Abstract 15677: Facilitated 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. It has been assumed that NO traverses the plasma membrane of the EC by simple diffusion owing to its relative lipophilicity, and that, in addition, aquaporin 1 (AQP1) facilitates NO transport. In this report, we employed other eNOS activators to rule out the membrane effect of A23187. We measured NO efflux by assaying NO released into the media by nitrite/nitrate determination, correcting for the fraction that was inhibited by L-NAME. To activate eNOS (without the membrane disruption accompanying calcium ionophore), we used acetylcholine (Ach) at 3x10-4 M, methacholine (Mch) at 3x10-4 M, serotonin (5HT) at 3x10-4 M, bradykinin (BK) at 3x10-5 M, substance P (SP) at 3x10-5 M, and 1.5 mM ATP. We found that none of them alone can induce detectable nitrite level over 6 hours of incubation. However, a combination of 3x10-5 M BK and 1.5 mM ATP generated 0.94 ± 0.44 nmol NO/mg protein/6 hrs. Given these results, we next used the combination of BK and ATP was used to induce NO efflux in bovine aortic ECs in order to assess the role of selected transporters in facilitating NO release. We knocked down AQP1, Glut 2 (facilitated glucose transporter 1), or UTB1 (urea transporter B1) with siRNA to each, and found that NO effluxes were 0.09 ± 0.14, 0.18 ± 0.29, and 0.22 ± 0.34 nmoles/mg protein/6 hrs, respectively, whereas SGLT1 (sodium-dependent glucose transporter 1), SGLT2, siRNA scrambled sequence, and buffer control were 0.81 ± 0.78, 0.54 ± 0.70, 0.68 ± 0.63, and 0.99 ± 0.17 nmoles/mg protein/6 hrs, respectively. Nitric oxide efflux at one hour was increased ~ 2-fold by overexpression of SGLT2 and UTB1 in BAECs, as compared to control. These results demonstrate that AQP1, Glut 2, and UTB1 play a role in NO flux in ECs, and suggest that the transfer of NO across the EC plasma membrane can be facilitated via specific membrane channel-dependent mechanisms.
- © 2011 by American Heart Association, Inc.