The novel distribution of phosphodiesterase-4 subtypes within the rat retina

Abstract

Phosphodiesterases (PDEs) are important regulators of signal transduction processes. While much is known about the function of cyclic GMP-specific PDEs in the retina, much less is known about the closely related, cyclic AMP-specific PDEs. The purpose of the present study is to characterize and localize PDE4 within the adult rat retina. We have used Western blotting, RT-PCR, and immunohistochemistry together with retrograde labeling to determine the presence and location of each PDE4 subtype. Western blot analysis revealed that multiple isoforms of PDE4A, B, and D subtypes are present within the retina, whereas the PDE4C subtype was absent. These data were confirmed by RT-PCR. Using immunohistochemistry we show that all three PDE4s are abundantly expressed within the retina where they all colocalize with retrograde-labeled retinal ganglion cells, as well as bipolar cells, horizontal cells, and cholinergic amacrine cells, whereas Müller cells lack PDE4 expression. Uniquely, PDE4B was expressed by the inner and outer segments of rod photoreceptors as well as their terminals within the outer plexiform layer. Collectively, our results demonstrate that PDE4s are abundantly expressed throughout the rodent retina and this study provides the framework for further functional studies.

Figure 1

PDE4 protein and mRNA expression in the rodent retina. PDE4A (A), PDE4B (B), and PDE4D (D) are represented within the retina with distinct known isoforms which are blocked with preadsorption of the antibody. PDE4C (C) protein was absent from the retina but abundant within lung, positive control tissue. Preadsorbtion with sequence specific blocking peptide p4A for PDE4A (A), p4B for PDE4B (B), and p4D for PDE4D (D) blocked all bands. RT-PCR analysis (E) reveals predicted bands of the proper size for all PDE4s. Interestingly PDE4C which was identified within lung also detects putative PDE4C mRNA within retina as well. Quantification of relative PDE4 mRNA expression (F) reveals that PDE4B and PDE4D are over 150 fold more abundant than PDE4A. Real-time PCR results of PDE4A, PDE4B, and PDE4D are expressed as fold change in mRNA. *p < .001.

Figure 2

PDE4 expression in the rodent retina. Immunofluorescent images of PDE4A (A), PDE4B (C), PDE4D (F) and preadsorbed p4A (B), p4B (D), and p4D (G) localize primarily to the inner retina, whereas PDE4C (E) immunolabeling was absent. Scale bar = 50μm

Figure 3

PDE4 expression in the photoreceptors. Double labeling of PDE4A (A), PDE4B (D), and PDE4D (G) with VGlut1 (B, E, H). VGlut1 is restricted to the outer OPL within photoreceptor terminals. In contrast, PDE4A (C) and PDE4D (I) are confined to the inner OPL and fail to colocalize with VGlut1 positive terminals. PDE4B (F) is present throughout the OPL and strongly colocalizes with VGlut1. Labeling with anti-PDE4B labels photoreceptor inner and outer segments (J) labeling with PNA specifically labels cone inner and outer segments (K) whereas double labeling reveals no colocalization (L). Labeling of PDE4B (M), rhodopsin (N), and Dapi nuclear stain (M, N, O) demonstrate PDE4B localization within outer segments (O). Scale bar = 25μm.

Figure 4

PDE4B expression pattern in the photoreceptors. Double labeling for PDE4B (A, D) and PNA (B, E) within a whole mounted retina. Labeling with PNA specifically labels cone photoreceptors (B, E). PDE4B labeling (A, D) is present within photoreceptors not labeled with PNA (C, F). Orthogonal views (D-F) demonstrates a close approximation of PNA and PDE4B labeling but no colocalization (F). Scale = 50μm.

Figure 5

PDE4 expression in the horizontal cells. Double labeling for PDE4A (A), PDE4B (G), and PDE4D (M) and calbindin (B, H, N). Calbindin specifically labels horizontal cells and their processes which ramify near the border of the OPL and INL. All 3 PDE4s (C, I, O) colocalize with calbindin positive somata (arrows). This is confirmed with orthogonal views through the z-axis (horizontal line). Scale bar = 8μm for orthogonal views and 25μm for all other figures.

Figure 6

PDE4 expression in the bipolar cells. Double labeling of PDE4A (A), PDE4B (E), and PDE4D (I) with PKCα (B, F, J) and Chx10 (C, G, K). PKCα immunoreactivity is present in rod bipolar cells, whereas Chx10 identifies all bipolar cells. All 3 PDE4s (D, H, L) colocalize with rod bipolar cells which are also Chx10 immunopositive (arrows) and presumptive cone bipolar cells (arrowheads) which are negative for PKCα labeling. Scale bar = 25μm.

Figure 7

PDE4 expression in the cholinergic amacrine cells. Double labeling of PDE4A (A), PDE4B (D), and PDE4D (G) with ChAT (B, E, H). ChAT labeling detects cholinergic amacrine cells and 2 strata within the IPL, an inner ON cholinergic layer and an outer OFF cholinergic layer. All 3 PDE4s (C, F, I) colocalize with ChAT positive cholinergic cells in the INL (arrows) and displaced amacrine cells (arrowheads). Scale bar = 25μm.

Figure 8

PDE4 expression in the retinal ganglion cells. Double labeling of PDE4A (A), PDE4B (D), and PDE4D (G) with CTB (B, E, H). Retrograde labeling from the superior colliclus with cholera toxin B subunit was detected within multiple ganglion cells. All 3 PDE4s (C, F, I) colocalized with CTB-labeled retinal ganglion cells (arrows). Scale bar = 25μm.

Figure 9

PDE4 expression in the Müller cells. Labeling of PDE4B (A) with GLAST (B, C, D) and GS (D) indicate the absence of PDE4B in Müller cells. While some approximation were found between PDE4B and GLAST (arrowhead), these cells were not positive for GS staining. Likewise cells with both GS and GLAST co-labeling (arrow) do not colocalize with PDE4B. Scale bar = 25μm.

Figure 10

Summary of PDE4 expression within the rodent retina. PDE4A, PDE4B, and PDE4D expression within various cell types where G - ganglion cells, Ch cholinergic amacrine cells, M - Müller cells, CB - cone bipolar cells, RB - rod bipolar cells, H - horizontal cells, C - cones, and R - rods.