Abstract
Purpose :
The Schlemm's canal inner wall endothelial cells (SCE) are the last barrier to the outflow of aqueous humor (AQH). SCE responds to shear stress and stretch resulting from IOP elevation by upregulating nitric oxide (NO) to increase the permeability of the Schlemm's canal inner wall. In endothelial cells, NOS3-mediated formation of NO from arginine paradoxically requires extracellular arginine even with sufficient intracellular arginine ("arginine paradox"). A resolution for this paradox in endothelial cells is the preferential delivery of arginine to NOS3 by its interaction with arginine transporter SLC7A1 within caveolae. Thus, SLC7A1 is likely to play a critical role in outflow physiology; hence we have determined the role of SLC7A1 in AQH outflow regulation.
Methods :
We first determined the localization of SLC7A1 in the Schelmm's canal in mice. Next, using the proximity ligation technique, we determined if NOS3 and SLC7A1 are associated with each other and in caveolae labeled with CAV1. We next determined the effect of inhibiting SLC7A1 arginine transport using lysine in wild-type and CAV1 null mouse eyes on aqueous humor outflow to determine if the transporter plays a role in outflow physiology and if caveolar sequestration of NOS3 and SLC7A1 affects efficient aqueous humor outflow.
Results :
Our results show that SLC7A1 is expressed in the Schlemm's canal and that a population is associated with NOS3( 40nm proximity) and CAV1, a proxy for caveolae. The facility measured in control eyes was 3.99±0.75 nl/min/mmHg (95%CI mean: 3.55-4.4) compared to lysine-treated eyes to inhibit arginine transport, which was 3.14±0.78 nl/min/mmHg (95%CImean: 2.71-3.57) showing a 21% reduction (p=0.003). Moreover, there is a pressure-dependent reduction in aqueous humor outflow. The pressure-dependent reduction in outflow is abrogated in the CAV1 null background.
Conclusions :
Together, our results suggest that SLC7A1 plays a role in regulating outflow. At a molecular level, this is likely due to the close association of NOS3 and SLC7A1 in caveolae to satisfy the paradigm to explain the "arginine paradox." We are currently determining the effect of the loss of caveolae on the association of NOS3 and SLC7A1 since this has a physiological effect on outflow. In addition, we are testing the requirement from the arginine transporter directly by knocking out SLC7A1 in the Schlemm's canal inner wall using CRIPSR-based methods.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.