Functional expression of B2 bradykinin receptors from Balb/c cell mRNA in Xenopus oocytes.
The murine BALB/c 3T3 fibroblast clone SV-T2 (3T3 cells) expresses receptors for the nonapeptide bradykinin. In these cells, bradykinin stimulates both inositol phosphate (InsP) formation and arachidonic acid release by independently activating phospholipase C and phospholipase A2, respectively. These actions of bradykinin are mediated by a receptor(s) coupled to pertussis toxin-insensitive guanine nucleotide-binding proteins. Bradykinin-stimulated increases in InsP lead to the mobilization of intracellular Ca2+. We examined the expression of 3T3 receptors for bradykinin in oocytes from Xenopus laevis, cells capable of in vitro expression of foreign mRNA for receptors coupled to the mobilization of Ca2+. Poly(A)+ mRNA was prepared from 3T3 cells and expression of receptors for bradykinin was demonstrated by agonist-mediated stimulation of 45Ca2+ efflux from oocytes injected with 50 ng of poly(A)+ RNA. Bradykinin-stimulated efflux of 45Ca2+ was dose dependent (EC50 = 15 nM) and blocked by the specific mixed B1,B2 bradykinin antagonist NPC 567 but not by the B1 antagonist desArg9[Leu8]bradykinin. Size fractionation of 3T3 poly(A)+ RNA on a sucrose gradient demonstrated a single peak of bradykinin-stimulated 45Ca2+ efflux, with an approximate mRNA size of 4.5 kilobases. Bradykinin-stimulated 45Ca2+ efflux in size-fractionated mRNA was clearly separable from response to [Arg]vasopressin at another receptor linked to InsP formation and Ca2+ mobilization in 3T3 cells.
Mahan
L C
LC
Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 20892.
Burch
R M
RM
D000818:Animals; D001920:Bradykinin / Q000031:analogs & derivatives / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D051379:Mice; D008807:Mice, Inbred BALB C; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics*; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics*; D014162:Transfection; D014982:Xenopus laevis / Q000235:genetics*
D012333:RNA, Messenger; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter; C053010:NPC 567; D001920:Bradykinin; D002118:Calcium