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.