Signal transduction mechanisms of recombinant bovine neurokinin-2 receptor stably expressed in baby hamster kidney cells.
The bovine neurokinin-2 (NK-2) receptor gene was stably transfected into Baby hamster kidney (BHK-21) fibroblasts and one recombinant clone expressing 17,700 high-affinity [125I]neurokinin A (NKA) binding sites/cell characterized further. [125I]NKA binding was displaced by unlabeled NKA with an IC50 of 8.26 +/- 2 nM (n = 5) and with the rank order of potency NKA > neurokinin B (NKB) > Substance P (SP) confirming pharmacological characteristics of an NK-2 receptor subtype. Stimulation with NKA resulted in a rapid and dose-dependent increase in inositol 1,4,5-trisphosphate (IP3) levels (EC50 = 32 +/- 10 nM; n = 7) which was paralleled by a transient biphasic rise in intracellular free calcium concentration [Ca2+]i (EC50 = 35 +/- 20 nM; n = 3). In addition to phosphoinositide (PI) hydrolysis and Ca2+ mobilization, NKA was found to stimulate both cyclic AMP formation (EC50 = 1.02 +/- 0.26 microM; n = 7) and [3H]arachidonic acid mobilization (EC50 = 0.65 +/- 0.45 microM; n = 4). Interestingly, cyclic AMP levels also rose after addition of an exogenous arachidonic acid metabolite, prostaglandin E2 (PGE2) (EC50 = 11.5 +/- 2 microM). Similar observations of NKA-induced IP3 production, Ca2+ mobilization, arachidonic acid liberation, and cAMP formation have been made previously following expression of the bovine NK-2 receptor in Chinese hamster ovary (CHO) epithelial cells. The present results suggest that activation of NK-2 receptors leads to characteristic and reproducible intracellular second messenger responses in a subclass of cell types which includes fibroblasts and epithelial cells irrespective of their genetic and phenotypic background.
Glaxo Institute for Molecular Biology S.A., Plan-les-Ouates, Geneva, Switzerland.
H R
HR
Eistetter
A
A
Mills
S J
SJ
Arkinstall
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D005347:Fibroblasts / Q000502:physiology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D018041:Receptors, Neurokinin-2; D017981:Receptors, Neurotransmitter / Q000096:biosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection
D018041:Receptors, Neurokinin-2; D017981:Receptors, Neurotransmitter; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D015544:Inositol 1,4,5-Trisphosphate; D000242:Cyclic AMP; D002118:Calcium