Ecto-5'-nucleotidase (CD73) inhibits nociception by hydrolyzing AMP to adenosine in nociceptive circuits

Abstract

Ecto-5'-nucleotidase (NT5E, CD73) is a membrane-anchored protein that hydrolyzes extracellular adenosine 5'-monophosphate (AMP) to adenosine in diverse tissues but has not been directly studied in nociceptive neurons. We found that NT5E was located on peptidergic and nonpeptidergic nociceptive neurons in dorsal root ganglia (DRG) and on axon terminals in lamina II (the substantia gelatinosa) of spinal cord. NT5E was also located on epidermal keratinocytes, cells of the dermis, and on nociceptive axon terminals in the epidermis. Following nerve injury, NT5E protein and AMP histochemical staining were coordinately reduced in lamina II. In addition, AMP hydrolytic activity was reduced in DRG neurons and spinal cord of Nt5e(-/-) mice. The antinociceptive effects of AMP, when combined with the adenosine kinase inhibitor 5-iodotubericidin, were reduced by approximately 50% in Nt5e(-/-) mice and were eliminated in Adenosine A(1) receptor (A(1)R, Adora1) knock-out mice. Additionally, Nt5e(-/-) mice displayed enhanced sensitivity in the tail immersion assay, in the complete Freund's adjuvant model of inflammatory pain and in the spared nerve injury model of neuropathic pain. Collectively, our data indicate that the ectonucleotidase NT5E regulates nociception by hydrolyzing AMP to adenosine in nociceptive circuits and represents a new molecular target for the treatment of chronic pain. Moreover, our data suggest NT5E is well localized to regulate nucleotide signaling between skin cells and sensory axons.

Figure 1.

NT5E is found on most nonpeptidergic and some peptidergic nociceptive neurons. A–U, Mouse L3–L5 DRG neurons were stained for sensory neuron markers (green) and with antibodies against NT5E (red). Arrowheads mark examples of double-labeled cells. Images were acquired by confocal microscopy. Scale bar: (in U) A–U, 50 μm.

Figure 2.

NT5E is colocalized with peptidergic and nonpeptidergic markers on axon terminals in dorsal spinal cord. Lumbar spinal cord sections were stained for selected axonal markers (A, D, G, J; green) and NT5E (B, E, H, K; red). C, F, I, L, Merged images. IB4, MrgprdΔ^EGFPf and PAP mark nonpeptidergic endings in lamina II. CGRP marks peptidergic endings in laminas I, II, and V. Images were acquired by confocal microscopy. Scale bar: (in L) A–L, 100 μm.

Figure 3.

NT5E is primarily found in nonpeptidergic nerve terminals in the epidermis. A–I, Confocal images of mouse glabrous skin immunostained for NT5E (red) and the indicated markers (green). C, F, I, Nuclei were pseudocolored gray to highlight stratification of the epidermis. Scale bar: (in I) A–I, 50 μm.

Figure 4.

NT5E protein and AMP hydrolytic activity are reduced in axon terminals following peripheral nerve injury. A–D, Sections of lumbar spinal cord from a mouse killed 14 d after ligation and transection of the sural and common peroneal branches of the sciatic nerve (the SNI model). A, Stained using AMP histochemistry (3 mm AMP; buffer pH was 7.0). B–D, An adjacent section labeled for the indicated markers. Arrowheads delimit the region of dorsal spinal cord where staining was reduced ipsilateral (ipsi) to the injured nerves. Contralateral (contra), uninjured side. Similar results were seen in n = 2 mice. Scale bar: (in D) A–D, 200 μm.

Figure 5.

AMP hydrolytic activity is reduced in nociceptive circuits of Nt5e^−/− mice. A–H, Lumbar DRG (A, B), lumbar spinal cord (C–F) and cultured DRG neurons (G, H) from WT and Nt5e^−/− adult mice stained using AMP histochemistry. Arrows point to epineurium (en). C, D, Arrowheads mark the location of axon terminals in dorsal spinal cord (lamina II). E, F, Higher magnification of C, D. G, H, The plasma membrane was not permeabilized so that extracellular AMP hydrolytic activity could be assayed. Arrowheads point to neurites emanating from cultured neurons. AMP (6 mm in A, B, G, H and 3 mm in C–F) was used as substrate, and buffer was pH 7.0. Scale bars: (in B, H) A, B, G, H, 50 μm; (in D) C, D, 500 μm; (in F) E, F, 200 μm.

Figure 6.

Nt5e^−/− mice show enhanced nociceptive responses following inflammation and nerve injury. A, B, CFA inflammatory pain model. WT and Nt5e^−/− mice were tested for (A) thermal and (B) mechanical sensitivity before (BL) and following injection of CFA (arrow) into one hindpaw. The contralateral hindpaw served as control. C, D, SNI neuropathic pain model. WT and Nt5e^−/− were tested for (C) thermal and (D) mechanical sensitivity before (BL) and after ligation and transaction of the sural and common peroneal branches of the sciatic nerve. A–D, Paired t tests were used to compare responses at each time point between WT and Nt5e^−/− mice (n = 10 per genotype); same paw comparisons. *p < 0.05, **p < 0.005, ***p < 0.0005. All data are presented as means ± SEM.

Figure 7.

The antinociceptive effects of AMP + ITU are reduced in Nt5e^−/− mice and are dependent on A₁R activation. A, B, Noxious thermal sensitivity was measured before (baseline, BL) and after intrathecal injection of AMP (200 nmol) (A), ITU (5 nmol) (B), or AMP (200 nmol) + ITU (5 nmol) (C). Paired t tests were used to compare responses at each time point to BL values (black asterisks) within a given genotype (n = 8 mice per genotype). Paired t tests were also used to compare responses at each time point between WT and Nt5e^−/− mice (red asterisks). D, Mice were tested for noxious thermal sensitivity before (BL) and after injection of CFA (arrowhead) into one hindpaw. The uninjected hindpaw served as control. One day later, all mice were injected intrathecally with AMP (200 nmol) + ITU (5 nmol) and thermal sensitivity was measured for several hours after injection. Paw withdrawal latencies were significantly elevated in both genotypes for up to 2 h following AMP + ITU injection, relative to the response 1 d after CFA injection (same paw comparisons). Paired t tests were used to compare responses at each time point between WT and Nt5e^−/− mice (red asterisks; same paw comparisons). n = 8 mice per genotype. *p < 0.05, **p < 0.005, ***p < 0.0005. All data are presented as means ± SEM.