title: Bradykinin binding to B2 kinin receptors and stimulation of phosphoinositide turnover and arachidonic acid release in primary cultures of cells from late pregnant rat myometrium.
abstract: Primary cultures of cells from late pregnant rat myometrium contain B2 kinin receptors through which bradykinin (BK) stimulates inositol phosphate (InsP) formation and arachidonic acid (20:4) release. Equilibrium binding at 4 degrees C revealed that [3H]BK identified a maximal number of cell surface B2 kinin receptor binding sites on rat myometrial cells of 308 +/- 78 fmol/10(6) cells with apparently a single equilibrium dissociation constant of 1.8 +/- 0.2 nM. At 37 degrees C, [3H]BK binding was associated with a time-dependent decrease in the reversibility of the binding. This decrease was due in part to formation of slowly dissociating cell surface receptor [3H]BK binding and in part to internalization of the receptor-bound [3H]BK. Exposure of labeled cells to BK resulted in dose-dependent increases in [3H]InsP3, [3H]InsP2 ([3H]Ins(1,4)P2), and [3H]InsP1 ([3H]Ins(1)P1) formation and [3H]20:4 release. Pretreatment with 100 ng/mL pertussis toxin did not perturb BK stimulation of [3H]InsP formation but partially (approximately 30%) inhibited BK stimulation of [3H]20:4 release. BK stimulation of [3H]20:4 release was directly proportional to the number of receptor sites occupied by BK. In contrast, stimulation of [3H]InsP formation required a threshold level of receptor occupancy, which decreased as a function of time of BK exposure. These results show that BK interacts with B2 kinin receptors on rat myometrial cells with apparently a single affinity through which BK stimulates [3H]InsP formation and [3H]20:4 release. BK stimulation of [3H]InsP formation requires a threshold BK concentration, which decreases with time, and we suggest that the decrease is due to a time-dependent formation of a BK receptor binding state from which BK slowly dissociates.
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
authors.forename: M M
authors.initials: MM
authors.lastname: Tropea
authors.forename: C M
authors.initials: CM
authors.lastname: Munoz
authors.forename: L M
authors.initials: LM
authors.lastname: Leeb-Lundberg
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D001920:Bradykinin / Q000378:metabolism*; D002478:Cells, Cultured; D005260:Female; D007295:Inositol Phosphates / Q000378:metabolism; D009215:Myometrium / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D011247:Pregnancy; D011270:Pregnancy, Animal / Q000378:metabolism*; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter / Q000378:metabolism*
chemical_list: D007295:Inositol Phosphates; D010716:Phosphatidylinositols; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter; D016718:Arachidonic Acid; D001920:Bradykinin