Mitochondrial potassium ATP channels and retinal ischemic preconditioning

Abstract

Purpose: To examine the mechanisms of ischemic preconditioning (IPC) related to the opening of mitochondrial KATP (mKATP) channels in the retina.

Methods: Rats were subjected to retinal ischemia after IPC, or retinas were rendered ischemic after pharmacological opening of mKATP channels. The effects of blocking mKATP channel opening, nitric oxide synthase (NOS) subtypes, or protein kinase C (PKC) on the protective effect of IPC or on the opening of mKATP channels were studied. Electroretinography assessed functional recovery after ischemia. Immunohistochemistry and image analysis were used to measure changes in levels of reactive oxygen species (ROS) and NOS subtypes and to determine their cellular localization.

Results: IPC was effectively mimicked by injection of the mKATP channel opener diazoxide. Both IPC and its mimicking by diazoxide were completely attenuated by the mKATP channel blocker 5-hydroxydecanoic acid (5-HD). Nonspecific blockade of NOS by N(omega)-nitro-L-arginine (L-NNA), but not by specific inducible (i)NOS or neuronal (n)NOS inhibitors, blunted IPC and IPC-mimicking, as did blockade of PKC. IPC and diazoxide IPC-mimicking significantly enhanced mitochondrial ROS production in the inner retina, an effect blocked by 5-HD. Mitochondrial ROS colocalized with e- and nNOS in retinal cells after stimulation with diazoxide.

Conclusions: The results showed that IPC in the retina requires opening of the mKATP channel, and that IPC could be effectively mimicked using the mKATP channel opener diazoxide. eNOS-generated nitric oxide, PKC, and ROS are activated by opening of the mKATP channel.

FIGURE 1

5-HD blocked the neuroprotective effects of IPC in a dose-dependent manner. IPC (8 minutes of increased intraocular pressure) was performed 24 hours before 45 minutes of ischemia. The b-wave was measured at baseline and at 7 days after ischemia; results shown at 7 days in the ischemic eye were normalized to baseline and for day-to-day variation in the control, nonischemic eye. The leftmost bar shows that IPC (n = 10 rats) resulted in nearly complete recovery of the ERG b-wave (y-axis) relative to preischemic baseline. Data are shown as the mean ± SEM. The ERG b-wave recovery was significantly reduced when 5-HD 1 mg/kg (P < 0.03, n = 4 rats) and 40 mg/kg (P < 0.005, n = 4 rats) was injected before IPC. The blunting of recovery after IPC by 5-HD approaches that of sham IPC (second bar from left).

FIGURE 2

The mKATP agonist, diazoxide, mimicked the neuroprotective effects of IPC in a dose-dependent manner. Diazoxide was injected IP 24 hours before 45 minutes of ischemia and compared with injection of DMSO vehicle (leftmost bar). See Figure 1 for a description of the method. The significant IPC-mimicking by diazoxide (P < 0.004) 40 mg/kg was inhibited by 5-HD (200 mg/kg, P < 0.0003 vs. diazoxide). n = 10 rats in the diazoxide 40-mg/kg group, and five to seven rats in the others.

FIGURE 3

The nonspecific NOS inhibitor L-NNA 30 mg/kg significantly attenuated the neuroprotective effect of IPC (P < 0.01). L-NNA was injected IP before IPC, which was followed 24 hours later by 45 minutes of ischemia. See Figure 1 for a description of the method. Sham IPC (n = 13 rats) shows no significant difference versus L-NNA+IPC (n = 5).

FIGURE 4

L-NNA 30 mg/kg significantly blocked (top, P < 0.002, n = 5 rats) the IPC-mimicking of diazoxide (40 mg/kg, n = 8 rats). In addition, the PKC inhibitors Bis1 or chelerythrine (bottom), injected into the vitreous at the time of IP injection of diazoxide, significantly diminished IPC mimicking (compared with diazoxide 40 mg/kg+PBS vehicle in vitreous, n = 9): Bis1 15 μM(P < 0.009, n = 3 rats) and 1.5 mM (P < 0.008, n = 4 rats), and chelerythrine 250 nM (P < 0.01, n = 11 rats), 25 μM(P < 0.0001, n = 6 rats), and 2.5 mM (P < 0.0001, n = 6 rats). See Figure 1 for a description of the method.

FIGURE 5

The IPC-mimicking effect of diazoxide was not affected by either the specific iNOS inhibitor 1400W or the specific nNOS antagonist 7-NI (n = 4 – 8 rats per group). Both inhibitors were injected IP before diazoxide, and ischemia was induced 24 hours later. See Figure 1 for a description of the method.

FIGURE 6

IPC increased both ROS generation and NOS subtype expression in the retina. Each time point after IPC represents the normalized intensity in the inner retina relative to the matched normal (sham IPC) paired eyes. The quantitation of the fluorescence-labeled (MitoTracker; Invitrogen) immunohistochemical retinal sections (n = 3–6 rats per group) shows that ROS generation was significantly (P < 0.05) increased at 1, 6, and 24 hours after IPC. The expression of both eNOS and iNOS was significantly increased at 15 minutes and 1 and 6 hours after IPC. nNOS expression was significantly increased at 1 hour after IPC. Representative images for the fluorescent labeling (MitoTracker; Invitrogen) and each NOS subtype are shown above each graph for a normal and IPC retinal section 1 hour after IPC.

FIGURE 7

Diazoxide (40 mg/kg) increased both ROS generation and NOS subtype expression in the retina. Each pair of bars represents absolute intensity in the inner retina for the matched time point after diazoxide or DMSO-vehicle injection. See Figure 6 for a description of the method. Quantitation of the fluorescence-labeled immunohistochemical retinal sections (n = 4 – 6 rats per group) showed that ROS generation was significantly (P < 0.05) increased at 15 minutes and 6 and 24 hours after diazoxide stimulation. The trend for increase at 1 hour was not significant. The expression of eNOS is significantly increased at all four time points after diazoxide. iNOS is significantly increased only at 15 minutes after diazoxide. nNOS expression was significantly increased at 15 minutes and 1 and 24 hours after diazoxide. Representative images of the fluorescent labeling and for each NOS subtype are shown above each graph for vehicle control and diazoxide retinal section at 1 hour.

FIGURE 8

The mKATP inhibitor 5-HD blocked IPC-induced ROS generation and NOS subtype expression (n = 3– 6 rats). Representative images of the fluorescent labeling and each NOS subtype are shown above each graph for a normal and IPC retinal section 1 hour after IPC with preceding 5-HD application. The fluorescent images were taken at different exposure times than those used for Figure 6.

FIGURE 9

The mKATP inhibitor 5-HD blocks the diazoxide-induced ROS generation and NOS subtype expression (n = 4 – 6 rats). Representative images of the fluorescent labeling and each NOS subtype are shown above each graph for a normal and IPC retinal section 1 hour after diazoxide and 5-HD application. The fluorescent images were taken at different exposure times than those used for Figure 7.

FIGURE 10

Images of 10-μm retinal cryosections. The mitochondrial inner membrane marker (COX IV; A), an RGC marker (Thy-1; D), a glial cell marker (GFAP; G) and a nuclear marker ( J; Sytox; Invitrogen) are depicted in the leftmost columns. The red fluorescent label (MitoTracker; Invitrogen) shows the generation of ROS in ischemic retinal sections (B, E, H, K). Combined images, in which yellow or orange indicate colocalization (white arrows). ROS localization in the mitochondria is confirmed by colocalization of red fluorescence and COX IV and intra-cellular localization to RGCs and Müller cells by Thy-1, GFAP, and nucleic acid staining (C, F, I, J). Bottom: nonischemic negative control section with minimal positive staining. Magnification, x63.

FIGURE 11

Shown are 63x images of 10-μm retinal cryosections. The mitochondrial marker (COX IV; A), an RGC marker (Thy-1; D), a glial cell marker (GFAP; G) and the nuclear marker (Sytox; J) are depicted in the leftmost columns. Dihydroethidium (HET) shows the generation of ROS in ischemic retinal sections (B, E, H, K)inthe middle columns. The combined images showing colocalization (yellow or orange; white arrows) are shown in the rightmost columns (C, F, I, J). A nonischemic negative control section with minimal positive staining appears at the bottom middle for comparison. The results with HET confirm those with the red fluorescent label (MitoTracker; Invitrogen) in Figure 10. Magnification, x63.

FIGURE 12

Red fluorescence (MitoTracker; Invitrogen) colocalized with COX IV (green) in the mitochondria of RGCs (right, orange arrow) and in their axonal projections (left, white arrows). Note the punctate staining of the mitochondria. Also note that not all mitochondria stained for both markers (right, green arrow). The IPL (left, brackets) stained intensely for both red fluorescence and COX IV. DAPI blue stained the cell nuclei (right, blue arrow). Left: 63x fluorescent image; right: the magnified image of an RGC in the left image’s boxed area.

FIGURE 13

Double-labeled 10-μm retinal cryosections, the colocalization of red fluorescent dye (Mito-Tracker; Invitrogen) and eNOS (left, arrows) and nNOS (right, arrows)is evident, whereas iNOS did not colocalize with the red label (middle). Magnification x63.

FIGURE 14

In 63x double-labeled 10-μm retinal cryosections, images (A—D) show the single immunohisto-chemical staining of eNOS (A) and the endothelial cell marker caveolin-1 (B). There was colocalization of eNOS and caveolin-1 (C, arrow) and, in a triple-labeled section, of red fluorescent dye (MitoTracker; Invitrogen), eNOS, and caveolin-1 (D, arrow). The expression of iNOS (E) and the microglial marker OX42 (F) show colocalization (G, arrows). The nNOS expression in RGCs is seen in (H), and DAPI nuclear staining in (I). The combined image of nNOS and DAPI demonstrating colocalization in RGCs is shown in ( J). A triple-labeled section (K), shows the colocalization of nNOS, DAPI, and red fluorescent label in RGCs (arrow).