Abstract 15975: Transmembrane Transport of Nitric Oxide in Endothelial Cells
Nitric oxide (NO) is a key endothelial cell (EC) signaling molecule that exerts its actions both within the EC and within heterologous target cells. Most investigators have assumed that NO traverses the plasma membrane of the EC by simple diffusion owing to its relative lipophilicity; however, the possibility of transmembrane channel-dependent transport has not been explored in any detail. In this study, we used bovine aortic ECs to test whether the water channel, aquaporin 1 (AQP1), and glucose transporters, could promote NO efflux from and influx into the EC. We measured NO efflux by activating EC NO synthase with 3 uM A23187, and assaying L-NAME-inhibited NO released into the media by nitrite/nitrate determination. We measured NO influx by incubating ECs with DPTA NONOate after loading the cells with the NO-sensitive intracellular fluorophore, DAF-FM. Using Western blot analysis, AQP1 mRNA was detected by quantitative RT-PCR (in human ECs), but protein expression was only detected with overexpression by transfection using a pCMV vector containing the human AQP1 cDNA. Following activation, EC released 24.6 ± 2.1 nmol NO/mg protein/6 hr; 20 uM HgCl2, an AQP1 inhibitor, and 20 uM phloretin, a facilitated glucose transporter inhibitor, reduced NO efflux to 2.3 ± 0.07 and 0.8 ± 0.2 nmol/mg protein/6 hr, respectively (p<0.05 for both treatments compared to their respective controls). Treatment of cells with siRNA to AQP1 and to GLT2 (the facilitated glucose transporter, glut 2) each reduced NO efflux by ∼50%, while overexpression of AQP1 increased efflux slightly to 27.7 ± 1.3 nmol/mg protein/6 hr. In response to treatment of EC with 300 uM DPTA NONOate, NO influx was 9.8 ± 1.8 arbitrary fluorescence units/104 cells/2 hr, which was reduced to 6.3 ± 1.2 with siRNA knockdown of AQP1 (p<0.05) and increased to 15.7 ± 2.5 with overexpression of AQP1 (p<0.05); siRNA knockdown of Glut2 had no effect on NO influx. These results demonstrate that both AQP1 and GLT2 participate in NO flux in ECs, and suggest that the transfer of NO across the EC plasma membrane can proceed via specific membrane channel mechanisms.
- © 2010 by American Heart Association, Inc.