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

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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 °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/106 cells with apparently a single equilibrium dissociation constant of 1.8 ± 0.2 nM. At 37 °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 (~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.Key words: bradykinin, receptors, myometrium, inositol phosphates, arachidonic acid.

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