Functional studies of bradykinin receptors in Chinese hamster ovary cells stably expressing the human B2 bradykinin receptor.
Bradykinin B1 and B2 receptors, members of the G-protein coupled receptor superfamily, are involved in inflammation and pain. Chinese hamster ovary (CHO) cells stably expressing the human B2 bradykinin receptor (CHO-B2) were used to characterize the signal transduction pathways associated with this receptor and its regulation. The selective B2 antagonist [3H]NPC17731 but not the selective B1 antagonist [3,4-prolyl-3,4-(3)H(N)]-[des-Arg10,Leu9]kallidin ([3H]DALKD) bound to CHO-B2 cell membranes with a Kd of 0.77 nM and a Bmax of 1087 fmol/mg protein. [3H]NPC17731 binding was inhibited by bradykinin ligands in the order: NPC17731 > bradykinin > kallidin >> DALKD > [des-Arg10] kallidin (DAKD), consistent with the pharmacological profile of B2 bradykinin receptors. The B2 agonist bradykinin and the B1/B2 agonist kallidin, but not the B1 agonist DAKD, increased [35S]GTP gamma S binding to the CHO-B2 cell membranes. The B2 bradykinin receptors were co-immunoprecipitated with G alpha q/11. In response to bradykinin stimulation, coupling of the B2 receptors to G alpha q/11 was increased by 10-fold. Bradykinin and kallidin, but not DAKD, induced intracellular calcium release in CHO-B2 cells, which was blocked by NPC17731 but not by DALKD. These results demonstrate that B2 bradykinin receptors directly coupled to G alpha q/11 to regulate intracellular calcium release. CHO-B2 cell is a useful system that can be applied to study the effect of potential agents that may influence the B2 receptor function.
Zhang
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SP
Drug Discovery Research, R.W. Johnson Pharmaceutical Research Institute, Welsh and McKean Roads, Spring House, PA 19477-0776, USA. spzhang@prius.jnj.com
Wang
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HY
Lovenberg
T W
TW
Codd
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EE
D000818:Animals; D065168:Bradykinin Receptor Antagonists; D016466:CHO Cells; D020013:Calcium Signaling / Q000187:drug effects; D002462:Cell Membrane / Q000502:physiology; D006224:Cricetinae; D015870:Gene Expression; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007609:Kallidin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000819:agonists / Q000235:genetics* / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects
D065168:Bradykinin Receptor Antagonists; D008024:Ligands; D009842:Oligopeptides; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; C082229:bradykinin, Lys-Leu(8)-desArg(9)-; C071327:NPC 17731; D007609:Kallidin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C052787:kallidin, des-Arg(10)-