Recombinant ecto-5'-nucleotidase (CD73) has long lasting antinociceptive effects that are dependent on adenosine A1 receptor activation

Abstract

Background: Ecto-5'-nucleotidase (NT5E, also known as CD73) hydrolyzes extracellular adenosine 5'-monophosphate (AMP) to adenosine in nociceptive circuits. Since adenosine has antinociceptive effects in rodents and humans, we hypothesized that NT5E, an enzyme that generates adenosine, might also have antinociceptive effects in vivo.

Results: To test this hypothesis, we purified a soluble version of mouse NT5E (mNT5E) using the baculovirus expression system. Recombinant mNT5E hydrolyzed AMP in biochemical assays and was inhibited by alpha,beta-methylene-adenosine 5'-diphosphate (alpha,beta-me-ADP; IC50 = 0.43 microM), a selective inhibitor of NT5E. mNT5E exhibited a dose-dependent thermal antinociceptive effect that lasted for two days when injected intrathecally in wild-type mice. In addition, mNT5E had thermal antihyperalgesic and mechanical antiallodynic effects that lasted for two days in the complete Freund's adjuvant (CFA) model of inflammatory pain and the spared nerve injury (SNI) model of neuropathic pain. In contrast, mNT5E had no antinociceptive effects when injected intrathecally into adenosine A1 receptor (A1R, Adora1) knockout mice.

Conclusion: Our data indicate that the long lasting antinociceptive effects of mNT5E are due to hydrolysis of AMP followed by activation of A1R. Moreover, our data suggest recombinant NT5E could be used to treat chronic pain and to study many other physiological processes that are regulated by NT5E.

Figure 1

Purification of recombinant mNT5E. (A) Diagram of the GST-mNT5E expression construct. (Top) Native mNT5E contains an N-terminal signal peptide (ss-cleavage) and GPI anchor site. (Bottom) The GST-mNT5E fusion construct contains the signal peptide from gp67 of baculovirus Autographica californica, GST, a thrombin cleavage site, the catalytic domain of mNT5E and (His)₆ tag. Translation start and stop codons are indicated. (B) GelCode blue-stained SDS-PAGE gel and (C) western blot of purified recombinant mNT5E protein (0.05 μg). The western blot was probed with an anti-mNT5E antibody.

Figure 2

mNT5E dephosphorylates AMP and can be inhibited by α,β-me-ADP. (A) Plot of initial velocity at the indicated concentrations of AMP at pH 7.0. Reactions (n = 3 per point) were stopped after 3 min. (B) The indicated concentrations of α,β-me-ADP were added to reactions (n = 3 per concentration) containing mNT5E (0.07 μg/μL) and 400 μM AMP at pH 7.0. (A, B) Inorganic phosphate was measured using malachite green. All data are presented as means ± s.e.m. Some error bars are obscured due to their small size. GraphPad Prism 5.0 was used to generate curves.

Figure 3

Dose-dependent antinociceptive effects of intrathecal mNT5E. Effects of the indicated amounts of mNT5E on (A) paw withdrawal latency to a radiant heat source and (B) paw withdrawal threshold (electronic von Frey apparatus). BL = Baseline. Injection (i.t.) volume was 5 μL. n = 10 wild-type mice were used per dose. Paired t tests were used to compare responses between BL values and later time points for each group. *P < 0.05, ** P < 0.005; *** P < 0.0005. All data are presented as means ± s.e.m.

Figure 4

mNT5E has antihyperalgesic and antiallodynic effects in WT mice following inflammation and nerve injury. WT and A₁R^-/- mice were tested for (A, C) noxious thermal and (B, D) mechanical sensitivity before (baseline, BL) and after injection of CFA into one hindpaw (A, B; arrow) or following nerve injury (C, D; SNI, arrow). (A, B) One or (C, D) six days later, mNT5E protein (1.7 U) was injected i.t. into all mice (arrowhead) then thermal and mechanical sensitivity was measured for several days. Inflamed/injured and non-inflamed/non-injured (control) hindpaws were tested. We used a 1.7 U dose to conserve protein and because it was nearly as effective as the 2.5 U dose (compare thermal antinociceptive effects in Figure 3A to panels A and C-control paws). Paired t testes were used to compare responses at each time point between genotypes (n = 10 animals per genotype). *P < 0.05, **P < 0.005, ***P < 0.0005. All data are presented as means ± s.e.m.