Tropea M M, Munoz C M, Leeb-Lundberg L M
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Can J Physiol Pharmacol. 1992 Oct;70(10):1360-71. doi: 10.1139/y92-191.
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.
来自妊娠晚期大鼠子宫肌层的细胞原代培养物含有B2激肽受体，缓激肽（BK）可通过该受体刺激肌醇磷酸（InsP）的形成和花生四烯酸（20:4）的释放。4℃下的平衡结合实验表明，[3H]BK可识别大鼠子宫肌层细胞表面最大数量的B2激肽受体结合位点，为308±78 fmol/10(6)细胞，其表观平衡解离常数为1.8±0.2 nM。在37℃时，[3H]BK结合与结合的可逆性随时间的下降有关。这种下降部分是由于形成了缓慢解离的细胞表面受体[3H]BK结合物，部分是由于受体结合的[3H]BK的内化。将标记细胞暴露于BK会导致[3H]InsP3、[3H]InsP2（[3H]Ins(1,4)P2）和[3H]InsP1（[3H]Ins(1)P1）的形成以及[3H]20:4的释放呈剂量依赖性增加。用100 ng/mL百日咳毒素预处理不会干扰BK对[3H]InsP形成的刺激，但会部分（约30%）抑制BK对[3H]20:4释放的刺激。BK对[3H]20:4释放的刺激与BK占据的受体位点数量成正比。相反，[3H]InsP形成的刺激需要一定的受体占据阈值水平，该阈值水平会随着BK暴露时间的延长而降低。这些结果表明，BK与大鼠子宫肌层细胞上的B2激肽受体相互作用，其亲和力明显单一，通过该受体BK刺激[3H]InsP的形成和[3H]20:4的释放。BK刺激[3H]InsP形成需要一定的BK浓度阈值，该阈值会随时间降低，我们认为这种降低是由于BK受体结合状态随时间形成，BK从该状态缓慢解离所致。
