Sphingosine‐1‐phosphate activates Rac in cultured endothelial cells using conditions that block acute inflammatory response in microvessels

Abstract

Elevation of endothelial cell intracellular cAMP using combined rolipram (10 µM) and forskolin (5 µM) (RF) blocked microvessel inflammation stimulated by platelet activating factor (PAF) or by bradykinin (Bk) (AJP Heart 294:H1188, 2008). Sphingosine‐1‐phosphate (S1P), believed to act through Rac activation, also attenuated PAF‐ or Bk‐induced acute inflammatory response. Direct activation of Rac using bacterial toxin reduced microvessel PAF response. Therefore we tested whether specific conditions known to attenuate PAF and Bk also induce Rac activation. Using cultured human dermal microvascular endothelial cells (HDMVEC) we compared the S1P dependent activation of Rac, and the Rac activation induced by treatment with RF with vehicle control. S1P (1 min) showed significant concentration (0.1 to 5 µM) dependent Rac activation. Rac activation (1 µM S1P) was maximal at 1 min and diminished over 30 min. In contrast, treatment with RF induced Rac activation greater at 10 to 30 min than at 1 to 5 min. Immunofluorescence demonstrated that both S1P and RF treatment strongly enhanced peripheral cortactin in HDMVEC and induced strong and continuous peripheral VE‐cadherin. The results demonstrate that S1P or RF treatments which we previously demonstrated to attenuate PAF‐ or Bk‐induced permeability in rat mesentery microvessels also significantly stimulate Rac activation and peripheral cortactin in HDMVEC. HL28607