[3H]Bradykinin Receptor‐Binding, Receptor‐Recycling, and Receptor‐Internalization of the B2 Bradykinin Receptor in the Murine Osteoblast‐like Cell Line MC3T3‐E1

• Published in: Journal of bone and mineral research 1997-10, Vol.12 (10), p.1615-1625
• Main Authors: Windischhofer, Werner, Leis, Hans J.
• Format: Article
• Language: English
• Subjects: 3T3 Cells - drug effects; Animals; Binding, Competitive; Biological and medical sciences; Bradykinin - analogs & derivatives; Bradykinin - antagonists & inhibitors; Bradykinin - metabolism; Bradykinin Receptor Antagonists; Calcium - metabolism; Cell receptors; Cell structures and functions; Cytosol - metabolism; Dinoprostone - metabolism; Fundamental and applied biological sciences. Psychology; Mice; Molecular and cellular biology; Neuropeptide receptors; Osteoblasts - drug effects; Osteoblasts - metabolism; Receptors, Bradykinin - metabolism; Structure-Activity Relationship; Tritium
• ISSN: 0884-0431; 1523-4681
• DOI: 10.1359/jbmr.1997.12.10.1615

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.