ATP-sensitive potassium (KATP) channel openers diazoxide and nicorandil lower intraocular pressure by activating the Erk1/2 signaling pathway.

Elevated intraocular pressure is the most prevalent and only treatable risk factor for glaucoma, a degenerative disease of the optic nerve. While treatment options to slow disease progression are available, all current therapeutic and surgical treatments have unwanted side effects or limited efficacy, resulting in the need to identify new options. Previous reports from our laboratory have established a novel ocular hypotensive effect of ATP-sensitive potassium channel (KATP) openers including diazoxide (DZ) and nicorandil (NCD). In the current study, we evaluated the role of Erk1/2 signaling pathway in KATP channel opener mediated reduction of intraocular pressure (IOP). Western blot analysis of DZ and NCD treated primary normal trabecular meshwork (NTM) cells, human TM (isolated from perfusion cultures of human anterior segments) and mouse eyes showed increased phosphorylation of Erk1/2 when compared to vehicle treated controls. DZ and NCD mediated pressure reduction (p0.1). Histologic evaluation of transmission electron micrographs from DZ + U0126 and NCD + U0126 treated eyes revealed no observable morphological changes in the ultrastructure of the conventional outflow pathway. Taken together, the results indicate that the Erk1/2 pathway is necessary for IOP reduction by KATP channel openers DZ and NCD

Correction: Ocular Hypotensive Effects of the ATP-Sensitive Potassium Channel Opener Cromakalim in Human and Murine Experimental Model Systems.

Elevated intraocular pressure (IOP) is the most prevalent and only treatable risk factor for glaucoma, a leading cause of irreversible blindness worldwide. Unfortunately, all current therapeutics used to treat elevated IOP and glaucoma have significant and sometimes irreversible side effects necessitating the development of novel compounds. We evaluated the IOP lowering ability of the broad spectrum KATP channel opener cromakalim. Cultured human anterior segments when treated with 2 μM cromakalim showed a decrease in pressure (19.33 ± 2.78 mmHg at 0 hours to 13.22 ± 2.64 mmHg at 24 hours; p<0.001) when compared to vehicle treated controls (15.89 ± 5.33 mmHg at 0 h to 15.56 ± 4.88 mmHg at 24 hours; p = 0.89). In wild-type C57BL/6 mice, cromakalim reduced IOP by 18.75 ± 2.22% compared to vehicle treated contralateral eyes (17.01 ± 0.32 mmHg at 0 hours to 13.82 ± 0.37 mmHg at 24 hours; n = 10, p = 0.002). Cromakalim demonstrated an additive effect when used in conjunction with latanoprost free acid, a common ocular hypotensive drug prescribed to patients with elevated IOP. To examine KATP channel subunit specificity, Kir6.2(-/-) mice were treated with cromakalim, but unlike wild-type animals, no change in IOP was noted. Histologic analysis of treated and control eyes in cultured human anterior segments and in mice showed similar cell numbers and extracellular matrix integrity within the trabecular meshwork, with no disruptions in the inner and outer walls of Schlemm's canal. Together, these studies suggest that cromakalim is a potent ocular hypotensive agent that lowers IOP via activation of Kir6.2 containing KATP channels, its effect is additive when used in combination with the commonly used glaucoma drug latanoprost, and is not toxic to cells and tissues of the aqueous humor outflow pathway, making it a candidate for future therapeutic development

U0126 inhibits ocular hypotensive effects of DZ but not of LFA in perfusion cultured human anterior segments.

<p>(A-C) Anterior segments treated with DZ showed decreased pressure (A, B) and increased outflow facility (C) which returned to baseline following treatment with DZ + U0126. In contrast, U0126 was unable to inhibit pressure reduction caused by LFA, indicating that utilization of the Erk1/2 pathway for lowering IOP was different between DZ and LFA. (D and E) Evaluation of histology using toluidine blue sections and transmission electron micrographs showed regular extracellular matrix deposition in JCT and intact trabecular meshwork beams and viable cells in the outflow pathway of vehicle (D) and treated (E) eyes. Representative micrographs are shown. Scale bar, 20 μm for toluidine blue sections; 10 μm for TEM; *p<0.05; TM, trabecular meshwork; SC, Schlemm’s canal; JCT, juxtacanalicular tissue.</p

Ocular hypotensive effects of DZ and NCD were inhibited by U0126 <i>in vivo</i>.

<p>(A) Mice treated with topical DZ eye drops showed an average IOP reduction of 14.9 ± 3.8% (n = 10, p<0.001) which was inhibited by addition of U0126. IOP returned to baseline after three days of DZ + U0126 treatment (vehicle control, 17.5 ± 0.5 mmHg; DZ + U0126, 17.4 ± 0.7 mmHg; n = 10, p = 0.7). (B) Similar to DZ treatment, U0126 treatment in the presence of the ocular hypotensive agent NCD returned IOP to baseline values within 3 days. (C-F) Assessment of the conventional outflow pathway gross morphology and tissue ultrastructure following treatment with vehicle (C) and DZ + U0126 (D) or vehicle (E) and NCD + U0126 (F) showed healthy living cells in the trabecular meshwork with an open and intact Schlemm’s canal. Scale bar, 20 μm for toluidine blue sections; 5 μm for TEM. SC, Schlemm’s canal; TM, trabecular meshwork.</p

Treatment with DZ and NCD increases Erk1/2 phosphorylation.

<p>(A) DZ and NCD caused upregulation of Erk1/2 phosphorylation in NTM cells (n = 2) within 6 hours of treatment. Phosphorylation of Erk1/2 was eliminated by the Erk1/2 pathway inhibitor U0126 either by itself or in the presence of DZ. (B) Human anterior segments treated with DZ for 6 h (n = 1) showed minimal change in phosphorylated Erk1/2. However, following 14 hours (n = 2) of DZ treatment, an increase in Erk1/2 phosphorylation was observed. (C) In vivo topical application of DZ to mouse eyes caused upregulation of Erk1/2 phosphorylation within 15 minutes of treatment. Mouse anterior segments from 6 DZ treated eyes and 6 vehicle treated eyes were pooled for this experiment. pErk1/2, phosphorylated Erk1/2.</p

DZ and NCD lower pressure by activating the Erk1/2 pathway in human anterior segments.

<p>(A-C) Addition of DZ caused significant reduction of pressure (A, B) and increase in outflow facility (C). However, addition of the Erk1/2 pathway inhibitor U0126 completely inhibited the pressure reduction (A, B) and outflow facility increase (C) caused by DZ. (D-F) Similar to results obtained with DZ, U0126 also inhibited the ocular hypotensive effects of NCD (D, E). Outflow facility increases caused by NCD treatment were reversed by U0126 (F). Graphs are representative images, *p<0.05.</p

Morphology of JCT and SC following DZ and NCD treatment with and without U0126.

<p>Cells and tissues of the conventional outflow pathway were histologically evaluated from toluidine blue stained sections and transmission electron micrographs of eyes treated with vehicle (A) or DZ and DZ + U0126 (B), vehicle (C) or NCD and NCD + U0126 (D) and vehicle (E) or U0126 by itself (F). Overall, all micrographs showed intact trabecular beams traversed by viable trabecular meshwork cells. Extracellular matrix deposition in the juxtacanalicular region was similar to that observed in corresponding vehicle controls. Schlemm’s canal inner and outer walls were also intact in control and treated groups. Representative images are shown. Scale bar, 20 μm for toluidine blue sections; 10 μm for transmission electron microscopy. TM, trabecular meshwork; SC, Schlemm’s canal; JCT, juxtacanalicular tissue.</p

U0126 does not affect pressure or outflow facility by itself.

<p>Human anterior segments were treated with the Erk1/2 pathway inhibitor U0126. U0126 or vehicle treatment had no effect on pressure (A, B) and outflow facility (C). Graph is representative of experiments performed in 7 separate eye pairs.</p