Independent coupling of the human tachykinin NK2 receptor to phospholipases C and A2 in transfected Chinese hamster ovary cells

Abstract

The human tachykinin NK₂ receptor stably expressed in Chinese hamster ovary cells (CHO-hNK₂R cells) was characterized by studying the effect of neurokinin A (NKA), the preferred natural ligand, and that of other agonists and antagonists in both binding experiments and functional assays. Competition experiments using [¹²⁵I]NKA showed that CHO-hNK₂R cells express binding sites which have high affinity for NKA (K _i=3.4±0.9 nM), GR 64349 (K _i=12±3 nM) and [βAla⁸]NKA(4–10) (K _i=21±8 nM) and for the antagonists MEN 10627 (K _i=0.55±0.2 nM), and MEN 11420 (K _i=2.4±0.8 nM). In contrast, the tachykinin NK₁ and NK₃ receptor agonists [Sar⁹,Met(O₂)¹¹]SP and senktide, respectively, were recognized with low affinity (K _i>10 μM). NKA (EC₅₀=68±18 nM) induced a rapid and concentration-dependent increase in the intracellular level of inositoltrisphosphate (IP₃). The concentration-response curve to GR 64349 (EC₅₀=155±14 nM) was close to that of NKA, whereas [βAla⁸]NKA(4–10) (EC₅₀=445±78 nM) and SP (EC₅₀=3197±669 nM) were 7- and 50-fold less potent, respectively. In addition, NKA stimulated the release of arachidonic acid and the production of prostaglandin E₂ (PGE₂) in a concentration-dependent manner. Also in this assay, NKA was found to be more potent than the other agonists tested (the EC₅₀ values were 3±0.3, 9±3, 7.8±0.9 and 217±37 nM for NKA, GR 64349, [βAla⁸]NKA(4–10) and SP, respectively). MEN 10627 and MEN 11420 were potent and competitive antagonists in blocking NKA-induced IP₃ formation and PGE₂ release: MEN 10627 and MEN 11420 displayed comparable potencies in blocking the two functional responses initiated by occupancy of the NK₂ receptor by NKA. Pretreatment of the cells with pertussis toxin (500 ng/ml for 18 h) did not significantly modify the basal or stimulated phosphatidylinositol turnover but reduced the basal and NKA-induced PGE₂ release by about 35%. The phospholipase C inhibitor U-73122 (10 μM) prevented the NKA-induced formation of IP₃ but did not affect PGE₂ release. Conversely, the phospholipase A₂ inhibitor quinacrine (100 μM) blocked the release of arachidonic acid and PGE₂ without affecting the NKA-stimulated formation of IP₃. Chelation of extracellular calcium with 3 mM EGTA inhibited the NKA-induced PGE₂ release by 81% but was without effect on basal and NKA-stimulated IP₃ production. The calcium channel blockers verapamil (10 μM) and ω-conotoxin GVIA (0.1 μM) did not modify the basal PGE₂ production and had no significant effect on the response to tachykinins while the blocker of non-selective cation channels, SKF-96365 (10 μM), inhibited the response to NKA by about 74%. SKF-96365 did not affect the basal or the NKA-induced IP₃ formation. In conclusion, our data demonstrate that the human tachykinin NK₂ receptor expressed in CHO cells displays binding affinity and functional properties which are those of a native NK₂ receptor. No pharmacological evidence for heterogeneity of the human NK₂ receptor was obtained in this study. Our findings indicate that the human tachykinin NK₂ receptor is independently coupled to both PLC and PLA₂ signaling pathways. Activation of the PLA₂ pathway may be linked to the opening of a voltage-independent cation channel which activates a Ca²⁺-dependent PLA₂.