Characterization of Recombinant Mouse ecto-5'-nucleotidase

Chronic pain is the most common medical complaint in the United States and the number of Americans affected by chronic pain is growing. Effectiveness of available treatments varies greatly and many have intolerable side effects. To address this need for pain treatments, more effective therapies are needed. Recently, our lab discovered an ectonucleotidase in the central nervous system (CNS) called Prostatic Acid Phosphatase (PAP). Spinal injections of PAP into live mice produced antinociceptive effects. The following thesis describes the properties of NT5E, an ectonucleotidase that is colocalized with PAP in the CNS. We have generated a recombinant mouse NT5E protein (mNT5E) and have shown that the protein is pure, catalytically active and capable of reducing nociceptive sensitivity in two animal models of chronic pain. We also show that mNT5E acts through A1-type adenosine receptors (A1R). This study provides an additional target for developing pain treatments and a new tool for studying purinergic signaling

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

<p>Abstract</p> <p>Background</p> <p>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 <it>in vivo</it>.</p> <p>Results</p> <p>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 α,β-methylene-adenosine 5'-diphosphate (α,β-me-ADP; IC₅₀= 0.43 μM), 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 A₁receptor (<it>A</it>₁<it>R, Adora1</it>) knockout mice.</p> <p>Conclusion</p> <p>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.</p