Implications of Methyltransferase on Prostacyclin Generation in Cultured Human Vascular Endothelial Cells

Abstract

The implications of phospholipid methyltransferase on prostacyclin (PGI₂) generation in cultured human vascular endothelial cells (HUVEC) were investigated. HUVECs were isolated and cultured from human umbilical cord veins. PGI₂ released from HUVEC was assayed by RIA, and cytosolic free Ca++ concentrations ([Ca⁺⁺]i) were measured using fura-2/AM. Results were as follows: Ca⁺⁺ ionophore A23187 (A23187, 10-6M), bradykinin (BK, 10-5M) or thrombin (Th, 10 unit/ml) increased [Ca⁺⁺]_i and PGI₂ generation. Pretreating HUVEC with EGTA had no effect on the basal [Ca⁺⁺]_i and PGI₂ levels, but suppressed their enhancement by A23187 or Th, and diminished them by BK. Pretreatment with 3-deazaadenosine (DAA, 10-4M, 30min) suppressed [Ca⁺⁺]_i increase and PGI₂ generation markedly by BK, and also inhibited effects induced by A23187 and Th.
These results suggest that basal PGI₂ generation was maintained mainly by mobilization from the Ca⁺⁺ pools, and its enhancement by BK was dependent on the influx of extracellular Ca⁺⁺. The activated phospholipid methyltransferase with the increase of [Ca⁺⁺]_i contributed to enhanced PGI₂ generation induced by A23187, BK and Th.