Functional expression of the bradykinin-B2 receptor cDNA in Chinese hamster lung CCL39 fibroblasts.
The bradykinin (BK) B2 receptor cDNA was synthesized by rt-PCR and transfected into the Chinese hamster lung fibroblasts, CCL39. The CCL39 do not contain the mRNA for this receptor and do not bind BK. Clones of transfected cells were screened for BK receptor mRNA, binding of BK, and for [Ca2+]i response to BK. The clones showed various levels of receptor mRNA. Scatchard analysis of three clones, B6, B5 and B1, each gave a Kd of approximately 1.0nM while the Bmax for each clone differed at 320, 38.7, and 5.39 fmoles per 10(6) cells respectively. The [Ca2+]i response of the three clones to BK decreased with the receptor number/cell. Thus, levels of mRNA, BK binding and [Ca2+]i response proved proportionally related in the transfected clones. The actions of BK and alpha-thrombin, which has an endogenous receptor in these cells, were assessed in clone B6. BK proved active but also distinct from thrombin. BK at 10nM and thrombin at 2units/ml both effectively increased cytosolic [Ca2+]i. BK at 10nM stimulated PGE2 production three fold over basal, while thrombin only marginally elevated PGE2 levels. Alone, BK stimulated a small increase in 3H-thymidine incorporation into DNA. However, in combination with insulin, BK stimulated DNA synthesis to 76% of thrombin, a potent mitogen in these cells. These results illustrate that the BK-B2 receptor cDNA can be stably transfected into a mammalian cell and can activate transmembrane signalling pathways.
D000818:Animals; D015152:Blotting, Northern; D001920:Bradykinin / Q000378:metabolism / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000096:biosynthesis; D015232:Dinoprostone / Q000096:biosynthesis; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D008168:Lung; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism; D014162:Transfection
D007328:Insulin; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter; D012313:RNA; D004247:DNA; D013917:Thrombin; D015232:Dinoprostone; D001920:Bradykinin; D002118:Calcium; D013936:Thymidine