[3H]bradykinin receptor-binding, receptor-recycling, and receptor-internalization of the B2 bradykinin receptor in the murine osteoblast-like cell line MC3T3-E1.

Abstract

Bradykinin (BK) has been demonstrated to induce inositol phosphate production, release of intracellular Ca2+, and prostaglandin E2 (PGE2) synthesis in the murine osteoblast-like cell line MC3T3-E1. Because cellular response to BK is a function of receptor affinity, receptor coupling, and receptor recycling, we investigated kinetic properties, specificity, and regulation at the BK-receptor level on intact, BK-sensitive MC3T3-E1 cells. Our results clearly demonstrate the existence of a single category of binding sites for [3H]BK (kD =366+/-98 pM; Bmax =45.3+/-6.6 fmol/mg of protein). Displacement studies with various BK analogs gave a rank order compatible with a B2 BK-receptor type (BK > Lys-BK > [Hyp3]-BK > Met-Lys-BK > HOE140 > Tyr-BK > Tyr8-BK > D-Arg, [Hyp3, Thi5,8, D-Phe7]-BK > [D-Phe7]-BK > des-Arg9-BK > des-Arg9, [Leu8]-BK = angiotensin II). No atypic high-affinity binding sites for the B1 receptor agonist des-Arg9-BK could be observed. Prestimulation of MC3T3-E1 cells with BK resulted in the disappearance of accessible B2 receptors at the cell surface by internalization. Postexposure of BK-pretreated cells to ligand-free medium resulted in almost complete receptor restoration within 30 minutes, exhibiting an intermediate state of two categories of binding sites (kD1 =444+/-37 pM, Bmax1 =9.2+/-0.3 fmol/mg of protein and kD2 =2.7+/-0.28 pM, Bmax2 =24.2+/-0.2 fmol/mg of protein), probably representing coupled and uncoupled B2 receptors. Prolonged stimulation with BK (2.5-5 h) also revealed the temporal occurrence of two categories of binding sites after 2.5 h (kD1 =228+/-3.5 pM; Bmax1 =15.6+/-0.6 fmol/mg of protein; kD2 =2.7+/-0.25 nM; Bmax2 =40.7+/-1.5 fmol/mg of protein), whereas low-affinity binding sites disappeared after 5 h.