Further identification of neurokinin receptor types and mechanisms of calcium signaling evoked by neurokinins in the murine neuroblastoma C1300 cell line.
It has been suggested that murine neuroblastoma C1300 cells express endogenous neurokinin NK2 receptors with features that differ from those of NK2 receptors characterized in other systems. In this study, we have further characterized the neurokinin receptor types present in this cell line. RNA blots showed that mRNAs of NK2 and NK3 receptors, but not of NK1 receptors, were expressed in C1300 cells. The increase in the cytosolic calcium concentration ([Ca2+]i) induced by 0.33 microM neurokinin A was completely inhibited by SR 48968, an NK2 receptor antagonist, whereas the partial response to 0.33 microM neurokinin B was unaffected, and the response was completely inhibited by SR 142801, and NK3 receptor antagonist. In addition, the [Ca2+]i increase by 0.33 microM senktide, an NK3 receptor agonist, was inhibited by SR 142801 but not by SR 48968. These findings indicated that C1300 cells endogenously express functional NK2 and NK3 receptors. It was also demonstrated that NK2 and NK3 receptors can be activated independently by 3.3 microM neurokinin A in the presence of 1.0 microM SR 142801 or 1.0 microM senktide, respectively. Therefore, the mechanisms of Ca2+ signaling mediated by endogenous NK2 and NK3 receptors were investigated. The independent activation of NK2 or NK3 receptors induced not only the [Ca2+]i increase, but also stimulated the formation of inositol trisphosphates; both these responses were inhibited by U73122, a phospholipase C (PLC) inhibitor. In addition, NK2 and NK3 receptor-mediated [Ca2+]i increase was partially attenuated in the absence of extracellular Ca2+ or in the presence of nickel, an inorganic Ca2+ influx blocker, but was unaffected by nifedipine and omega-conotoxin, L- and N-type voltage-dependent Ca2+ channel blockers, respectively. Furthermore, the depolarization by 60 mM K+ did not affect the [Ca2+]i. These findings suggested that the NK2 and NK3 receptor-mediated [Ca2+]i increase was due to the activation of PLC and was dependent on the mobilization of internal Ca2+ and the entry of extracellular Ca2+ through voltage-independent channels. This study showed that the C1300 cell line is a useful system with which to investigate pharmacological functions and signaling pathways of endogenous NK2 and NK3 receptors.
Fukuhara
S
S
Institute of Applied Biochemistry, University of Tsukuba, Ibaraki, Japan.
Mukai
H
H
Kako
K
K
Nakayama
K
K
Munekata
E
E
D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002118:Calcium / Q000502:physiology*; D004963:Estrenes / Q000494:pharmacology; D015870:Gene Expression / Q000502:physiology; D006868:Hydrolysis; D051379:Mice; D009447:Neuroblastoma; D010716:Phosphatidylinositols / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D018041:Receptors, Neurokinin-2 / Q000737:chemistry / Q000235:genetics*; D018042:Receptors, Neurokinin-3 / Q000737:chemistry / Q000235:genetics*; D015398:Signal Transduction / Q000502:physiology*; D015320:Tachykinins / Q000502:physiology; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000502:physiology
D004963:Estrenes; D010716:Phosphatidylinositols; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D012333:RNA, Messenger; D018041:Receptors, Neurokinin-2; D018042:Receptors, Neurokinin-3; D015320:Tachykinins; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D002118:Calcium