Adenosine protected against pulmonary edema through transporter- and receptor A2-mediated endothelial barrier enhancement

Abstract

We have previously demonstrated that adenosine plus homocysteine enhanced endothelial basal barrier function and protected against agonist-induced barrier dysfunction in vitro through attenuation of RhoA activation by inhibition of isoprenylcysteine-O-carboxyl methyltransferase. In the current study, we tested the effect of elevated adenosine on pulmonary endothelial barrier function in vitro and in vivo. We noted that adenosine alone dose dependently enhanced endothelial barrier function. While adenosine receptor A₁ or A₃ antagonists were ineffective, an adenosine transporter inhibitor, NBTI, or a combination of DPMX and MRS1754, antagonists for adenosine receptors A_2A and A_2B, respectively, partially attenuated the barrier-enhancing effect of adenosine. Similarly, inhibition of both A_2A and A_2B receptors with siRNA also blunted the effect of adenosine on barrier function. Interestingly, inhibition of both transporters and A_2A/A_2B receptors completely abolished adenosine-induced endothelial barrier enhancement. The adenosine receptor A_2A and A_2B agonist, NECA, also significantly enhanced endothelial barrier function. These data suggest that both adenosine transporters and A_2A and A_2B receptors are necessary for exerting maximal effect of adenosine on barrier enhancement. We also found that adenosine enhanced Rac1 GTPase activity and overexpression of dominant negative Rac1 attenuated adenosine-induced increases in focal adhesion complexes. We further demonstrated that elevation of cellular adenosine by inhibition of adenosine deaminase with Pentostatin significantly enhanced endothelial basal barrier function, an effect that was also associated with enhanced Rac1 GTPase activity and with increased focal adhesion complexes and adherens junctions. Finally, using a non-inflammatory acute lung injury (ALI) model induced by α-naphthylthiourea, we found that administration of Pentostatin, which elevated lung adenosine level by 10-fold, not only attenuated the development of edema before ALI but also partially reversed edema after ALI. The data suggest that adenosine deaminase inhibition may be useful in treatment of pulmonary edema in settings of ALI.