INHIBITION OF ANNEXIN V-DEPENDENT CA2-BENZOTHIAZEPINE DERIVATIVE( MOVEMENT IN LARGE UNILAMELLAR VESICLES BY K201, A NEW 1,4)

Examination was made of the effect of annexin V on Ca2+ movement into large unilamellar vesicles (LUV) using fura-2, a calcium-sensitive flu orescent dye. To avoid the possible difficulties relating to the addit ion of annexin V and/or Ca2+ in fura-2-loaded LUV, the burst method wa s used. LUV, preincubated with rat annexin V in the presence of Ca2+ w ere collected by centrifugation and resuspended, and then burst with T riton X-100 in the presence of fura-2. Inward Ca2+ movement across the artificial lipid membrane was measured by determination of fura-2 flu orescence due to the leaked Ca2+ from ruptured LUV. The observed Ca2signal increased dependent on annexin V and Ca2+ concentrations, where as bovine serum albumin did not affect this signal up to 1 mu M. Thus, annexin V shows Ca2+ channel activity in LUV. K201, a novel 1,4-benzo thiazepine, inhibited inward Ca2+ movement into LUV caused by annexin V in a dose-dependent manner. In the presence of 50 nM annexin V and 4 00 mu M Ca2+, 3 mu M K201 showed significant inhibition of Ca2+ moveme nt due to annexin V, and 50% inhibition was achieved at 25 mu M K201. On the other hand, diltiazem had no such effect even at 30 mu M. K201 is thus shown to have inhibitory activity on inward Ca2+ movement due to annexin V in artificial vesicles and may prove useful as a probe fo r elucidating the functions of annexin V in vivo. (C) 1997 Elsevier Sc ience B.V.