Immunohistochemical localization of phosphodiesterase 2A in multiple mammalian species

Abstract

Phosphodiesterases (PDEs) comprise a family of enzymes that regulate the levels of cyclic nucleotides, key second messengers that mediate a diverse array of functions. PDE2A is an evolutionarily conserved cGMP-stimulated cAMP and cGMP PDE. In the present study, the regional and cellular distribution of PDE2A in tissues of rats, mice, cynomolgus monkeys, dogs, and humans was evaluated by immunohistochemistry. A polyclonal antibody directed to the C-terminal portion of PDE2A specifically detected PDE2A by Western blotting and by immunohistochemistry. The pattern of PDE2A immunoreactivity (ir) was consistent across all species. Western blot analysis demonstrated that PDE2A was most abundant in the brain relative to peripheral tissues. PDE2A ir was heterogeneously distributed within brain and was selectively expressed in particular peripheral tissues. In the brain, prominent immunoreactivity was apparent in components of the limbic system, including the isocortex, hippocampus, amygdala, habenula, basal ganglia, and interpeduncular nucleus. Cytoplasmic PDE2A staining was prominent in several peripheral tissues, including the adrenal zona glomerulosa, neurons of enteric ganglia, endothelial cells in all organs, lymphocytes of spleen and lymph nodes, and pituitary. These studies suggest that PDE2A is evolutionarily conserved across mammalian species and support the hypothesis that the enzyme plays a fundamental role in signal transduction.

Figure 1

Western blot analysis of human, cynomolgus monkey, rat, and dog brain extracts probed with anti-PDE2A antibody. Left lane is molecular mass standards, whereas the right lane represents the positive control. Each additional lane represents tissue from two individual subjects loaded in adjacent lanes, with the exception of rat, which represented a single animal in one lane. Additional rat samples are illustrated in Figure 2. Recombinant PDE2A with His tag expressed in sf21 cells (rPDE2A) was loaded as a positive control. A primary band of ∼100 kDa is detected in 10 μg of striatal extract from human, with a stronger signal in equally loaded extracts from cynomolgus monkey (Cyno), rat, and dog brain. Equivalent loading was demonstrated by probing the same blot with an anti-actin antibody.

Figure 2

Western blot analysis of rat central nervous system (CNS) and peripheral tissue extracts probed with anti-PDE2A antibody. Twenty micrograms of total protein per lane was loaded from the various highlighted tissue specimens. Left lane is molecular mass standards, and right lane is PDE2A-positive control. pAb PD2A-101AP recognizes a primary band of ∼100 kDa in CNS tissues that is far less detectable in peripheral tissue extracts. Recombinant PDE2A with His tag expressed in sf21 cells was loaded as a positive control. Equivalent loading was demonstrated by probing the same blot with an anti-actin antibody. Positive control was loaded with less protein to avoid overloading the lane.

Figure 3

Western blot analysis of human brain extracts probed with anti-PDE2A antibody with (B) and without (A) antigenic peptide competition. Molecular mass standards are shown in left and right lanes and recombinant PDE2A-positive control is shown in the near far-right lane. pAb PD2A-101AP recognizes a primary band of ∼100 kDa in human brain tissues (A, 20 μg sample loaded per lane). The 100-kDa band is prominently observed in human striatum, cingulate cortex, prefrontal cortex, and hippocampus, with less abundance in the substantia nigra and virtually no detectable signal in cerebellum. (B) Identical blot probed with PDE2A-101AP in the presence of the antigenic peptide at a 300× molar excess. Equivalent loading was demonstrated by probing the same blot with an anti-actin antibody, shown on bottom panel.

Figure 4

PDE2A immunoreactivity (ir) is abundantly expressed in mammalian forebrain. (A) Sagittal section of rat brain stained with isotype control showing hematoxylin counterstain and a lack of PDE2A immunoperoxidase staining. (B) Section of rat brain near-adjacent to that shown in A demonstrates strong PDE2A ir prominently in forebrain structures including the cortex, striatum, hippocampus, and substantia nigra. Bar = 2.5 mm.

Figure 5

PDE2A staining is not detected in PDE2A knockout embryos. Developing mouse cortex from wild-type (A,B) and PDE2A knockout (C) embryonic day–15 mouse fetus stained with anti-PDE2A antibody (A,C) or negative control antibody (B). In wild-type embryos, PDE2A staining is present in cerebrovascular endothelium (arrows) and in neurons and neuropil of the superficial layers of the developing neuroepithelium. No detectable immunoperoxidase reaction product is observed in corresponding regions from PDE2A knockout fetuses (C) or in sections incubated with negative-control antibody (B). Bar = 40 μm.

Figure 6

PDE2A is highly expressed in rat habenula. (A) Coronal section of rat forebrain incubated with anti-PDE2A antibody immunoadsorbed with PDE2A peptide immunogen. (B) Near-adjacent coronal section demonstrating PDE2A immunoperoxidase staining in the rat habenula. (C) Chromogenic in situ hybridization of PDE2A in rat habenula. Blue reaction product is abundant in neurons of the medial habenula. Sections are counterstained with hemotoxylin (A,B) or Nuclear Fast Red (C). Bar = 100 μm.

Figure 7

PDE2A ir is abundantly expressed in rat brain, with discrete staining patterns in many regions. (A) PDE2A ir in the somatosensory cortex shows a distinctive laminar staining distribution (laminae I–VI are indicated). (B) In layer III of the isocortex, fine neuropil staining of terminals and varicosities is present, and isolated immunoreactive non-pyramidal neurons show PDE2A ir in the neuronal cytoplasm. (C) Dorsal hippocampus demonstrates neuropil staining throughout multiple layers, including stratum oriens (so), stratum radiatum (sr), and dentate gyrus (dg). Neuron cell bodies within the CA pyramidal neuron layers (sp) as well as dentate gyrus granule neurons (gc) lack PDE2A ir. (D) High magnification of PDE2A ir in CA3 hippocampal mossy fibers. (E) Low magnification image of midbrain showing intense PDE2A ir fibers with the substantia nigra pars reticulata (SNPr) and neuropil staining in the amygdala and thalamus (th). (F) A subset of substantia nigra pars compacta neurons (SNPc) express intraneuronal PDE2A ir, and strong fiber staining is present in the SNPr. Bars: A,C = 250 μm; B = 40 μm; D,F = 50 μm; E = 500 μm.

Figure 8

PDE2A ir is observed in rat dorsal root ganglion (A) and dorsal horn of the spinal cord (B). Dorsal root ganglion shows PDE2A ir associated with nearly all ganglion cells in rat. No immunoreactivity is observed in dorsal root and non-ganglionic tissue. (B) Dorsal horn of rat spinal cord exhibiting a diffuse punctate staining pattern throughout the superficial spinal cord laminae. Bars: A = 150 μm; B = 100 μm.

Figure 9

PDE2A ir is present in endothelium of many tissue types. (A) Rat heart shows lack of cardiac myocyte staining but labeling of endothelial cells. (B) Rat liver shows the presence of PDE2A ir in central vein and sinusoidal endothelium but not in hepatocytes. (C) Rat kidney section shows endothelial staining in glomerulus (arrow) and in neighboring veins. Note the lack of endothelial staining in a small arteriole indicated by the arrowhead. (D) Specialty types of endothelium, such as high endothelial venules, were also shown to express PDE2A ir, as seen in section of human gut-associated lymphoid tissue (arrow). Bars: A–C = 50 μm; D =100 μm.

Figure 10

The distribution of PDE2A ir is conserved across mammalian species. Adrenal cortex from rat (A), mouse (C), dog (E), cynomolgus monkey (G), and human (I) demonstrates strong immunoreaction product within the zona glomerulosa. Brain PDE2A ir in dorsolateral striatum adjacent to the corpus callosum from rat (B), mouse (D), dog (F), cynomolgus monkey (H), and human (J). PDE2A ir is similar in rat, mouse, dog, cynomolgus monkey, and human tissue. Bar = 50 μm.