ATP-sensitive potassium (K(ATP)) channel openers diazoxide and nicorandil lower intraocular pressure in vivo

Abstract

Purpose: To evaluate the expression of ATP-sensitive potassium (K(ATP)) channel subunits and study the effect of K(ATP) channel openers diazoxide and nicorandil on intraocular pressure (IOP) in an in vivo mouse model.

Methods: Expression of K(ATP) channel subunits in normal C57BL/6 mouse eyes was studied by immunohistochemistry and confocal microscopy. Wild-type C57BL/6 mice were treated with K(ATP) channel openers diazoxide (n = 10) and nicorandil (n = 10) for 14 days. Similar treatments with diazoxide were performed on K(ir)6.2((-/-)) mice (n = 10). IOP was recorded with a handheld tonometer 1 hour, 4 hours, and 23 hours following daily treatment. Posttreatment histology was examined by light and transmission electron microscopy.

Results: The K(ATP) channel subunits SUR2B, K(ir)6.1, and K(ir)6.2 were identified in all tissues within mouse eyes. Treatment with diazoxide in wild-type mice decreased IOP by 21.5 ± 3.2% with an absolute IOP reduction of 3.9 ± 0.6 mm Hg (P = 0.002). Nicorandil also decreased IOP (18.9 ± 1.8%) with an absolute IOP reduction of 3.4 ± 0.4 mm Hg (P = 0.002). Treatment with diazoxide in K(ir)6.2((-/-)) mice had no effect on IOP. No morphological abnormalities were observed in diazoxide- or nicorandil-treated eyes.

Conclusions: K(ATP) channel openers diazoxide and nicorandil are effective regulators of IOP in mouse eyes. K(ir)6.2 appears to be a major K(ATP) channel subunit through which IOP is lowered following treatment with diazoxide.

Keywords: anterior segment; intraocular pressure; potassium channel.

Figure 1

Expression profile of K_ATP channel subunits SUR1, SUR2B, K_ir6.1, and K_ir6.2. SUR1 was not present in mouse eye tissues (B, G, L). SUR2B, K_ir6.1, and K_ir6.2 were present in the trabecular meshwork (including inner and outer walls of Schlemm's canal) (C–E); corneal epithelium, endothelium, and parts of the stroma (H–J); and in cell layers of the retina (M–O). Fluorescent intensity for the images was set based on the negative controls, which were incubated with secondary antibodies only (A, F, K). C. Epi, corneal epithelium; C. Endo, corneal endothelium; SC, Schlemm's canal; TM, trabecular meshwork; CB, ciliary body; I, iris; AC, anterior chamber; GC, ganglion cells; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segments; OS, outer segments. Red fluorescence: SUR2B. Green fluorescence: SUR1, K_ir6.1, and K_ir6.2. Blue fluorescence: DAPI. Scale bar: 20 μm for all micrographs.

Figure 2

Expression profile of K_ATP channel subunit SUR2A. SUR2A was not present in the anterior angle (B), cornea (D), or retina (F). Some background autofluorescence was observed in the angle and the retinal IPL and OPL. The negative controls were incubated with secondary antibody only and used to set the fluorescent intensity. Red fluorescence: SUR2A. Blue fluorescence: DAPI. Scale bar: 20 μm for all micrographs.

Figure 3

Effect of diazoxide on IOP in C57BL/6 wild-type and K_ir6.2^(−/−) mice. IOP was lowered within 48 hours of diazoxide treatment and maintained an average reduction of 3.9 ± 0.6 mm Hg during the 14-day treatment period (black line). In contrast, K_ir6.2^(−/−) mice showed no change in IOP on any of the treatment days following diazoxide treatment (gray line). *P = 0.002.

Figure 4

Effect of diazoxide treatment on ocular histology of C57BL/6 wild-type and K_ir6.2^(−/−) mice. Histologic examination of treated and control eyes in both wild type (A, B, E, F) and Kir6.2^(−/−) mice (C, D, G, H) did not show any observable change in cell and tissue appearance within the conventional outflow pathway following diazoxide treatment as evident from H&E staining (A–D) and transmission electron microscopy (E–H). Scale bar: 50 μm for H&E and 5 μm for transmission electron microscopy.

Figure 5

Effect of nicorandil treatment on IOP of wild-type C57BL/6 mice. Treatment with nicorandil caused a decrease in IOP reaching near maximal reduction levels within 48 hours. Reduction in IOP was maintained throughout the 14-day treatment period with an average reduction of 3.4 ± 0.4 mm Hg compared with control eyes. Following cessation of treatment, IOP returned to baseline within 72 hours. *P = 0.002.

Figure 6

Histology of nicorandil-treated C57BL/6 mice eyes. Ocular morphology of the tissues and cells of the conventional outflow pathway appeared similar between treated and control eyes by either H&E (A, B) or transmission electron microscopy (C, D). Scale bar: 50 μm for H&E and 5 μm for transmission electron microscopy.