The authors have declared that no competing interests exist.
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 (p<0.02) in human anterior segments (n = 7 for DZ, n = 4 for NCD) was abrogated by U0126 (DZ + U0126: -9.7 ± 11.5%, p = 0.11; NCD + U0126: -0.1 ± 11.5%, p = 1.0). In contrast, U0126 had no effect on latanoprostfree acid-induced pressure reduction (-52.5 ± 6.8%, n = 4, p = 0.001). In mice, DZ and NCD reduced IOP (DZ, 14.9 ± 3.8%, NCD, 16.9 ± 2.5%, n = 10, p<0.001), but the pressure reduction was inhibited by U0126 (DZ + U0126, 0.7 ± 3.0%; NCD + U0126, 0.9 ± 2.2%, n = 10, p>0.1). 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.
Glaucoma is a neurodegenerative disease affecting over 60 million people worldwide.[
ATP-sensitive potassium (KATP) channels are hetero-octamers formed with 4 regulatory sulphonylurea receptor (SUR) subunits (SUR1, SUR2A or SUR2B) and 4 inwardly rectifying potassium channel subunits (Kir6.1 or Kir6.2).[
Our laboratory has established a novel ocular hypotensive property of several KATP channel openers.[
Three NTM cell lines (passage 3 to 7) derived from independent human donor eyes (age 3 months, 32 years and 57 years) were established as previously described.[
Confluent NTM cells were serum starved in DMEM containing antibiotics for 24 hours and treated with 20 μM DZ or NCD (Sigma-Aldrich, St. Louis, MO; diluted in serum-free DMEM from a 20 mM stock in DMSO). Concentration of 20 μM was chosen as it was the concentration that lowered pressure in previous human anterior segment perfusion culture studies.[
To establish specificity of DZ action, primary NTM cells were pretreated with U0126 (Calbiochem, Billerica, MA; 20 μM, diluted in serum free DMEM from a 20 mM stock solution in DMSO), an Erk1/2 pathway inhibitor that targets MAPK/ERK (Mek) kinase, for 15 minutes followed by addition of DZ (20 μM) + U0126 (20 μM). Cells were lysed and total protein concentration was determined using Bradford’s assay.
Cell lysates were mixed with 5X reducing sample buffer (Thermo Fisher Scientific, Waltham, MA) containing 15% 2-mercaptoethanol (Sigma-Aldrich). Total protein (12–15 μg) was loaded and separated on 4–15% gradient SDS-PAGE gels (Bio-Rad, Hercules, CA), transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA), and blocked in 2% non-fat dried milk as previously described.[
Use of human donor eyes for this study was approved by the Mayo Clinic Institutional Review Board and adhered to the tenets of the Declaration of Helsinki. A total of 18 pairs of human eyes (age 73.8 ± 11.3 years, range 51 to 88 years) were used for this study. All eyes were obtained from the Minnesota Lions Eye Bank within 13.1 ± 2.6 hours of death. None of the donors had a documented history of eye disease and were not on any topical eye medications. For culture preparation, the eyes were bisected at the equator and the ciliary body, iris and lens were removed as previously described.[
In 3 pairs of human eyes (age 49, 81 and 81 years), one anterior segment of each pair was perfused with 20 μM DZ (diluted with DMEM from a 20 mM stock in DMSO) while the contralateral anterior segment was perfused with vehicle for 6 hours (n = 1) and 14 hours (n = 2). TM tissue was dissected from these anterior segments and homogenized individually in 120 μl lysis buffer with protease and phosphatase inhibitors (described above) [
In 7 pairs of eyes (age 76.4 ± 12.3 years, range 54 to 86 years), one anterior segment was perfused for 24 hours with 20 μM DZ while the contralateral anterior segment received vehicle.[
In 4 pairs of eyes (age 62.5 ± 9.7 years, range 51 to 71 years) one anterior segment received NCD (20 μM; prepared as described for DZ) for 24 hours while the contralateral eye received vehicle. Following NCD treatment, 3 out of the 4 anterior segments that received NCD only, were treated with NCD (20 μM) + U0126 (20 μM) for an additional 24 hours while contralateral eyes received appropriate vehicle.
In 4 pairs of eyes (age 79.0 ± 7.8 years, range 69 to 88 years) one anterior segment from each pair received 20 μM DZ for 24 hours followed by DZ (20 μM) + U0126 (0.5 mM) for an additional 24 hours. Subsequently, LFA (0.1 μM) + U0126 (0.5 mM) was added for another 24 hours. Contralateral eye was treated with appropriate vehicle at designated drug treatment times. Final LFA (Cayman Chemical, Ann Arbor, MI) concentration was prepared by diluting a 100 mM stock dissolved in ethanol with DMEM.
In 7 pairs of eyes (age 68.3 ± 11.0 years; range 51 to 84 years), 0.5 mM U0126 was added to one anterior segment while the fellow eye received vehicle.
Use of animals and experimental protocols were pre-approved by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC) and adhered to the tenets of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Wild type C57BL/6 mice (retired breeders, age >8 months) were purchased from Charles River Laboratories (Wilmington, MA) and maintained at the Mayo Clinic animal care facility under a 12 hour light and dark cycle. Animals received standard rodent chow and water
One eye of 6 mice were treated with 5 mM DZ [(prepared by diluting a 100 mM stock (in DMSO) in 10% polyethoxylated castor oil (Cremophor EL; Sigma-Aldrich); delivered as a 5 μl bolus, equivalent to 25 nmol)] while the contralateral eye received vehicle. Following 15 minute treatment, animals were euthanized by CO2 asphyxiation, eyes were enucleated, and micro-dissection was performed to remove the cornea along with the TM and SC from each animal. Anterior segment tissues from all 6 animals were pooled into appropriate treatment and control groups, incubated in cell lysis buffer (described above for western blots) and homogenized using an ultrasonic cell disruptor (Misonix). Total protein was assayed by Bradford’s method. Western blot for phosphorylated Erk1/2 and total Erk1/2 was performed as described above.
Prior to addition of drugs, baseline IOP was measured 3 times daily for 3 consecutive days with a handheld rebound tonometer (Icare Tonolab: Colonial Medical Supply, Franconia, NH) in live non-anesthetized mice as previously described.[
At the end of each study, two tissue wedges containing the TM and SC were dissected 180° apart from each anterior segment and fixed in 10% neutral buffered formalin. Tissue wedges were post fixed in 2% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA) in 0.1 M phosphate buffer followed by dehydration in ascending ethanol concentrations. Tissues were subjected to a clearing agent (acetone, Sigma-Aldrich), embedded in epoxy resin blocks, and 500 nm and 100 nm sections were obtained using an ultramicrotome (Leica Microsystems, Buffalo Grove, IL). 500 nm sections were stained with toluidine blue and gross morphology was assessed by light microscopy. 100 nm sections were placed on copper grids and stained with 2% uranyl acetate (Electron Microscopy Sciences) followed by lead citrate (Mager Scientific, Dexter, MI). Sections were imaged using a JEOL 1400 transmission electron microscope (JEOL USA, Peabody, MA) for evaluation of cell and tissue ultrastructure.
Enucleated mouse eyes were placed in 10% neutral buffered formalin for at least 24 hours. Whole eyes were processed as described for human anterior segments. Whole eyes were cut longitudinally and 500 nm and 100 nm sections were used for toluidine blue staining and transmission electron microscopy as described above.
All IOP data are represented as absolute IOP or as absolute change compared to control (ΔIOP in mmHg) and are obtained by subtracting the average IOP of the treated eye from that of the control eye at any given time point. Values are represented as mean ± standard deviation. Data between control and treated groups were compared using Student’s paired t test. Differences were considered significant when p≤0.05.
To evaluate the effect of DZ and NCD on phosphorylated Erk1/2, we treated primary cultures of human NTM cells (in vitro), anterior human segments (ex vivo), and mouse eyes (in vivo) with DZ and NCD. In primary NTM cells, elevated levels of phosphorylated Erk1/2 were identified within 6 hours of treatment with DZ (7.0 ± 2.4 fold; n = 2) and NCD (4.2 ± 3.8 fold; n = 2) (
(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.
To determine whether the Erk1/2 pathway was the main signaling axis through which DZ and NCD lowered pressure, we evaluated DZ in the presence of U0126 in the human anterior segment perfusion culture model. Similar to a previously published report,[
(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.
Study number | Treatment | n | 0 h (μl/min/mmHg) | 24 h (μl/min/mmHg) |
---|---|---|---|---|
DZ | 7 | 0.13 ± 0.03 | 0.27 ± 0.14 |
|
DZ+U0126 | 7 | 0.13 ± 0.03 | 0.15 ± 0.02 | |
Vehicle | 7 | 0.16 ± 0.07 | 0.16 ± 0.07 | |
2 | NCD | 4 | 0.17 ± 0.03 | 0.33 ± 0.14 |
NCD+U0126 | 3 | 0.17 ± 0.03 | 0.17 ± 0.04 | |
Vehicle | 4 | 0.15 ± 0.04 | 0.16 ± 0.04 | |
3 | U0126 | 7 | 0.18 ± 0.03 | 0.18 ± 0.03 |
Vehicle | 7 | 0.17 ± 0.04 | 0.15 ± 0.03 | |
4 | DZ | 4 | 0.14 ± 0.03 | 0.38 ± 0.14 |
DZ+U0126 | 4 | 0.14 ± 0.03 | 0.12 ± 0.03 | |
LFA + U0126 | 4 | 0.13 ± 0.03 | 0.30 ± 0.19 |
|
Vehicle | 4 | 0.25 ± 0.06 | 0.29 ± 0.09 |
* p<0.05
To verify results obtained with DZ, we investigated the effect of NCD, a separate KATP channel opener, on pressure and outflow facility of cultured human anterior segments. When anterior segments (n = 4) were treated with NCD alone, pressure was reduced from 15.0 ± 2.8 mmHg at 0 hour to 8.8 ± 4.4 mmHg at 24 hours (p = 0.01) (
To determine if Erk1/2 was essential for IOP, we treated human anterior segments with U0126 alone. No change in pressure (vehicle 15.6 ± 4.6 mmHg at 0 hour and 17.1 ± 4.1 mmHg at 24 hours, p = 0.17; U0126, 14.1 ± 2.6 mmHg at 0 h and 14.3 ± 2.6 mmHg at 24 hours, p = 0.69) or outflow facility (vehicle, 0.17 ± 0.04 μl/min/mmHg at 0 hour to 0.15 ± 0.03 μl/min/mmHg at 24 hours; U0126, 0.18 ± 0.03 μl/min/mmHg at 0 hour and 0.18 ± 0.03 at 24 hours, p = 0.72) was noted in these eyes (
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.
To assess the effect of DZ, NCD and U0126 on conventional outflow morphology, we sectioned various tissue wedges isolated from treated anterior segments and processed them for toluidine blue staining and transmission electron microscopy. Treatment with DZ, NCD and U0126, in various combinations did not cause detrimental side effects to the overall morphology and the ultrastructure of the cells in the TM and inner and outer walls of SC in comparison to vehicle treated controls (
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.
To evaluate the association of IOP reduction by KATP channel openers and Erk1/2 phosphorylation in vivo, C57Bl/6 mice were treated with DZ (25 nmol) and NCD (25 nmol) in the presence or absence of U0126 (2.5 nmol). DZ lowered IOP by 2.6 ± 0.7 mmHg (range of 1.2 ± 0.9 mmHg to 3.2 ± 0.8) which resulted in a 14.9 ± 3.8% decrease in IOP compared to fellow vehicle treated eyes (
(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.
To determine whether the Erk1/2 pathway was unique to KATP channel openers or was a more generalized pathway utilized in pressure reduction, we evaluated the effect of U0126 on anterior segments (n = 4) treated with DZ and the prostaglandin analog LFA. Anterior segments treated with DZ alone reduced pressure from 18.8 ± 3.8 mmHg at 0 hour to 7.5 ± 2.9 mmHg at 24 hours (p = 0.002). This pressure reduction was inhibited by addition of U0126 (baseline, 18.8 ± 3.8 mmHg; DZ + U0126, 21.5 ± 5.3 mmHg; p = 0.37) (
(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.
Our previous studies have elucidated a novel ocular hypotensive property of several KATP channel openers, including DZ and NCD.[
The MAP kinase pathway is a highly conserved mammalian second messenger system. Of the various MAP kinase pathways, the Ras-Raf-Mek-Erk pathway (also known as the Erk1/2 or p42/p44 MAP kinase pathway) is the best characterized.[
Various kinds of cellular insults (e.g. hypoxia, reactive oxygen species and oxidative stress, etc.) that have been linked to glaucoma [
One of the hallmark pathological changes in glaucoma is the damage to retinal ganglion cells.[
The intraocular pressure reduction that we observed in human and mouse eyes following treatment with DZ and NCD was similar to that noted in our previous reports.[
In summary, data from the current study elucidates that the KATP channel openers DZ and NCD lower IOP by specifically activating the Erk1/2 signaling pathway in relevant ocular cells. The Erk1/2 signaling axis is one of the single most important pathways involved in survival and proliferation of various cells. The fact that KATP channel openers can target the Erk1/2 signaling pathway suggests these molecules may be candidate therapeutic agents for management of ocular hypertensive diseases like glaucoma.
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