The NHE-1 member of the family of six Na
+/H
+ exchanger (NHE) transporters is known to be the major basis for antiport activity at the basolateral surface of the PE cells facing the stromal surface.
11 DMA, an amiloride analogue with a highly selective inhibitory effect on the NHE-1 antiport,
22 produced a concentration-dependent lowering of IOP (
Fig. 2 ,
Table 1 ). The precise values are uncertain for the threshold droplet concentrations of the drugs used, but DMA was clearly effective at a droplet concentration of 1 mM (2.94 μg,
n = 23,
Table 1 ), and a greater lowering of IOP (by 5.0 ± 0.7 mm Hg) was obtained with a droplet concentration of 3 mM (8.82 μg,
n = 4;
Table 1 ). Another amiloride analogue, EIPA, displayed the same minimally effective droplet concentration and enhanced lowering of IOP at 3 mM (300 ng; by 4.1 ± 1.0 mm Hg,
Table 1 ). A third acylguanidine antiport inhibitor, BIIB723, produced a maximal hypotensive effect at 3 mM (16.0 μg) of 4.9 ± 1.7 mm Hg, similar to that of DMA (
n = 4,
Table 1 ), but displayed a lower minimally effective droplet concentration (100 μM [554 ng]),
n = 4,
Table 1 ). The similarity of the effects of BIIB723 at 1 mM (5.34 μg; −4.5 ± 0.5 mm Hg) and 3 mM (16.0 μg; −4.9 ± 1.7 mm Hg) and the similar reductions produced by all three NHE-1 inhibitors at 3 mM suggest that a maximal IOP reduction was achieved of 4.1 to 5.0 mm Hg. We were unable to increase the delivered droplet concentration without substantially increasing the DMSO level, thereby triggering a vehicle-induced change in IOP.
Carbonic anhydrase inhibition reduces the rate of production of H
+ and HCO
3 −, which in turn must slow the rate of delivery of H
+ and HCO
3 − to all cell sites, including the antiports. We have already reported that inhibiting carbonic anhydrase with intraperitoneal acetazolamide lowers mouse IOP (by 11.9 ± 1.3 mm Hg).
16 We have now found that topical application of dorzolamide also reduces IOP, albeit to a lesser extent at the droplet concentration applied
(Table 1) .
We also tested the effects of amiloride which inhibits NHE-1 antiports at a potency 1 to 2 orders of magnitude lower than the amiloride analogues DMA and EIPA.
11 Consistent with this information, amiloride itself exerted no significant effect on mouse IOP at a droplet concentration of 1 mM (2.30 μg,
n = 7, data not shown). To reach a 10-mM concentration, it was necessary to solubilize amiloride in 30% DMSO. After pretreatment with vehicle containing 30% DMSO, subsequent application of 10 mM amiloride in the same concentration of vehicle did not alter that IOP (ΔIOP = −1.0 ± 0.7 mm Hg,
n = 4,
P > 0.2). Thus, at a concentration 10 times higher than EIPA’s minimal effective concentration, amiloride had no effect, consistent with the known ratio of the potency of these inhibitors (3.9:0.07 μM, or ∼56) when applied to PE cells.
11
In contrast to the IOP reductions triggered by the three selective inhibitors of the NHE-1 antiport at droplet concentrations of 0.1 to 3 mM
(Table 1) , blockage of the Na
+-K
+-2Cl
− symport with droplet concentrations of 0.1 to 10 mM (364 ng to 36.4 μg) bumetanide had no significant effect on IOP (
Fig. 2 ,
Table 1 ).