Hamada E, Nakajima T, Ota S, Terano A, Omata M, Nakade S, Mikoshiba K, Kurachi Y
Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
J Gen Physiol. 1993 Oct;102(4):667-92. doi: 10.1085/jgp.102.4.667.
The effects of acetylcholine (ACh) and histamine (His) on the membrane potential and current were examined in JR-1 cells, a mucin-producing epithelial cell line derived from human gastric signet ring cell carcinoma. The tight-seal, whole cell clamp technique was used. The resting membrane potential, the input resistance, and the capacitance of the cells were approximately -12 mV, 1.4 G ohms, and 50 pF, respectively. Under the voltage-clamp condition, no voltage-dependent currents were evoked. ACh or His added to the bathing solution hyperpolarized the membrane by activating a time- and voltage-independent K+ current. The ACh-induced hyperpolarization and K+ current persisted, while the His response desensitized quickly (< 1 min). These effects of ACh and His were mediated predominantly by m3-muscarinic and H1-His receptors, respectively. The K+ current induced by ACh and His was inhibited by charybdotoxin, suggesting that it is a Ca(2+)-activated K+ channel current (IK.Ca). The measurement of intracellular Ca2+ ([Ca2+]i) using Indo-1 revealed that both agents increased [Ca2+]i with similar time courses as they increased IK.Ca. When EGTA in the pipette solution was increased from 0.15 to 10 mM, the induction of IK.Ca by ACh and His was abolished. Thus, both ACh and His activate IK.Ca by increasing [Ca2+]i in JR-1 cells. In the Ca(2+)-free bathing solution (0.15 mM EGTA in the pipette), ACh evoked IK.Ca transiently. Addition of Ca2+ (1.8 mM) to the bath immediately restored the sustained IK.Ca. These results suggest that the ACh response is due to at least two different mechanisms; i.e., the Ca2+ release-related initial transient activation and the Ca2+ influx-related sustained activation of IK.Ca. Probably because of desensitization, the Ca2+ influx-related component of the His response could not be identified. Intracellularly applied inositol 1,4,5-trisphosphate (IP3), with and without inositol 1,3,4,5-tetrakisphosphate (IP4), mimicked the ACh response. IP4 alone did not affect the membrane current. Under the steady effect of IP3 or IP3 plus IP4, neither ACh nor His further evoked IK.Ca. Intracellular application of heparin or of the monoclonal antibody against the IP3 receptor, mAb18A10, inhibited the ACh and His responses in a concentration-dependent fashion. Neomycin, a phospholipase C (PLC) inhibitor, also inhibited the agonist-induced response in a concentration-dependent fashion. Although neither pertussis toxin (PTX) nor N-ethylmaleimide affected the ACh or His activation of IK,Ca, GDP beta S attenuated and GTP gamma S enhanced the agonist response.(ABSTRACT TRUNCATED AT 400 WORDS)
在JR - 1细胞（一种源自人胃印戒细胞癌的黏蛋白分泌上皮细胞系）中研究了乙酰胆碱（ACh）和组胺（His）对膜电位和电流的影响。采用了紧密封接的全细胞钳技术。细胞的静息膜电位、输入电阻和电容分别约为 - 12 mV、1.4 GΩ和50 pF。在电压钳制条件下，未诱发电压依赖性电流。添加到浴液中的ACh或His通过激活一种时间和电压非依赖性的K⁺电流使膜超极化。ACh诱导的超极化和K⁺电流持续存在，而His反应迅速脱敏（<1分钟）。ACh和His的这些作用分别主要由M3 - 毒蕈碱受体和H1 - 组胺受体介导。ACh和His诱导的K⁺电流被蝎毒素抑制，表明它是一种Ca²⁺激活的K⁺通道电流（IK.Ca）。使用Indo - 1测量细胞内Ca²⁺（[Ca²⁺]i）表明，两种试剂增加[Ca²⁺]i的时间进程与它们增加IK.Ca的时间进程相似。当移液管溶液中的乙二醇双乙醚二胺四乙酸（EGTA）从0.15 mM增加到10 mM时，ACh和His对IK.Ca的诱导被消除。因此，ACh和His都通过增加JR - 1细胞中的[Ca²⁺]i来激活IK.Ca。在无Ca²⁺的浴液中（移液管中为0.15 mM EGTA），ACh短暂诱发IK.Ca。立即向浴液中添加Ca²⁺（1.8 mM）可恢复持续的IK.Ca。这些结果表明，ACh反应至少归因于两种不同机制；即，Ca²⁺释放相关的初始瞬时激活和Ca²⁺内流相关的IK.Ca持续激活。可能由于脱敏作用，His反应中与Ca²⁺内流相关的成分无法识别。细胞内应用肌醇1,4,5 - 三磷酸（IP3），无论有无肌醇1,3,4,5 - 四磷酸（IP4），都模拟了ACh反应。单独的IP4不影响膜电流。在IP3或IP3加IP4的稳定作用下，ACh和His都不再诱发IK.Ca。细胞内应用肝素或抗IP3受体的单克隆抗体mAb18A10以浓度依赖性方式抑制ACh和His反应。新霉素，一种磷脂酶C（PLC）抑制剂，也以浓度依赖性方式抑制激动剂诱导的反应。尽管百日咳毒素（PTX）和N - 乙基马来酰亚胺都不影响ACh或His对IK.Ca的激活，但GDPβS减弱而GTPγS增强激动剂反应。（摘要截断于400字）
