Prostatic acid phosphatase is expressed in peptidergic and nonpeptidergic nociceptive neurons of mice and rats

Abstract

Thiamine monophosphatase (TMPase, also known as Fluoride-resistant acid phosphatase or FRAP) is a classic histochemical marker of small- to medium-diameter dorsal root ganglia (DRG) neurons and has primarily been studied in the rat. Previously, we found that TMPase was molecularly identical to Prostatic acid phosphatase (PAP) using mice. In addition, PAP was expressed in a majority of nonpeptidergic, isolectin B4-binding (IB4+) nociceptive neurons and a subset of peptidergic, calcitonin gene-related peptide-containing (CGRP+) nociceptive neurons. At the time, we were unable to determine if PAP was present in rat DRG neurons because the antibody we used did not cross-react with PAP in rat tissues. In our present study, we generated a chicken polyclonal antibody against the secretory isoform of mouse PAP. This antibody detects mouse, rat and human PAP protein on western blots. Additionally, this antibody detects PAP in mouse and rat small- to medium-diameter DRG neurons and axon terminals in lamina II of spinal cord. In the rat, 92.5% of all PAP+ cells bind the nonpeptidergic marker IB4 and 31.8% of all PAP+ cells contain the peptidergic marker CGRP. Although PAP is found in peptidergic and nonpeptidergic neurons of mice and rats, the percentage of PAP+ neurons that express these markers differs between species. Moreover, PAP+ axon terminals in the rat partially overlap with Protein kinase Cgamma (PKCgamma+) interneurons in dorsal spinal cord whereas PAP+ axon terminals in the mouse terminate dorsal to PKCgamma+ interneurons. Collectively, our studies highlight similarities and differences in PAP localization within nociceptive neurons of mice and rats.

Figure 1. Chicken antibody detects mouse, rat and human PAP on western blots.

(A) Western blot containing purified recombinant mPAP protein and pure hPAP protein probed with chicken (Ck) anti-PAP antibody. (B) Duplicate gel stained with GelCode blue to confirm that equivalent amounts of protein were loaded. (C) Western blot of cell lysates from untransfected HEK 293 cells and HEK 293 cells transfected with rTM-PAP or mTM-PAP.

Figure 2. Chicken antibody detects PAP in mouse DRG neurons and dorsal spinal cord.

(A–D) Sections from mouse L4-L6 DRG and (E–L) lumbar spinal cord were stained with chicken anti-PAP antibodies (red) and with antibodies against various sensory neuron markers and spinal interneuron marker PKCγ (blue, green). (D, H, L) Merged images. All images were acquired by confocal microscopy. Scale bar in (D) 50 µm, (H) 100 µm.

Figure 3. Chicken antibody detects PAP in rat DRG neurons and dorsal spinal cord.

(A–D) Sections from rat L4-L6 DRG and (E–L) lumbar spinal cord were stained with chicken anti-PAP antibodies (red) and with antibodies against various sensory neuron markers and spinal interneuron marker PKCγ (blue, green). (D, H, L) Merged images. All images were acquired by confocal microscopy. Scale bar in (D) 50 µm, (H) 100 µm.