Further Identification of Neurokinin Receptor Types and Mechanisms of Calcium Signaling Evoked by Neurokinins in the Murine Neuroblastoma C1300 Cell Line

Abstract: 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 µM neurokinin A was completely inhibited by SR 48968, an NK₂ receptor antagonist, whereas the partial response to 0.33 µM neurokinin B was unaffected, and the response was completely inhibited by SR 142801, an NK₃ receptor antagonist. In addition, the Ca²⁺]_i increase by 0.33 µM 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 µM neurokinin A in the presence of 1.0 µM SR 142801 or 1.0 µM 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 ω-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.