Zini J M, Schmaier A H, Cines D B
Department of Medicine (Hematology/Oncology), Hospital of the University of Pennsylvania, Philadelphia 19104.
Blood. 1993 Jun 1;81(11):2936-46.
The vasoactive compound bradykinin (BK) is liberated by proteolytic cleavage from high molecular weight kininogen (HK) and low molecular weight kininogen (LK). Expression of kininogens on cell surface receptors may affect the delivery of BK at sites of inflammation. Therefore, we investigated whether BK itself alters the expression of binding sites for its parent molecules, HK and LK, on the surface of cultured human umbilical vein endothelial cells (HUVEC). 125I-LK and 125I-HK each bind to a single class of sites on HUVEC in reactions that are saturable, reversible, and zinc-dependent (Bmax = 9.7 +/- 0.2 x 10(5) sites/cell; kd = 43.3 +/- 8 nmol/L; n = 5 and Bmax = 10.3 +/- 0.4 x 10(5) sites/cell; kd = 40.3 +/- 0.9 nmol/L; n = 3 for LK and HK, respectively). HK and LK compete for the same binding site (Ki = 19.4 +/- 5 nmol/L HK v 125I-LK; Ki = 24.5 +/- 4 nmol/L LK v 125I-HK, n = 3). Moreover, 50-fold molar excess light chain of HK inhibits 125I-LK binding 51% and 50-fold molar excess LK and the heavy chain of HK inhibit 125I-light chain of HK binding 92% and 76%, respectively. Preincubation of HUVEC with BK produces a transient, concentration-dependent increase in the binding of HK and LK, reaching a maximum 3 to 4 hours after addition of BK (46% increase over control for HK; 57% increase over control for LK; P < .005 for each ligand). Des-Arg9-bradykinin, a B1 receptor agonist, increases kininogen binding to the same extent as BK; the upregulation of kininogen binding sites by BK is partially blocked by a B1 but not by a B2 receptor antagonist. The protein kinase C inhibitors (PKC), sphingosine and H7, completely block the induction of HK receptors by BK. Phorbol 12-myristate 13-acetate (PMA), which also activates PKC, stimulates the binding of HK and the purified light chain of HK to HUVEC as well. However, unlike HK and its light chain, binding of LK and the heavy chain of HK are increased by PMA only in the presence of added calcium ion. These studies show that BK upregulates a common binding site for HK, LK, and each chain of HK on HUVEC. Induction of kininogen receptors on endothelial cells by BK may modulate the generation of this vasoactive compound at sites of vascular injury.
血管活性化合物缓激肽（BK）通过蛋白水解从高分子量激肽原（HK）和低分子量激肽原（LK）中裂解产生。激肽原在细胞表面受体上的表达可能会影响BK在炎症部位的递送。因此，我们研究了BK本身是否会改变其母体分子HK和LK在培养的人脐静脉内皮细胞（HUVEC）表面的结合位点表达。125I-LK和125I-HK在HUVEC上均与一类单一的位点结合，反应具有饱和性、可逆性且依赖锌（Bmax = 9.7 +/- 0.2 x 10(5) 个位点/细胞；kd = 43.3 +/- 8 nmol/L；n = 5，对于LK和HK，Bmax分别为10.3 +/- 0.4 x 10(5) 个位点/细胞；kd = 40.3 +/- 0.9 nmol/L；n = 3）。HK和LK竞争相同的结合位点（Ki = 19.4 +/- 5 nmol/L HK对125I-LK；Ki = 24.5 +/- 4 nmol/L LK对125I-HK，n = 3）。此外，50倍摩尔过量的HK轻链抑制125I-LK结合51%，50倍摩尔过量的LK和HK重链分别抑制125I-HK轻链结合92%和76%。用BK预孵育HUVEC会使HK和LK的结合产生短暂的、浓度依赖性增加，在添加BK后3至4小时达到最大值（HK比对照增加46%；LK比对照增加57%；每种配体的P <.005）。去-精氨酸9-缓激肽，一种B1受体激动剂，与BK以相同程度增加激肽原结合；BK对激肽原结合位点的上调被B1受体拮抗剂部分阻断，但不被B2受体拮抗剂阻断。蛋白激酶C抑制剂（PKC）、鞘氨醇和H7完全阻断BK对HK受体的诱导。佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA），它也激活PKC，也刺激HK和HK纯化轻链与HUVEC的结合。然而，与HK及其轻链不同，LK和HK重链的结合仅在添加钙离子的情况下被PMA增加。这些研究表明，BK上调了HUVEC上HK-LK以及HK各链的共同结合位点。BK在内皮细胞上诱导激肽原受体可能会调节这种血管活性化合物在血管损伤部位的产生。
