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[3H]bradykinin receptor-binding, receptor-recycling, and receptor-internalization of the B2 bradykinin receptor in the murine osteoblast-like cell line MC3T3-E1.
J Bone Miner Res. 1997 Oct; 12(10):1615-25.JB

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.

Authors+Show Affiliations

University Childrens Hospital, Department of Biochemical Analysis and Mass Spectrometry, University of Graz, Austria.No affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9333122

Citation

Windischhofer, W, and H J. Leis. "[3H]bradykinin Receptor-binding, Receptor-recycling, and Receptor-internalization of the B2 Bradykinin Receptor in the Murine Osteoblast-like Cell Line MC3T3-E1." Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research, vol. 12, no. 10, 1997, pp. 1615-25.
Windischhofer W, Leis HJ. [3H]bradykinin receptor-binding, receptor-recycling, and receptor-internalization of the B2 bradykinin receptor in the murine osteoblast-like cell line MC3T3-E1. J Bone Miner Res. 1997;12(10):1615-25.
Windischhofer, W., & Leis, H. J. (1997). [3H]bradykinin receptor-binding, receptor-recycling, and receptor-internalization of the B2 bradykinin receptor in the murine osteoblast-like cell line MC3T3-E1. Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research, 12(10), 1615-25.
Windischhofer W, Leis HJ. [3H]bradykinin Receptor-binding, Receptor-recycling, and Receptor-internalization of the B2 Bradykinin Receptor in the Murine Osteoblast-like Cell Line MC3T3-E1. J Bone Miner Res. 1997;12(10):1615-25. PubMed PMID: 9333122.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [3H]bradykinin receptor-binding, receptor-recycling, and receptor-internalization of the B2 bradykinin receptor in the murine osteoblast-like cell line MC3T3-E1. AU - Windischhofer,W, AU - Leis,H J, PY - 1997/10/23/pubmed PY - 1997/10/23/medline PY - 1997/10/23/entrez SP - 1615 EP - 25 JF - Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research JO - J Bone Miner Res VL - 12 IS - 10 N2 - 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. SN - 0884-0431 UR - https://www.unboundmedicine.com/medline/citation/9333122/[3H]bradykinin_receptor_binding_receptor_recycling_and_receptor_internalization_of_the_B2_bradykinin_receptor_in_the_murine_osteoblast_like_cell_line_MC3T3_E1_ L2 - https://doi.org/10.1359/jbmr.1997.12.10.1615 DB - PRIME DP - Unbound Medicine ER -