6, and Orai1 in pancreatic acinar cells, whereas these expressions showed no difference in whole brains of wild-type and Homer2^-/- mice. Furthermore, the response of Ca²⁺ entry by carbachol also showed significant changes to the patterns regulated by specific blockers of SOCE and ROCE in pancreatic acinar cells of Homer2^-/- mice. Thus, these results suggest that Homer2 plays a critical role in the regulatory action of the [Ca²⁺]_i via SOCE and ROCE in mouse pancreatic acinar cells.

• The Korean Journal of Physiology and Pharmacology
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• 제23권3호
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• pp.191-201
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• 2019

The transient receptor potential canonical (TRPC) 5 channel, known as a nonselective cation channel, has a crucial role in calcium influx. TRPC5 has been reported to be activated by muscarinic receptor activation and extracellular pH change and inhibited by the protein kinase C pathway. Recent studies have also suggested that TRPC5 is extracellularly activated by englerin A (EA), but the mechanism remains unclear. The purpose of this study is to identify the EA-interaction sites in TRPC5 and thereby clarify the mechanism of TRPC5 activation. TRPC5 channels are over-expressed in human embryonic kidney (HEK293) cells. TRPC5 mutants were generated by site-directed mutagenesis. The whole-cell patch-clamp configuration was used to record TRPC5 currents. Western analysis was also performed to observe the expression of TRPC5 mutants. To identify the EA-interaction site in TRPC5, we first generated pore mutants. When screening the mutants with EA, we observed the EA-induced current increases of TRPC5 abolished in K554N, H594N, and E598Q mutants. The current increases of other mutants were reduced in different levels. We also examined the functional intactness of the mutants that had no effect by EA with TRPC5 agonists, such as carbachol or $GTP{\gamma}S$. Our results suggest that the three residues, Lys-554, His-594, and Glu-598, in TRPC5 might be responsible for direct interaction with EA, inducing the channel activation. We also suggest that although other pore residues are not critical, they could partly contribute to the EA-induced channel activation.

• 한국수정란이식학회지
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• 제20권3호
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• pp.191-200
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• 2005

체외 배양 과정 중에 나타나는 생쥐 초기 2-세포 배의 "in vitro 2-cell block" 현상은 세포내 $Ca^{2+}$ 농도 변화와 밀접한 관련이 있다. 다양한 종류의 세포에서 acetylcholine은 세포막에 존재하는 muscarnic acetylcholine receptor를 통해 세포내 $Ca^{2+}$ 농도 증가를 유도한다. 본 실험에서는 생쥐 "in vitro 2-cell block" 현상에 있어서 ACh의 영향을 알아보기 위해 세포 내 $Ca^{2+}$ 농도 조절 물질을 처리한 후, 공초점 현미경을 이용하여 세포 내 $Ca^{2+}$ 농도 변화를 기록하였다. ACh은 세포 내에서 농도 의존적으로 $Ca^{2+}$ 농도 증가를 유도하며, "in Vitro 2-cell block" 현상을 극복하여 포배기로 발생을 유도하였다. ACh에 의한 $Ca^{2+}$ 농도 증가가 세포막에 존재하는 ACh receptor를 경유하여 나타나는 반응인지를 알아보기 위해 ACh receptor의 저해제인 atropine을 전처리한 결과, ACh에 의한 $Ca^{2+}$ 농도 증가가 완전히 저해되었다. 초기 2-세포 배에서 ACh이 결합하는 receptor의 종류를 확인하기 위하여 carbachol과 nicotin tartrate를 처리 하였다. Nicotinic AChR의 agonist인 nicotine tartrate 1 mM은 세포내 $Ca^{2+}$ 농도 증가를 보이지 않았다. 따라서 초기 2-세포 배의 세포막에는 muscarnic AChR가 기능적으로 작용함을 알 수 있다. ACh에 의한 세포내 $Ca^{2+}$ 농도 증가가 $Ca^{2+}$이 제거된 배양액에서도 나타나는 것으로 보아 ACh에 의한 세포내 $Ca^{2+}$ 변화는 주로 소포체와 같은 세포내 $Ca^{2+}$ 저장고로부터 분비됨을 알 수 있었다. 이러한 세포내 $Ca^{2+}$ 저장고로부터의 $Ca^{2+}$ 분비가 어떤 신호전달체계를 통해 나타나는 지를 조사하였다. 세포막의 PLC 저해제인 U73122를 전처리한 배는 ACh에 의한 $Ca^{2+}$ 농도 증가가 나타나지 않았으며, 세포 내 $Ca^{2+}$ 통로인 IP3R와 RyR의 저해제인 xestospongin과 heparin 혹은 dantrolene을 전처리한 결과 dantrolene에 의해 세포내 $Ca^{2+}$ 농도 증가가 억제되었다. 그리고 세포내 반복적인 $Ca^{2+}$ 농도 증가에 의해 활성도가 변화는 CaMKII의 작용을 확인하기 위하여 Ca MKII의 저해제인 KN-93을 전처리한 결과 $Ca^{2+}$ 농도 증가가 억제되는 것을 확인하였다. 이상의 결과로부터 ACh은 생쥐 초기 2-세포 배에서 ryano-dine receptor를 통하여 세포내 $Ca^{2+}$ 저장고로부터 $Ca^{2+}$ 분비를 유도하며, CaM KII에 의해서도 영향을 받는 것으로 보여진다. 생쥐 초기 2-세포 배에서 "in vitro 2-cell block"의 극복은 ACh에 의해 유도된 신호전달체계를 통해 세포내에 증가하는 $Ca^{2+}$ 농도 및 이에 따른 세포내 대사 작용의 활성화에 의하여 나타나는 것으로 생각된다.

• The Korean Journal of Physiology and Pharmacology
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• 제1권6호
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• pp.783-796
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• 1997

The relaxation induced by stimulation of the inhibitory non-adrenergic, non-cholinergic (iNANC) nerve is mediated by the release of iNANC neurotransmitters such as nitric oxide (NO), vasoactive intestinal peptide (VIP) and adenosine triphosphate (ATP). The mechanisms of NO, VIP or ATP-induced relaxation have been partly determined in previous studies, but the detailed mechanism remains unknown. We tried to identify the nature of iNANC neurotransmitters in the smooth muscle of guinea pig ileum and to determine the mechanism of the inhibitory effect of nitric oxide. We measured the effect of NO-donors VIP and ATP on the intracellular $Ca^{2+}$ concentration$([Ca^{2+}]_i)$, by means of a fluorescence dye(fura 2) and tension simultaneously in the isolated guinea pig ileal smooth muscle. Following are the results obtained. 1. Sodium nitroprusside $(SNP:10^{-5}\;M)$ or S -nitro-N-acetyl-penicillamine $(SNP:10^{-5}\;M)$ decreased resting $[Ca^{2+}]_i$ I and tension of muscle. SNP or SNAP also inhibited rhythmic oscillation of $[Ca^{2+}]_i$ and tension. In 40mM $K^+$ solution or carbachol ($(CCh:10^{-6}\;M)$-induced precontracted muscle, SNP decreased muscle tension. VIP did not change $[Ca^{2+}]_i$ and tension in the resting or precontracted muscle, but ATP increased resting $[Ca^{2+}]_i$ and tension in the resting muscle. 2. 1H-[1,2,4]oxadiazol(4,3-a)quinoxalin-1-one $(ODQ:1\;{\mu}M)$, a specific inhibitor of soluble guanylate cyclase, limited the inhibitory effect of SNP 3. Glibenclamide $(10\;{\mu}M)$, a blocker of $K_{ATP}$ channel, and 4-aminopyridine (4-AP:5 mM), a blocker of delayed rectifier K channel, apamin $(0.1\;{\mu}M)$, a blocker of small conductance $K_{Ca}$ channel had no effect on the inhibitory effect of SNP. Iberiotoxin $(0.1\;{\mu}M)$, a blocker of large conductance $K_{Ca}$ channel, significantly increased the resting $[Ca^{2+}]_i$, and tension, and limited the inhibitory effect of SNP. 4. Nifedipine $(1\;{\mu}M)$ or elimination of external $Ca^{2+}$ decreased not only resting $[Ca^{2+}]_i$ and tension but also oscillation of $[Ca^{2+}]_i$ and tension. Ryanodine $(5\;{\mu}M)$ and cyclopiazonic acid $(10\;{\mu}M)$ decreased oscillation of $[Ca^{2+}]_i$ and tension. 5. SNP decreased $Ca^{2+}$ sensitivity of contractile protein. In conclusion, these results suggest that 1) NO is an inhibitory neurotransmitter in the guinea pig ileum, 2) the inhibitory effect of SNP on the $[Ca^{2+}]_i$ and tension of the muscle is due to a decrease in $[Ca^{2+}]_i$ by activation of the large conductance $K_{Ca}$ channel and a decrease in the sensitivity of contractile elements to $Ca^{2+}$ through activation of G-kinase.

• 대한수의학회지
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• 제43권3호
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• pp.415-421
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• 2003

It is now generally accepted that acupuncture is effective in diarrhea caused by bacterial infection. However, its effect on the intestinal smooth muscle dysfunction is not clear. Therefore, we investigated the effect of acupuncture therapy at Jiao-chao (GV-1) on the intestinal muscle dysfunction in weaning piglets orally infected by Escherichia coli. The animals are divided into four groups; 1) E. coli + no-treatment, 2) E. coli + antibiotic, 3) E. coli + acupuncture, 4) normal group. In the three E. coli infected groups, low frequency electrical field stimulation (EFS, 1 Hz) provoked triphasic responses composed of initial relaxation followed by on-contraction and off-contraction. While in the normal group, EFS (1Hz) induced biphasic responses composed of relaxation during the stimulation and off-contraction. At the high frequency (16Hz) EFS, both on-contraction and off-contraction of the E. coli + antibiotic, E. coli + acupuncture and the normal group were larger than those of the E. coli + no-treatment group. In the non-adrenergic non-cholinergic (NANC) condition, only biphasic responses occurred to EFS in all experimental groups and the off-contraction of E. coli + antibiotic, E. coli + acupuncture and the normal group were larger than those of the E. coli + no-treatment group. The response to carbachol of those three groups was also significantly greater than that of the E. coli + no-treatment group. These results suggest that acupuncture is as effective as antibiotic in the dysfunction of colonic circular muscle caused by E. coli infection. The maintenance of contractile neuromuscular transmission seems to be involved in the mechanism of the acupuncture effects on diarrhea.