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[ 3 H]Bradykinin Receptor‐Binding, Receptor‐Recycling, and Receptor‐Internalization of the B 2 Bradykinin Receptor in the Murine Osteoblast‐like Cell Line MC3T3‐E1
Author(s) -
Windischhofer Werner,
Leis Hans J.
Publication year - 1997
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1359/jbmr.1997.12.10.1615
Subject(s) - bradykinin , receptor , chemistry , internalization , bradykinin receptor , agonist , kallidin , microbiology and biotechnology , endocrinology , biochemistry , biology , kinin
Bradykinin (BK) has been demonstrated to induce inositol phosphate production, release of intracellular Ca 2+ , and prostaglandin E 2 (PGE 2 ) 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 [ 3 H]BK ( k D = 366 ± 98 pM; B max = 45.3 ± 6.6 fmol/mg of protein). Displacement studies with various BK analogs gave a rank order compatible with a B 2 BK‐receptor type (BK > Lys‐BK > [Hyp 3 ]‐BK > Met‐Lys‐BK > HOE140 > Tyr‐BK > Tyr 8 ‐BK > D‐Arg, [Hyp 3 , Thi 5,8 , D‐Phe 7 ]‐BK > [D‐Phe 7 ]‐BK > des‐Arg 9 ‐BK > des‐Arg 9 , [Leu 8 ]‐BK = angiotensin II). No atypic high‐affinity binding sites for the B 1 receptor agonist des‐Arg 9 ‐BK could be observed. Prestimulation of MC3T3‐E1 cells with BK resulted in the disappearance of accessible B 2 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 ( k D1 = 444 ± 37 pM, B max1 = 9.2 ± 0.3 fmol/mg of protein and k D2 = 2.7 ± 0.28 pM, B max2 = 24.2 ± 0.2 fmol/mg of protein), probably representing coupled and uncoupled B 2 receptors. Prolonged stimulation with BK (2.5–5 h) also revealed the temporal occurrence of two categories of binding sites after 2.5 h ( k D1 = 228 ± 3.5 pM; B max1 = 15.6 ± 0.6 fmol/mg of protein; k D2 = 2.7 ± 0.25 nM; B max2 = 40.7 ± 1.5 fmol/mg of protein), whereas low‐affinity binding sites disappeared after 5 h.