Characterization of bradykinin receptors in a human osteoblastic cell line.

Bradykinin receptor subtypes linked to prostaglandin release have been assessed in a human osteosarcoma cell line with osteoblastic phenotype (MG-63). Bradykinin (BK; 1 micromol/l) caused a burst of prostaglandin E(2) release that was maximal at 10 min. When the effect on the burst of PGE(2) and PGI(2) release by a variety of kinins and kinin analogues was assessed, the following rank order of response was found: Lys-BK>BK> or =Met-Lys-BK>Ile-Ser-BK>[Tyr(8)]-BK> or =[Hyp(3)]-BK>des-Arg(9)-BK=des-Arg(10)-Lys-BK=des-Arg(1)-BK, [Thi(5,8),D-Phe(7)]-BK=Sar-[D-Phe(8)]-des-Arg(9)-BK=Tyr-Gly-Lys-Aca-Lys-des-Arg(9)-BK. The rapid effect of BK on PGE(2) and PGI(2) release was unaffected by des-Arg(9)-[Leu(8)]-BK, des-Arg(10)-[Leu(9)]-Lys-BK and des-Arg(10)-[Hoe 140], but strongly inhibited by Hoe 140 in a concentration-dependent manner. When the incubation time was extended to 48 h, it was found that des-Arg(9)-BK and des-Arg(10)-Lys-BK caused a delayed enhancement of the formation of PGE(2). When PGE(2) formation was assessed in 24-h experiments, the following rank order of response was obtained: Tyr-Gly-Lys-Aca-Lys-des-Arg(9)-BK>BK=Lys-BK>des-Arg(10)-Lys-BK>Sar[D-Phe(8)]-des-Arg(9)-BK>des-Arg(9)-BK. The stimulatory effect of BK at 24 h was unaffected by des-Arg(9)-[Leu(8)]-BK, des-Arg(10)-[Leu(9)]-Lys-BK and des-Arg(10)-[Hoe 140] but inhibited by Hoe 140. The stimulatory effect of des-Arg(10)-Lys-BK in 24-h experiments was inhibited by des-Arg(9)-[Leu(8)]-BK, des-Arg(10)-[Leu(9)]-Lys-BK and des-Arg(10)-[Hoe 140]. Similarly, the stimulatory effects of Sar[D-Phe(8)]-des-Arg(9)-BK and Tyr-Gly-Lys-Aca-Lys-des-Arg(9)-BK was inhibited by des-Arg(10)-[Hoe 140]. The following rank order of response was seen for inhibition of [3H]-BK binding to MG-63 cells: Lys-BK=BK=Hoe 140>des-Arg(10)-Hoe 140=des-Arg(10)-Lys-BK=des-Arg(9)-BK=Tyr-Gly-Lys-Aca-Lys-des-Arg(9)-BK. Using [3H]-des-Arg(10)-Lys-BK, the following rank order of response for inhibition of binding was seen: des-Arg(10)-Lys-BK=Tyr-Gly-Lys-Aca-Lys-des-Arg(9)-BK>des-Arg(10)-Hoe 140>des-Arg(9)-BK=Lys-BK=BK=Hoe 140. MG-63 cells expressed mRNAs for BK B1 and B2 receptors, as assessed by RT-PCR. These data indicate that the human osteoblastic osteosarcoma cell line MG-63 is equipped with functional BK receptors of both B1 and B2 receptor subtypes. The B2 receptors are linked to a burst of prostanoid release, whereas the B1 receptors mediate a delayed prostaglandin response, indicating that the two receptor subtypes are linked to different signal transducing mechanisms or that the molecular mechanisms involved in prostaglandin release are different.