Intraocular pressure was higher and pressure-dependent outflow in NOS3 KO mouse was lower than WT control (
Figs. 1,
2). It is consistent with results in WT mice perfused with NOS inhibitor L-NAME, which reduced C
con by 36% and the eNOS inhibitor cavtratin reduced the C
con in WT mice by 24%.
28 The authors also showed that eNOS is only expressed by SC and that cavtratin an eNOS inhibitor knocks down outflow facility equivalent to L-NAME. This suggests that eNOS protein is an important regulator of conventional aqueous humor outflow. This is consistent with findings using eNOS selective immunocytochemistry which showed that eNOS is the primary isozyme expressed in both SC cells and ciliary muscle fibers, which by ciliary tendon contraction and relaxing regulate the opening and compressing of TM/SC and aqueous humor outflow.
8,28 We previously demonstrated in a strain of eNOS overexpressed transgenic mice that an approximately 2-fold overexpression of eNOS resulted in 57% increase in conventional outflow facility.
1 eNOS is present in the particulate and cytosolic fractions of the endothelial cells.
29 It binds to caveolin-1 (CAV1) in the microdomains of the plasma membrane called caveolae. eNOS activity is inhibited by interaction with CAV1, which is regulated by Ca
2+/calmodulin.
30 In the eye, shear stress generated by aqueous humor outflow could either stimulate SC endothelial cells to release NO,
31 or induce phosphorylation of Ser1177/1179 of eNOS through phosphatidylinositol-3 (PI3) kinase and the downstream serine/threonine protein kinase Akt (protein kinase B), resulting in enhanced NO synthesis without any increase in intracellular Ca
2+.
32–34 However, with eNOS KO its regulatory role is significantly reduced as shown here in IOP and outflow facility (
Figs. 1,
2). Our immunostaining results (
Fig. 1) showed that in the eNOS KO mice there is still some expression of iNOS and nNOS in the TM and SC. Immunoreactivity against nNOS antibody in the epidermis showed that although eNOS was absent nNOS was still present in the tissue, and nNOS may play an compensatory role for eNOS in the brain
35 and vasculature.
36 Consistent with current literature findings in human donor eyes,
24 our data confirmed that NO-donor SNP and SNAP induced a greater pressure-dependent drainage in both WT and KO mouse eyes compared with vehicle-treated eyes (
Figs. 4,
5). In vivo study in monkey showed that NO donor SNP caused 77% increase in outflow facility.
23 In mouse eyes, the increase was greater (2.5- and 2.4-fold in WT and KO mice, respectively). The smaller magnitude of increase in conventional outflow in monkey might be due to difference in drug concentration, species, and dosing method. In the monkey study, SNP was given topically as an eye drop to the central cornea, which was almost equivalent to a drug concentration of 10
−3 M assuming 1% cornea penetration. We perfused mouse eyes using the same concentration as that of the monkey study of 10
−3 M, and with this drug concentration, outflow rate increased significantly at pressures greater than 15 mm Hg but there was little affect at 8 mm Hg. NO has a short half-life in the circulation of approximately 2 minutes. It is rapidly distributed to the extracellular space and is cleared from intraerythrocytic reaction with hemoglobin. However, the aqueous humor is free of hemoglobin, and the half-life of SNP would be considerably longer. From the flow readings, it had no signs of degradation or metabolism during the experiment. The flow rate was measured three times at each pressure level for 10 minutes each time and the readings were generally consistent. The effect of NO donor SNP and SNAP on IOP is variable between species and studies. In our study, topical application of SNP reduced IOP, whereas SNAP did not have a significant IOP-lowering effect. This is consistent with findings in a monkey study.
23 Kotikoski et al.
37 showed that SNP lowered IOP in normotensive rabbits after either topical or intravitreal dosing, and the maximal IOP reduction was between 2 and 5 hours. However, others found that SNP had a tendency to increase IOP in anesthetized rabbit.
38 Intravitreal or intracameral injection of the NO donors SNAP induced a marked IOP-lowering effect.
20,21 Together with our results of SNAP increasing outflow in perfused eyes, it suggest that dosing method is important for SNAP and topical application might not give it enough corneal penetration to induce IOP changes.