INDEPENDENT COUPLING OF THE HUMAN TACHYKININ NK2 RECEPTOR TO PHOSPHOLIPASE-C AND PHOSPHOLIPASE-A(2) IN TRANSFECTED CHINESE-HAMSTER OVARY CELLS

The human tachykinin NK2 receptor stably expressed in Chinese hamster ovary cells (CHO-hNK(2)R cells) was characterized by studying the effe ct of neurokinin A (NKA), the preferred natural ligand, and that of ot her agonists and antagonists in both binding experiments and functiona l assays. Competition experiments using [I-125]NKA showed that CHO-hNK (2)R 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 [beta Ala(8)]NKA(4-10) (K -i=21+/-8 nM) and for the antagonists MEN 10627 (K-i=0.55+/-0.2 nM), a nd MEN 11420 (K-i=2.4+/-0.8 nM). In contrast, the tachykinin NK1 and N K3 receptor agonists [Sar(9),Met(O-2)(11)]SP and senktide, respectivel y, were recognized with low affinity (K-i>10 mu M) NKA (EC50=68+/-18 n M) induced a rapid and concentration-dependent increase in the intrace llular level of inositoltrisphosphate (IP3). The concentration-respons e curve to GR 64349 (EC50=155+/-14 nM) was close to that of NKA, where as [beta Ala(8)]NKA(4-10) (EC50=445+/-78 nM) and SP (EC50=3197+/-669 n M) were 7- and 50-fold less potent, respectively. In addition, NKA sti mulated the release of arachidonic acid and the production of prostagl andin E-2 (PGE(2)) in a concentration-dependent manner. Also in this a ssay, 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, [beta Ala(8)]NKA(4-10) and SP, respectively). MEN 10627 an d MEN 11420 were potent and competitive antagonists in blocking NKA-in duced IP3 formation and PGE(2) release: MEN 10627 and MEN 11420 displa yed comparable potencies in blocking the two functional responses init iated by occupancy of the NK2 receptor by NKA. Pretreatment of the cel ls with pertussis toxin (500 ng/ml for 18 h) did not significantly mod ify the basal or stimulated phosphatidylinositol turnover but reduced the basal and NKA-induced PGE(2) release by about 35%. The phospholipa se C inhibitor U-73122 (10 mu M) prevented the NKA-induced formation o f IP3, but did not affect PGE(2) release. Conversely, the phospholipas e A(2) inhibitor quinacrine (100 mu M) blocked the release of arachido nic acid and PGE(2) without affecting the NKA-stimulated formation of IP3. Chelation of extracellular calcium with 3 mM EGTA inhibited the N KA-induced PGE(2) release by 81% but was without effect on basal and N KA-stimulated IP3 production. The calcium channel blockers verapamil ( 10 mu M) and omega-conotoxin GVIA (0.1 mu M) did not modify the basal PGE(2) production and had no significant effect on the response to tac hykinins while the blocker of non-selective cation channels, SKF-96365 (10 CIM), inhibited the response to NKA by about 74%. SKF-96365 did n ot affect the basal or the NKA-induced IP3 formation. In conclusion, o ur data demonstrate that the human tachykinin NK2 receptor expressed i n CHO cells displays binding affinity and functional properties which are those of a native NK2 receptor. No pharmacological evidence for he terogeneity of the human NK2 receptor was obtained in this study. Our findings indicate that the human tachykinin NK2 receptor is independen tly coupled to both PLC and PLA(2) signaling pathways. Activation of t he PLA(2) pathway may be linked to the opening of a voltage-independen t cation channel which activates a Ca2+-dependent PLA(2).