title: Activation of Ca(2+)-dependent K+ current by acetylcholine and histamine in a human gastric epithelial cell line.
abstract: 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)
authors.affiliation: Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
authors.forename: E
authors.initials: E
authors.lastname: Hamada
authors.forename: T
authors.initials: T
authors.lastname: Nakajima
authors.forename: S
authors.initials: S
authors.lastname: Ota
authors.forename: A
authors.initials: A
authors.lastname: Terano
authors.forename: M
authors.initials: M
authors.lastname: Omata
authors.forename: S
authors.initials: S
authors.lastname: Nakade
authors.forename: K
authors.initials: K
authors.lastname: Mikoshiba
authors.forename: Y
authors.initials: Y
authors.lastname: Kurachi
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D004553:Electric Conductivity; D019204:GTP-Binding Proteins / Q000502:physiology; D005753:Gastric Mucosa / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D006632:Histamine / Q000494:pharmacology*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000502:physiology; D007425:Intracellular Membranes / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D011188:Potassium / Q000502:physiology*; D015290:Second Messenger Systems
chemical_list: D006632:Histamine; D015544:Inositol 1,4,5-Trisphosphate; D019204:GTP-Binding Proteins; D000109:Acetylcholine; D011188:Potassium; D002118:Calcium