多药耐药逆转剂与血脑屏蔽上P—糖蛋白ATP酶活性间的相互作用

目的：进一步探讨P－糖蛋白（P-gp)与其多种耐药逆转剂量ATP依赖性相互作用的机制。方法：从牛脑灰质中分离得到微血管内皮细胞（BCEC）,制成细胞膜,定磷法测定BCEC膜上P-gp ATPas活性,结果：维拉帕米（Ver),长春新碱（VCR）,阿霉素（Dox),粉防己碱（Tet),蝙蝠葛碱（DRC）,小檗胺（BBM）以及蝙蝠葛苏林碱（DRS）增加基础P-gp ATPas活性,其Km值分别约为17,5．9,41,2．3,11,23和22μmol/L,小檗碱（BBR）仅有轻微的激活作用,延胡索乙素（dl-THP)和左旋四氢巴马汀（l-THP)不改变基础P-gp ATPas活性。环孢素A（CsA)抑制基因P-gp ATPas活性;竞争性抑制Ver或VCR激活的P-gp ATPas活性;非竞争性抑制Dox或Tet激活的P-gp ATPas活性。Dox非竞争性抑制Tet-激活的P-gp ATPas活性。结论：各种多药耐药逆转剂与P-gp 相互作用的机制及其对P-gp ATPas活性的影响各不相同,CsA,Ver和VCR在血脑屏蔽P-gp 上的结合部位可能是重叠的或者是有相互联系的,而CsA,Dox和Tet与P－gp的结合是相互独立的,并存在各自不同的结合部位。

Interaction of multidrug resistance reversal agents with P-glycoprotein ATPase activity on blood-brain barrier

AIM: To gain further insights into the mechanism of the ATP-dependent interaction of P-glycoprotein (P-gp) with various multidrug resistance (MDR) reversal agents. METHODS: Bovine brain capillary endothelial cells (BCEC) were isolated from cerebral gray matter using modifications of the mechanical homogenization technique. Plasma membranes were prepared from BCEC. The P- gp adenosine triphosphatase (ATPase) activity of the isolated BCEC membranes was estimated by measuring inorganic phosphate liberation. RESULTS: The basal P-gp ATPase activity was increased by verapamil (Ver), vincristine (VCR), doxorubicin (Dox), tetrandrine (Tet), dauricine (DRC), berbamine (BBM), and daurisoline (DRS), with respective half-maximal activity concentrations Km of about 17, 5.9, 41, 2.3, 11, 23, and 22 micromol/L. Berberine (BBR) produced a relatively slight activation. dl-Tetrahydropalmatine (dl-THP) and l-tetrahydropalmatine (l-THP ) does not alter the basal P-gp ATPase activity. Cyclosporin A (CsA) inhibited both the basal and the drug-stimulated ATPase activity of P-gp with high affinity. Kinetic analysis indicated a competitive inhibition of Ver- or VCR-stimulated ATPase activity and a noncompetitive inhibition of Dox- or Tet-activated ATPase activity by CsA. Moreover, Dox inhibited Tet-activated P-gp ATPase activity in a noncompetitive manner. CONCLUSION: Various MDR reversal agents could interact with P-gp and alter its ATPase activity in different manners. This is the result of the b road molecular recognition specificity of P-gp. CsA, Ver, and VCR could bin d P-gp either on overlapping sites or distant but interacting sites, while CsA, Dox, and Tet could independently bind P-gp on separated sites on blood-brain barrier.