ATP metabolizing enzymes ENPP1, 2 and 3 are localized in sensory neurons of rat dorsal root ganglion

Abstract

In dorsal root ganglion (DRG) neurons, ATP is an important neurotransmitter in nociceptive signaling through P2 receptors (P2Rs) such as P2X2/3R, and adenosine is also involved in anti-nociceptive signaling through adenosine A1R. Thus, the clearance system for adenine nucleotide/nucleoside plays a critical role in regulation of nociceptive signaling, but there is little information on it, especially ectoenzyme expression profiles in DRG. In this study, we examined expression and localization of ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPPs), by which ATP is metabolized to AMP, in rat DRG. The mRNA expression levels of ENPP2 were greater than those of ENPP1 and ENPP3 in rat DRGs. On immunohistochemical analysis, ENPP1, 2 and 3 were found in soma of DRG neurons. Immunopositive rate of ENPP3 was greater than that of ENPP1 and ENPP2 in all DRG neurons. ENPP3, as compared with ENPP1 and ENPP2, was expressed mainly by isolectin B4-positive cells, and slightly by neurofilament 200-positive ones. In this way, the expression profile of ENPP1, 2 and 3 was different in DRGs, and they were mainly expressed in small/medium-sized DRG neurons. Moreover, ENPP1-, 2- and 3-immunoreactivities were colocalized with P2X2R, P2X3R and prostatic acid phosphatase (PAP), as an ectoenzyme for metabolism from AMP to adenosine. Additionally, PAP-immunoreactivity was colocalized with equilibrative nucleoside transporter (ENT) 1, as an adenosine uptake system. These results suggest that the clearance system consisted of ENPPs, PAP and ENT1 plays an important role in regulation of nociceptive signaling in sensory neurons.

Keywords: ATP, ectoenzyme; Ecto-nucleotide pyrophosphatase/phosphodiesterase; clearance system; dorsal root ganglion; sensory neuron..

Figure 1.

Expression of mRNA for NTPDases and ENPPs in rat DRG. mRNA expression of NTPDases (A) and ENPPs (B) in rat DRG was quantitatively analyzed by real-time PCR. The amount of mRNA was normalized by comparison with that of mRNA for NTPDase1 (A) or ENPP1 (B). Each bar represents the mean + SD (N=3-7).

Figure 2.

Immunohistochemical analysis of NTPDase2 in rat DRG. Immunostaining for NTPDase2 (red) with GFAP (green)-positive satellite cells and nucleus marker Hoechst 33258 (blue) was performed. Arrowheads indicate the colocalization of NTPDase2 and GFAP. The lower panel shows a magnified image of the square area in the upper panel. Representative images from three independent experiments are shown. Scale bar: 50 m.

Figure 3.

Immunohistochemical analysis of ENPP1, ENPP2 and ENPP3 in rat DRG. A-C) Representative images of ENPP1, ENPP2 and ENPP3 in rat DRG are shown (n=4); nuclei were stained with Hoechst 33258; the insets show magnified images of the square areas; scale bar: 50 m. D-F) Distribution of ENPP1-, ENPP2- and ENPP3-immunofluoresent intensity in the DRG neuronal cell body (soma) vs cell size for all DRG neurons is shown in panels D, E and F, respectively. G-I) Size distribution histograms of ENPP1-, ENPP2- and ENPP3-positive DRG neurons, respectively. DRG neurons were classified as ENPP1-, ENPP2- or ENPP3-positive when the cytoplasmic intensity was three SDs above the background, divided into small- (less than 750 m), medium- (750-1750 m), and large- (larger than 1750 m) DRG neurons. Each bar represents the mean + SD (n=4).

Figure 4.

Distribution of ENPP1 in rat DRG neurons. Double-immunostaining for a neuronal marker (green) and ENPP1 (red) is shown. IB4 (A), CGRP (B), NF200 (C), P2X2R (D), P2X3R (E), and PAP (F) were used as neuronal markers. Representative images from three to four independent experiments are shown. Scale bar: 50 m.

Figure 5.

Distribution of ENPP2 in rat DRG neurons. Double-immunostaining for a neuronal marker (green) and ENPP2 (red) is shown. IB4 (A), CGRP (B), NF200 (C), P2X2R (D), P2X3R (E), and PAP (F) were used as neuronal markers. Representative images from four to five independent experiments are shown. Scale bar: 50 m.

Figure 6.

Distribution of ENPP3 in rat DRG neurons. Double-immunostaining for a neuronal marker (green) and ENPP3 (red) is shown. IB4 (A), CGRP (B), NF200 (C), P2X2R (D), P2X3R (E), and PAP (F) were used as neuronal markers. Representative images from three to four independent experiments are shown. Scale bar: 50 m.

Figure 7.

Immunohistochemical analysis of ENT1 and PAP in rat DRG. Double-immunostaining for ENT1 (red) and PAP (green) is shown. Representative images from four independent experiments are shown. Scale bar: 50 m.