Inhibition of Type 4 Phosphodiesterase by Rolipram and Ginkgo biloba Extract (EGb 761) Decreases Agonist-Induced Rises in Internal Calcium in Human Endothelial Cells

Abstract

Abstract —The effects of Gingko biloba extract EGb 761 on 5 isolated, vascular, cyclic nucleotide phosphodiesterase (PDE) isoforms were evaluated. EGb 761 preferentially inhibited PDE4 (IC 50 =25.1 mg/L), the isoform that is mainly present in endothelial cells, in a competitive manner ( K i =12.5 mg/L). Because changes in cyclic nucleotide levels may affect intracellular calcium ([Ca 2+ ] i ) levels in endothelial cells, we examined the effects of EGb 761 on both resting [Ca 2+ ] i levels and agonist-induced rises in [Ca 2+ ] i in single human umbilical vein endothelial cells (HUVECs) in culture. The effects of EGb 761 were compared with those of rolipram, a selective PDE4 inhibitor that increases cellular cAMP levels, and the cAMP analogue dibutyryl cAMP (db-cAMP). EGb 761 (20 and 100 mg/L), rolipram (50 μmol/L), and db-cAMP (100 μmol/L) significantly inhibited histamine-, ATP-, and thrombin-induced [Ca 2+ ] i increases in HUVECs without modifying resting [Ca 2+ ] i levels. Similar results were obtained by using a Ca 2+ -free bath solution. EGb 761 (100 mg/L), but not rolipram (50 μmol/L) or db-cAMP (100 μmol/L), also inhibited Ca 2+ influx into cells having thapsigargin-depleted internal Ca 2+ stores and bathed in a Ca 2+ -free external solution. Our results are consistent with an inhibition of PDE activity that causes a reduction of agonist-induced increases in [Ca 2+ ] i in HUVECs, mainly by inhibition of Ca 2+ mobilization from internal stores. It thus may be that the cardiovascular effects of EGb 761 involve inhibition of PDE4 activity and subsequent modification of Ca 2+ signaling in endothelial cells.