Inhibition of type 4 phosphodiesterase by rolipram and Ginkgo biloba extract (EGb 761) decreases agonist-induced rises in internal calcium in human endothelial cells

The effects of Gingko biloba extract EGb 761 on 5 isolated, vascular, cyclic nucleotide phosphodiesterase (PDE) isoforms were evaluated. EGb 761 preferentially inhibited PDE4 (IC50=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 ([Ca2+](i)) levels in endothelial cells, we examined the effects of EGb 761 on both resting [Ca2+](i) levels and agonist-induced rises in [Ca2+](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 mu mol/L), and db-cAMP (100 mu mol/L) significantly inhibited histamine-, ATP-, and thrombin-induced [Ca2+](i) increases in HUVECs without modifying resting [Ca2+](i) levels. Similar results were obtained by using a Ca2+-free bath solution. EGb 761 (100 mg/L), but not rolipram (50 mu mol/L) or db-cAMP (100 mu mol/L), also inhibited Ca2+ influx into cells having thapsigargin-depleted internal Ca2+ stores and bathed in a Ca2+-free external solution. Our results are consistent with an inhibition of PDE activity that causes a reduction of agonist-induced increases in[Ca2+](i) in HUVECs, mainly by inhibition of Ca2+ mobilization from internal stores. It thus may be that the cardiovascular effects of EGb 761 involve inhibition of PDE4 activity and subsequent modification of Ca2+ signaling in endothelial cells.