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

To gain further insights into the mechanism of the ATP-dependent interaction of P-glycoprotein (P-gp) with various multidrug resistance (MDR) reversal agents.

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.

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.

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.