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

Recombinant ecto-5'-nucleotidase (CD73) has long lasting antinociceptive effects that are... 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 α,β-methylene-adenosine 5'-diphosphate (α,β-me-ADP; IC = 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 A receptor (A R, Adora1) knockout 1 1 mice. Conclusion: Our data indicate that the long lasting antinociceptive effects of mNT5E are due to hydrolysis of AMP followed by activation of A R. 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. Background known as ACPP, Fluoride-resistant acid phosphatase or Ecto-5'-nucleotidase (NT5E) is a glycosyl phosphati- thiamine monophosphatase) in nociceptive neurons. Like dylinositol (GPI)-anchored membrane protein that cata- NT5E, PAP functions as an ectonucleotidase in nocicep- lyzes the hydrolysis of extracellular AMP to adenosine tive neurons by hydrolyzing AMP to adenosine [10,11]. -/- -/- [1]. NT5E regulates diverse physiological processes that Both Pap and Nt5e mice show enhanced thermal are modulated by adenosine, including hypoxia, inflam- hyperalgesia in animal models of inflammatory pain and mation and epithelial ion transport [2-8]. Recently, we neuropathic pain as well as enhanced mechanical allo- -/- found that NT5E is expressed in peptidergic and nonpep- dynia following inflammation [9,10]. Interestingly, A R tidergic nociceptive (pain-sensing) neurons and their mice also show enhanced sensitization following inflam- axon terminals in spinal cord and skin [9]. Based on mation and nerve injury [12]. Thus, deficiencies in ade- -/- experiments with Nt5e mice, we established that NT5E nosine production or A R signaling cause similar accounts for ~50% of all AMP hydrolytic activity in noci- behavioral phenotypes. ceptive neurons [9]. In support of an A R-dependent mechanism, we found In addition, we observed that NT5E was extensively co- that intrathecal (i.t.) injection of secretory PAP protein localized with Prostatic acid phosphatase (PAP, also (from mouse or human) into wild-type mice had long lasting antinociceptive, antihyperalgesic and antiallo- * Correspondence: zylka@med.unc.edu dynic effects that were entirely dependent on A R activa- Department of Cell and Molecular Physiology, UNC Neuroscience Center, tion [10,11]. These data suggested that spinal delivery of University of North Carolina, CB #7545, Chapel Hill, North Carolina 27599, USA Full list of author information is available at the end of the article © 2010 Sowa et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in BioMed Central any medium, provided the original work is properly cited. Sowa et al. Molecular Pain 2010, 6:20 Page 2 of 8 http://www.molecularpain.com/content/6/1/20 PAP protein could be used therapeutically to generate adenosine and activate A R over an extended time period. Likewise, direct activation of A R with adenosine or selective A R agonists had antinociceptive effects in rodents and humans [13-31]. Considering that both PAP and NT5E generate adenos- ine, we hypothesized that NT5E protein might also have A R-dependent antinociceptive effects. However, we were unable to test this hypothesis because mammalian NT5E protein was not available. As emphasized in a recent review by Colgan and colleagues, this lack of a reli- able source of purified protein has hindered studies with NT5E [2]. Others used 5'-nucleotidase protein purified from rattlesnake (Crotalus atrox) venom instead [4,32,33]. Crotalus 5'-nucleotidase had no deleterious effects when injected intraperitoneally and rescued phe- -/- notypes in Nt5e mice; however, possible toxicity from venom contaminants remains a concern--especially if this venom-derived protein were to be used in the nervous system. To overcome these toxicity concerns, we purified and Figure 1 Purification of recombinant mNT5E. (A) Diagram of the characterized (in vitro and in vivo) a secretory version of GST-mNT5E expression construct. (Top) Native mNT5E contains an N- recombinant mouse NT5E. Our present study builds terminal signal peptide (ss-cleavage) and GPI anchor site. (Bottom) The upon work by Servos and colleagues who purified a GST-mNT5E fusion construct contains the signal peptide from gp67 of baculovirus Autographica californica, GST, a thrombin cleavage site, the recombinant but non-secretory version of rat NT5E catalytic domain of mNT5E and (His) tag. Translation start and stop using the baculovirus expression system [34]. codons are indicated. (B) GelCode blue-stained SDS-PAGE gel and (C) western blot of purified recombinant mNT5E protein (0.05 μg). The Results western blot was probed with an anti-mNT5E antibody. Purification of soluble mouse NT5E using the baculovirus expression system cleavage site to permit removal of GST and a C-terminal NT5E is anchored to the membrane via a GPI linkage on hexahistidine (His) tag (Figure 1A). Ser523 [35]. In an effort to produce a secreted and soluble Two days after infecting Hi5 insect cells with recombi- (non-membrane anchored) version of NT5E, Servos and nant baculovirus we detected GST-mNT5E protein in the colleagues generated a baculovirus expression construct tissue culture supernatant at approximately 10 mg/L. of rat NT5E that reportedly lacked the GPI-anchor at This observation suggested that exclusion of Ser523 was Ser523 [34]. While Servos and colleagues successfully important for producing a soluble version of NT5E. used their construct to purify a catalytically active version Additionally, based on SDS-PAGE and western blotting, of rat NT5E, they did not detect NT5E in the tissue cul- the GST-mNT5E found in the medium was largely intact ture medium as would be expected if the protein were whereas cell lysates contained truncated and intact ver- secreted and soluble. Instead, NT5E was only present in sions of mNT5E-GST (data not shown). We next purified cell lysates. Upon re-examination of the cloning strategy mNT5E from the culture supernatant in two steps (see used by Servos and colleagues we noticed that their Methods). We reasoned that GST, a protein that binds expression construct included Ser523 but excluded glutathione, might interfere with physiological or behav- neighboring Ser526. Using GPI prediction software [36], ioral studies if administered in vivo. So as part of our we confirmed that Ser523 (but not Ser526) was the most purification procedure, the GST fusion was removed by likely GPI anchor site. Inclusion of this GPI anchor thrombin cleavage. Protein purity was analyzed under sequence could explain why Servos and colleagues did denaturing conditions with GelCode Blue protein stain not detect NT5E in the culture medium. We thus gener- (Figure 1B) and western blotting with an anti-NT5E anti- ated a mouse NT5E (Trp29-Phe522) expression construct body (Figure 1C). We observed a single band at ~62 kDa, that was truncated just before Ser523 (Figure 1A). Our corresponding to the calculated molecular weight of ung- construct was otherwise identical to the one used by Ser- lycosylated mNT5E with a (His) tag (61.7 kDa). No addi- vos and colleagues--our construct contained a gp67 sig- nal peptide, glutathione S-transferase (GST), a thrombin Sowa et al. Molecular Pain 2010, 6:20 Page 3 of 8 http://www.molecularpain.com/content/6/1/20 tional bands were observed, indicating that mNT5E found that α,β-me-ADP (0.01-500 μM) did not inhibit protein was intact. recombinant mouse PAP when AMP was used as sub- The activity of secreted mNT5E was initially deter- strate (data not shown). Production of recombinant mined in vitro. Purified recombinant mNT5E protein mouse PAP was described previously [11]. dephosphorylated AMP with a K of 26 μM (Figure 2A). mNT5E has long lasting antinociceptive effects that are A R This K value is in agreement with the previously dependent reported range of 1-50 μM using AMP as substrate at pH We previously found that a single intrathecal injection of 7.0 [8,34]. Recombinant mNT5E was also inhibited by PAP had antinociceptive, antihyperalgesic and antiallo- α,β-me-ADP (IC = 0.43 μM; Figure 2B), a commonly dynic effects that lasted for three days and that were used inhibitor of NT5E [1]. Although recombinant NT5E dependent on A R activation [10,11]. To empirically iden- was not used, others obtained a slightly higher IC value tify an effective dose of mNT5E for in vivo studies and to of 3.6 μM with this inhibitor [37]. For comparison, we determine if mNT5E had long lasting antinociceptive effects, we intrathecally injected wild-type mice with increasing doses of recombinant mNT5E protein (Figure 3). Time points were based on our previous studies with PAP [10,11]. We then measured noxious thermal and mechanical sensitivity before (baseline, BL) and after mNT5E injection. Six hours post i.t. injection, paw with- drawal latency to the noxious thermal stimulus was sig- nificantly increased relative to controls and remained elevated for two days at all doses tested (Figure 3A). Intrathecal injection of mNT5E did not alter mechanical sensitivity (Figure 3B) nor did it cause paralysis or seda- tion at any of the doses tested. We previously found that PAP (from human, cow and mouse) also had selective thermal but not mechanical antinociceptive effects in naïve mice and had no obvious motor side effects [10,11]. We next evaluated the antinociceptive effects of mNT5E in the CFA model of inflammatory pain and the SNI model of neuropathic pain. We used wild-type (WT) -/- and A R mice, to evaluate dependence on A R activa- 1 1 tion. We used the contralateral (non-inflamed/non- injured) paw as a control. As seen previously [9,10,12], -/- A R mice displayed enhanced thermal hyperalgesia after CFA injection and after nerve injury relative to WT mice (Figure 4A, C). In both chronic pain models, a single i.t. injection of mNT5E had long lasting (at least 2 days) thermal antihyperalgesic and mechanical antiallodynic effects in the inflamed/injured paw of WT mice but not -/- A R mice (Figure 4A-D). Indeed, thermal sensitivity transiently returned to baseline levels in the injured/ inflamed paws following mNT5E injection whereas mechanical sensitivity approached but did not reach baseline levels. Consistent with our dose-response study above, mNT5E had thermal but not mechanical antinoci- Figure 2 mNT5E dephosphorylates AMP and can be inhibited by ceptive effects in the control (non-inflamed/non-injured) α,β-me-ADP. (A) Plot of initial velocity at the indicated concentrations paws of WT mice. mNT5E had no antinociceptive effects of AMP at pH 7.0. Reactions (n = 3 per point) were stopped after 3 min. -/- in A R mice, highlighting a critical dependence on A R (B) The indicated concentrations of α,β-me-ADP were added to reac- 1 1 tions (n = 3 per concentration) containing mNT5E (0.07 μg/μL) and 400 activation. We previously found that control injections μM AMP at pH 7.0. (A, B) Inorganic phosphate was measured using had no effect on thermal or mechanical sensitivity in malachite green. All data are presented as means ± s.e.m. Some error these mouse models [10,11]. When combined with bars are obscured due to their small size. GraphPad Prism 5.0 was used numerous studies of NT5E by others (reviewed by [2]), to generate curves. Sowa et al. Molecular Pain 2010, 6:20 Page 4 of 8 http://www.molecularpain.com/content/6/1/20 Figure 3 Dose-dependent antinociceptive effects of intrathecal mNT5E. Effects of the indicated amounts of mNT5E on (A) paw withdrawal la- tency 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. our data suggest that all the antinociceptive effects of explain why saturating doses of PAP or NT5E protein do mNT5E are due to production of adenosine and activa- not paralyze mice. Moreover, adenosine can be elimi- tion of A R on DRG neurons and/or spinal neurons nated from the extracellular space by nucleoside trans- porters and metabolic enzymes, including adenosine [38,39]. deaminase and adenosine kinase [40,44]. These compet- ing processes of adenosine production and elimination Discussion place an upper limit on how much extracellular adenos- Adenosine and selective A R agonists have well-studied ine is available to activate A R in animals. In support of antinociceptive effects in rodents and humans [26,40,41]. 1 this, adenosine deaminase inhibitors and adenosine These agonists are often delivered intrathecally to avoid kinase inhibitors prolong the extracellular bioavailability side effects associated with peripheral A R activation of adenosine and have antinociceptive effects in animals [42]. However, A R agonists do have side effects when [17,21,45,46]. delivered intrathecally at high doses, including overt Unexpectedly, our studies revealed that two molecu- motor paralysis [10,43]. These motor side effects are larly distinct ectonucleotidases (PAP and NT5E) have likely due to activation of A R on motor neurons [39]. pronounced antinociceptive effects that persist for an Our present study indicates that NT5E, an ectonucleoti- extended time period (2-3 days). This long duration of dase that hydrolyzes AMP to adenosine, provides an action in vivo could not have been predicted from the alternative means of activating A R for therapeutic pur- biochemical properties of these enzymes alone. More poses without causing overt motor paralysis. Although importantly, our findings suggest ectonucleotidases rep- PAP also has A R-dependent antinociceptive effects resent a new class of antinociceptive drugs with potential when injected intrathecally [10,11], PAP shares no therapeutic advantages over adenosine, A R agonists and sequence similarity to NT5E. This lack of similarity made inhibitors of adenosine metabolism. For example, the it impossible for us to determine a priori if recombinant long duration of action of ectonucleotidases could be use- mNT5E would be catalytically active, stable and effective ful in situations where there is a need to provide a sus- when tested in live animals. tained level of A R activation. Moreover, recombinant As previously discussed [11], ectonucleotidases are cat- NT5E could be used to further study the role of NT5E in alytically restricted in that they generate adenosine in pain mechanisms and to study many other physiological proportion to substrate availability. Catalytic restriction processes that are regulated by NT5E [2,9]. limits the amount of adenosine produced and could Sowa et al. Molecular Pain 2010, 6:20 Page 5 of 8 http://www.molecularpain.com/content/6/1/20 -/- Figure 4 mNT5E has antihyperalgesic and antiallodynic effects in WT mice following inflammation and nerve injury. WT and A R 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. Methods fusion tag, with a single thrombin cleavage site between Molecular biology the GST tag and the coding sequence of mNT5E. The The GST-mNT5E baculovirus expression plasmid was final plasmid was sequence verified. generated by PCR amplification of mouse NT5E (nt 131- mNT5E protein purification 1696 from GenBank accession # NM_011851.3) using The GST-mNT5E plasmid was used to generate recombi- Phusion polymerase and a full-length expression con- nant mNT5E protein using the BD BaculoGold Expres- struct of mNT5E as template. Primer sequences con- sion System (BD Biosciences). Briefly, we infected Hi5 tained EcoRI sites (in bold italics) to facilitate cloning into insect cells with high-titer recombinant baculovirus, pAcSecG2T (BD Biosciences). N-terminal primer: 5'- incubated the cells for 48 hours at 27°C and then removed cgcgaattcattgggagctcacgatcctgcacaca. C-terminal (His) the cells from the supernatant by centrifugation. The tag primer: 5'-gcggaattcttaatgatgatgatgatgatggaacttgatc- supernatant containing secreted GST-mNT5E was fil- cgcccttcaacg. These primers amplify a product that con- tered (0.45 μm pore size, Millipore) and concentrated in tains the catalytic domain of mNT5E fused to the (His) PBS (10 mM sodium phosphate, 140 mM NaCl, pH 7.4) epitope tag but that lacks the signal peptide and GPI using a Millipore cartridge with a 10 k retention cutoff. anchor sequence (located at Ser523). This mNT5E PCR The concentrated supernatant was loaded onto a 5 mL product was subcloned in frame with an N-terminal GST GSTrap FF column (GE Healthcare) using a peristaltic Sowa et al. Molecular Pain 2010, 6:20 Page 6 of 8 http://www.molecularpain.com/content/6/1/20 pump at 4°C. Loading was performed overnight at a slow least three days before testing. To further reduce variabil- flow rate (0.4 mL/min. for 14-16 hours) to optimize bind- ity in behavioral studies, mice were almost exclusively ing of the GST-tagged protein. The column was then tested when in the resting or light sleep behavioral state washed with 50 mL PBS. Purified thrombin (GE Health- [50]. The experimenter was blind to genotype during care, Cat. # 27-0846-01) was added to 2 mL of PBS (250 U behavioral testing. thrombin/L of expression culture) and loaded onto the Thermal sensitivity was measured by heating one hind- GSTrap column using a syringe. The on-column cleavage paw with a Plantar Test apparatus (IITC) following the reaction proceeded for 16 hours at room temperature. Hargreaves method [51]. The radiant heat source inten- The pre-loaded GSTrap column was then attached to an sity was calibrated so that a paw withdrawal reflex was ÄKTA Explorer chromatography system with UV moni- evoked in ~10 s., on average, in wild-type C57BL/6 mice. toring. Cleaved mNT5E and thrombin were eluted in PBS Cutoff time was 20 s. One measurement was taken from while the GST tag remained bound to the column. Frac- each paw per time point to determine paw withdrawal tions were monitored by SDS-PAGE to estimate purity, latency. Mechanical sensitivity was measured using a mNT5E concentration and cleavage efficiency (~80%). semi-flexible tip attached to an Electronic von Frey appa- The cleaved mNT5E was separated from thrombin using ratus (IITC) as described elsewhere [52,53]. The force a Superdex75 10/300 GL column attached to the ÄKTA values obtained with this apparatus are higher than the Explorer system. Proteins were eluted in PBS at a flow force values obtained using calibrated von Frey filaments rate of 0.5 mL/min. A maximum of 500 μL was injected [53]. Three measurements were taken from each paw per run. Fractions containing cleaved mNT5E were then averaged to determine paw withdrawal threshold in pooled, concentrated and then dialyzed against 0.9% grams. To induce inflammatory pain, 20 μL complete Fre- saline. Protein purity was confirmed by SDS-PAGE, und's adjuvant (MP Biomedicals) was injected into one staining for total protein with GelCode Blue (Pierce/ hindpaw, centrally beneath glabrous skin, with a 30G nee- Thermo Scientific, Cat. # 24590) and western blotting dle. We performed spared nerve injury surgeries as with anti-NT5E antibody (R&D Systems, AF4488). described by Shields and colleagues [54]. mNT5E protein Amersham full-range rainbow molecular weight markers was diluted in 0.9% saline for intrathecal injection (5 μL/ (GE Healthcare) were used for SDS-PAGE and western mouse) using the direct lumbar puncture method [55]. blots. Recombinant mNT5E was kept at 4°C for short- None of the mNT5E-injected mice displayed reduced term (1-2 months) use and at -80°C for long term storage. mobility or paralysis following injection, as assessed by visually observing motor activity following injections. Enzyme assays Competing interests Enzymatic reactions (50 μL final) were carried out with The authors declare that they have no competing interests. recombinant mNT5E at 37°C for 3 minutes in 100 mM HEPES, pH 7.0, 4 mM MgCl with adenosine 5'-mono- Authors' contributions NAS carried out the behavioral studies and drafted figure legends, MKV carried phosphate disodium salt (Fluka, 01930) as substrate. out molecular and biochemical studies and drafted portions of the manuscript, Reactions were terminated by adding 950 μL of the mala- MJZ designed the mNT5E expression construct, conceived of and designed chite green color reagent [0.03% (w/v) malachite green the study and wrote the manuscript. All authors read and approved the final manuscript. oxalate, 0.2% (w/v) sodium molybdate, 0.05% (v/v) Triton X-100, dissolved in 0.7 M HCl] followed by incubation at Acknowledgements room temperature for 30 minutes. Inorganic phosphate We thank Yvette Chuang for excellent technical assistance, Jost Vielmetter, Inderjit Nangiana and Michael Anaya at the Caltech Protein Expression Center was quantified by measuring OD and comparing to an for purifying mNT5E protein. This work was supported by grants to MJZ from inorganic phosphate (KH PO ) standard curve [47]. Unit 2 4 The Searle Scholars Program, The Klingenstein Foundation, The Whitehall Foundation, Rita Allen Foundation and NINDS (R01NS060725, R01NS067688). (U) definition: 1 U hydrolyzes 1 nmol of AMP per minute NAS was supported by NINDS (F30NS063507) and a MSTP grant at 37°C at pH 7.0. α,β-me-ADP was purchased from ( T32GM008719). MJZ is a Rita Allen Foundation Milton E. Cassel Scholar. Sigma (M3763). Author Details Behavior Department of Cell and Molecular Physiology, UNC Neuroscience Center, University of North Carolina, CB #7545, Chapel Hill, North Carolina 27599, USA All behavioral experiments involving vertebrate animals were approved by the Institutional Animal Care and Use Received: 14 February 2010 Accepted: 14 April 2010 Published: 14 April 2010 Committee at the University of North Carolina at Chapel T © T M h h o 2 i is s l0 e arti i1 c su 0 an la S cle o r P O w i p a s ain e ava et n 2010, A aicce llable ; lic ss arti 6 e f n :20 r so ee m cl B :e h i d o ttp:/ M ist ed C ri/bu ww e te n w d t.m r u aln o Lt d le e d. c r th u larpai e term n.co s o m f th /co en C te re n a tti /6 ve /1 C /2 o0 mmons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hill. 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Adv Drug Deliv Rev 2003, 55(8):1007-1041. doi: 10.1186/1744-8069-6-20 Cite this article as: Sowa et al., Recombinant ecto-5'-nucleotidase (CD73) has long lasting antinociceptive effects that are dependent on adenosine A1 receptor activation Molecular Pain 2010, 6:20 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Pain Springer Journals

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

Sowa, Nathaniel; Voss, Meagen; Zylka, Mark
Molecular Pain , Volume 6 (1) – Apr 14, 2010
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Springer Journals
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Copyright © 2010 by Sowa et al; licensee BioMed Central Ltd.
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Medicine & Public Health; Pain Medicine; Molecular Medicine; Neurobiology
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1744-8069
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10.1186/1744-8069-6-20
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20398264
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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 α,β-methylene-adenosine 5'-diphosphate (α,β-me-ADP; IC = 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 A receptor (A R, Adora1) knockout 1 1 mice. Conclusion: Our data indicate that the long lasting antinociceptive effects of mNT5E are due to hydrolysis of AMP followed by activation of A R. 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. Background known as ACPP, Fluoride-resistant acid phosphatase or Ecto-5'-nucleotidase (NT5E) is a glycosyl phosphati- thiamine monophosphatase) in nociceptive neurons. Like dylinositol (GPI)-anchored membrane protein that cata- NT5E, PAP functions as an ectonucleotidase in nocicep- lyzes the hydrolysis of extracellular AMP to adenosine tive neurons by hydrolyzing AMP to adenosine [10,11]. -/- -/- [1]. NT5E regulates diverse physiological processes that Both Pap and Nt5e mice show enhanced thermal are modulated by adenosine, including hypoxia, inflam- hyperalgesia in animal models of inflammatory pain and mation and epithelial ion transport [2-8]. Recently, we neuropathic pain as well as enhanced mechanical allo- -/- found that NT5E is expressed in peptidergic and nonpep- dynia following inflammation [9,10]. Interestingly, A R tidergic nociceptive (pain-sensing) neurons and their mice also show enhanced sensitization following inflam- axon terminals in spinal cord and skin [9]. Based on mation and nerve injury [12]. Thus, deficiencies in ade- -/- experiments with Nt5e mice, we established that NT5E nosine production or A R signaling cause similar accounts for ~50% of all AMP hydrolytic activity in noci- behavioral phenotypes. ceptive neurons [9]. In support of an A R-dependent mechanism, we found In addition, we observed that NT5E was extensively co- that intrathecal (i.t.) injection of secretory PAP protein localized with Prostatic acid phosphatase (PAP, also (from mouse or human) into wild-type mice had long lasting antinociceptive, antihyperalgesic and antiallo- * Correspondence: zylka@med.unc.edu dynic effects that were entirely dependent on A R activa- Department of Cell and Molecular Physiology, UNC Neuroscience Center, tion [10,11]. These data suggested that spinal delivery of University of North Carolina, CB #7545, Chapel Hill, North Carolina 27599, USA Full list of author information is available at the end of the article © 2010 Sowa et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in BioMed Central any medium, provided the original work is properly cited. Sowa et al. Molecular Pain 2010, 6:20 Page 2 of 8 http://www.molecularpain.com/content/6/1/20 PAP protein could be used therapeutically to generate adenosine and activate A R over an extended time period. Likewise, direct activation of A R with adenosine or selective A R agonists had antinociceptive effects in rodents and humans [13-31]. Considering that both PAP and NT5E generate adenos- ine, we hypothesized that NT5E protein might also have A R-dependent antinociceptive effects. However, we were unable to test this hypothesis because mammalian NT5E protein was not available. As emphasized in a recent review by Colgan and colleagues, this lack of a reli- able source of purified protein has hindered studies with NT5E [2]. Others used 5'-nucleotidase protein purified from rattlesnake (Crotalus atrox) venom instead [4,32,33]. Crotalus 5'-nucleotidase had no deleterious effects when injected intraperitoneally and rescued phe- -/- notypes in Nt5e mice; however, possible toxicity from venom contaminants remains a concern--especially if this venom-derived protein were to be used in the nervous system. To overcome these toxicity concerns, we purified and Figure 1 Purification of recombinant mNT5E. (A) Diagram of the characterized (in vitro and in vivo) a secretory version of GST-mNT5E expression construct. (Top) Native mNT5E contains an N- recombinant mouse NT5E. Our present study builds terminal signal peptide (ss-cleavage) and GPI anchor site. (Bottom) The upon work by Servos and colleagues who purified a GST-mNT5E fusion construct contains the signal peptide from gp67 of baculovirus Autographica californica, GST, a thrombin cleavage site, the recombinant but non-secretory version of rat NT5E catalytic domain of mNT5E and (His) tag. Translation start and stop using the baculovirus expression system [34]. codons are indicated. (B) GelCode blue-stained SDS-PAGE gel and (C) western blot of purified recombinant mNT5E protein (0.05 μg). The Results western blot was probed with an anti-mNT5E antibody. Purification of soluble mouse NT5E using the baculovirus expression system cleavage site to permit removal of GST and a C-terminal NT5E is anchored to the membrane via a GPI linkage on hexahistidine (His) tag (Figure 1A). Ser523 [35]. In an effort to produce a secreted and soluble Two days after infecting Hi5 insect cells with recombi- (non-membrane anchored) version of NT5E, Servos and nant baculovirus we detected GST-mNT5E protein in the colleagues generated a baculovirus expression construct tissue culture supernatant at approximately 10 mg/L. of rat NT5E that reportedly lacked the GPI-anchor at This observation suggested that exclusion of Ser523 was Ser523 [34]. While Servos and colleagues successfully important for producing a soluble version of NT5E. used their construct to purify a catalytically active version Additionally, based on SDS-PAGE and western blotting, of rat NT5E, they did not detect NT5E in the tissue cul- the GST-mNT5E found in the medium was largely intact ture medium as would be expected if the protein were whereas cell lysates contained truncated and intact ver- secreted and soluble. Instead, NT5E was only present in sions of mNT5E-GST (data not shown). We next purified cell lysates. Upon re-examination of the cloning strategy mNT5E from the culture supernatant in two steps (see used by Servos and colleagues we noticed that their Methods). We reasoned that GST, a protein that binds expression construct included Ser523 but excluded glutathione, might interfere with physiological or behav- neighboring Ser526. Using GPI prediction software [36], ioral studies if administered in vivo. So as part of our we confirmed that Ser523 (but not Ser526) was the most purification procedure, the GST fusion was removed by likely GPI anchor site. Inclusion of this GPI anchor thrombin cleavage. Protein purity was analyzed under sequence could explain why Servos and colleagues did denaturing conditions with GelCode Blue protein stain not detect NT5E in the culture medium. We thus gener- (Figure 1B) and western blotting with an anti-NT5E anti- ated a mouse NT5E (Trp29-Phe522) expression construct body (Figure 1C). We observed a single band at ~62 kDa, that was truncated just before Ser523 (Figure 1A). Our corresponding to the calculated molecular weight of ung- construct was otherwise identical to the one used by Ser- lycosylated mNT5E with a (His) tag (61.7 kDa). No addi- vos and colleagues--our construct contained a gp67 sig- nal peptide, glutathione S-transferase (GST), a thrombin Sowa et al. Molecular Pain 2010, 6:20 Page 3 of 8 http://www.molecularpain.com/content/6/1/20 tional bands were observed, indicating that mNT5E found that α,β-me-ADP (0.01-500 μM) did not inhibit protein was intact. recombinant mouse PAP when AMP was used as sub- The activity of secreted mNT5E was initially deter- strate (data not shown). Production of recombinant mined in vitro. Purified recombinant mNT5E protein mouse PAP was described previously [11]. dephosphorylated AMP with a K of 26 μM (Figure 2A). mNT5E has long lasting antinociceptive effects that are A R This K value is in agreement with the previously dependent reported range of 1-50 μM using AMP as substrate at pH We previously found that a single intrathecal injection of 7.0 [8,34]. Recombinant mNT5E was also inhibited by PAP had antinociceptive, antihyperalgesic and antiallo- α,β-me-ADP (IC = 0.43 μM; Figure 2B), a commonly dynic effects that lasted for three days and that were used inhibitor of NT5E [1]. Although recombinant NT5E dependent on A R activation [10,11]. To empirically iden- was not used, others obtained a slightly higher IC value tify an effective dose of mNT5E for in vivo studies and to of 3.6 μM with this inhibitor [37]. For comparison, we determine if mNT5E had long lasting antinociceptive effects, we intrathecally injected wild-type mice with increasing doses of recombinant mNT5E protein (Figure 3). Time points were based on our previous studies with PAP [10,11]. We then measured noxious thermal and mechanical sensitivity before (baseline, BL) and after mNT5E injection. Six hours post i.t. injection, paw with- drawal latency to the noxious thermal stimulus was sig- nificantly increased relative to controls and remained elevated for two days at all doses tested (Figure 3A). Intrathecal injection of mNT5E did not alter mechanical sensitivity (Figure 3B) nor did it cause paralysis or seda- tion at any of the doses tested. We previously found that PAP (from human, cow and mouse) also had selective thermal but not mechanical antinociceptive effects in naïve mice and had no obvious motor side effects [10,11]. We next evaluated the antinociceptive effects of mNT5E in the CFA model of inflammatory pain and the SNI model of neuropathic pain. We used wild-type (WT) -/- and A R mice, to evaluate dependence on A R activa- 1 1 tion. We used the contralateral (non-inflamed/non- injured) paw as a control. As seen previously [9,10,12], -/- A R mice displayed enhanced thermal hyperalgesia after CFA injection and after nerve injury relative to WT mice (Figure 4A, C). In both chronic pain models, a single i.t. injection of mNT5E had long lasting (at least 2 days) thermal antihyperalgesic and mechanical antiallodynic effects in the inflamed/injured paw of WT mice but not -/- A R mice (Figure 4A-D). Indeed, thermal sensitivity transiently returned to baseline levels in the injured/ inflamed paws following mNT5E injection whereas mechanical sensitivity approached but did not reach baseline levels. Consistent with our dose-response study above, mNT5E had thermal but not mechanical antinoci- Figure 2 mNT5E dephosphorylates AMP and can be inhibited by ceptive effects in the control (non-inflamed/non-injured) α,β-me-ADP. (A) Plot of initial velocity at the indicated concentrations paws of WT mice. mNT5E had no antinociceptive effects of AMP at pH 7.0. Reactions (n = 3 per point) were stopped after 3 min. -/- in A R mice, highlighting a critical dependence on A R (B) The indicated concentrations of α,β-me-ADP were added to reac- 1 1 tions (n = 3 per concentration) containing mNT5E (0.07 μg/μL) and 400 activation. We previously found that control injections μM AMP at pH 7.0. (A, B) Inorganic phosphate was measured using had no effect on thermal or mechanical sensitivity in malachite green. All data are presented as means ± s.e.m. Some error these mouse models [10,11]. When combined with bars are obscured due to their small size. GraphPad Prism 5.0 was used numerous studies of NT5E by others (reviewed by [2]), to generate curves. Sowa et al. Molecular Pain 2010, 6:20 Page 4 of 8 http://www.molecularpain.com/content/6/1/20 Figure 3 Dose-dependent antinociceptive effects of intrathecal mNT5E. Effects of the indicated amounts of mNT5E on (A) paw withdrawal la- tency 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. our data suggest that all the antinociceptive effects of explain why saturating doses of PAP or NT5E protein do mNT5E are due to production of adenosine and activa- not paralyze mice. Moreover, adenosine can be elimi- tion of A R on DRG neurons and/or spinal neurons nated from the extracellular space by nucleoside trans- porters and metabolic enzymes, including adenosine [38,39]. deaminase and adenosine kinase [40,44]. These compet- ing processes of adenosine production and elimination Discussion place an upper limit on how much extracellular adenos- Adenosine and selective A R agonists have well-studied ine is available to activate A R in animals. In support of antinociceptive effects in rodents and humans [26,40,41]. 1 this, adenosine deaminase inhibitors and adenosine These agonists are often delivered intrathecally to avoid kinase inhibitors prolong the extracellular bioavailability side effects associated with peripheral A R activation of adenosine and have antinociceptive effects in animals [42]. However, A R agonists do have side effects when [17,21,45,46]. delivered intrathecally at high doses, including overt Unexpectedly, our studies revealed that two molecu- motor paralysis [10,43]. These motor side effects are larly distinct ectonucleotidases (PAP and NT5E) have likely due to activation of A R on motor neurons [39]. pronounced antinociceptive effects that persist for an Our present study indicates that NT5E, an ectonucleoti- extended time period (2-3 days). This long duration of dase that hydrolyzes AMP to adenosine, provides an action in vivo could not have been predicted from the alternative means of activating A R for therapeutic pur- biochemical properties of these enzymes alone. More poses without causing overt motor paralysis. Although importantly, our findings suggest ectonucleotidases rep- PAP also has A R-dependent antinociceptive effects resent a new class of antinociceptive drugs with potential when injected intrathecally [10,11], PAP shares no therapeutic advantages over adenosine, A R agonists and sequence similarity to NT5E. This lack of similarity made inhibitors of adenosine metabolism. For example, the it impossible for us to determine a priori if recombinant long duration of action of ectonucleotidases could be use- mNT5E would be catalytically active, stable and effective ful in situations where there is a need to provide a sus- when tested in live animals. tained level of A R activation. Moreover, recombinant As previously discussed [11], ectonucleotidases are cat- NT5E could be used to further study the role of NT5E in alytically restricted in that they generate adenosine in pain mechanisms and to study many other physiological proportion to substrate availability. Catalytic restriction processes that are regulated by NT5E [2,9]. limits the amount of adenosine produced and could Sowa et al. Molecular Pain 2010, 6:20 Page 5 of 8 http://www.molecularpain.com/content/6/1/20 -/- Figure 4 mNT5E has antihyperalgesic and antiallodynic effects in WT mice following inflammation and nerve injury. WT and A R 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. Methods fusion tag, with a single thrombin cleavage site between Molecular biology the GST tag and the coding sequence of mNT5E. The The GST-mNT5E baculovirus expression plasmid was final plasmid was sequence verified. generated by PCR amplification of mouse NT5E (nt 131- mNT5E protein purification 1696 from GenBank accession # NM_011851.3) using The GST-mNT5E plasmid was used to generate recombi- Phusion polymerase and a full-length expression con- nant mNT5E protein using the BD BaculoGold Expres- struct of mNT5E as template. Primer sequences con- sion System (BD Biosciences). Briefly, we infected Hi5 tained EcoRI sites (in bold italics) to facilitate cloning into insect cells with high-titer recombinant baculovirus, pAcSecG2T (BD Biosciences). N-terminal primer: 5'- incubated the cells for 48 hours at 27°C and then removed cgcgaattcattgggagctcacgatcctgcacaca. C-terminal (His) the cells from the supernatant by centrifugation. The tag primer: 5'-gcggaattcttaatgatgatgatgatgatggaacttgatc- supernatant containing secreted GST-mNT5E was fil- cgcccttcaacg. These primers amplify a product that con- tered (0.45 μm pore size, Millipore) and concentrated in tains the catalytic domain of mNT5E fused to the (His) PBS (10 mM sodium phosphate, 140 mM NaCl, pH 7.4) epitope tag but that lacks the signal peptide and GPI using a Millipore cartridge with a 10 k retention cutoff. anchor sequence (located at Ser523). This mNT5E PCR The concentrated supernatant was loaded onto a 5 mL product was subcloned in frame with an N-terminal GST GSTrap FF column (GE Healthcare) using a peristaltic Sowa et al. Molecular Pain 2010, 6:20 Page 6 of 8 http://www.molecularpain.com/content/6/1/20 pump at 4°C. Loading was performed overnight at a slow least three days before testing. To further reduce variabil- flow rate (0.4 mL/min. for 14-16 hours) to optimize bind- ity in behavioral studies, mice were almost exclusively ing of the GST-tagged protein. The column was then tested when in the resting or light sleep behavioral state washed with 50 mL PBS. Purified thrombin (GE Health- [50]. The experimenter was blind to genotype during care, Cat. # 27-0846-01) was added to 2 mL of PBS (250 U behavioral testing. thrombin/L of expression culture) and loaded onto the Thermal sensitivity was measured by heating one hind- GSTrap column using a syringe. The on-column cleavage paw with a Plantar Test apparatus (IITC) following the reaction proceeded for 16 hours at room temperature. Hargreaves method [51]. The radiant heat source inten- The pre-loaded GSTrap column was then attached to an sity was calibrated so that a paw withdrawal reflex was ÄKTA Explorer chromatography system with UV moni- evoked in ~10 s., on average, in wild-type C57BL/6 mice. toring. Cleaved mNT5E and thrombin were eluted in PBS Cutoff time was 20 s. One measurement was taken from while the GST tag remained bound to the column. Frac- each paw per time point to determine paw withdrawal tions were monitored by SDS-PAGE to estimate purity, latency. Mechanical sensitivity was measured using a mNT5E concentration and cleavage efficiency (~80%). semi-flexible tip attached to an Electronic von Frey appa- The cleaved mNT5E was separated from thrombin using ratus (IITC) as described elsewhere [52,53]. The force a Superdex75 10/300 GL column attached to the ÄKTA values obtained with this apparatus are higher than the Explorer system. Proteins were eluted in PBS at a flow force values obtained using calibrated von Frey filaments rate of 0.5 mL/min. A maximum of 500 μL was injected [53]. Three measurements were taken from each paw per run. Fractions containing cleaved mNT5E were then averaged to determine paw withdrawal threshold in pooled, concentrated and then dialyzed against 0.9% grams. To induce inflammatory pain, 20 μL complete Fre- saline. Protein purity was confirmed by SDS-PAGE, und's adjuvant (MP Biomedicals) was injected into one staining for total protein with GelCode Blue (Pierce/ hindpaw, centrally beneath glabrous skin, with a 30G nee- Thermo Scientific, Cat. # 24590) and western blotting dle. We performed spared nerve injury surgeries as with anti-NT5E antibody (R&D Systems, AF4488). described by Shields and colleagues [54]. mNT5E protein Amersham full-range rainbow molecular weight markers was diluted in 0.9% saline for intrathecal injection (5 μL/ (GE Healthcare) were used for SDS-PAGE and western mouse) using the direct lumbar puncture method [55]. blots. Recombinant mNT5E was kept at 4°C for short- None of the mNT5E-injected mice displayed reduced term (1-2 months) use and at -80°C for long term storage. mobility or paralysis following injection, as assessed by visually observing motor activity following injections. Enzyme assays Competing interests Enzymatic reactions (50 μL final) were carried out with The authors declare that they have no competing interests. recombinant mNT5E at 37°C for 3 minutes in 100 mM HEPES, pH 7.0, 4 mM MgCl with adenosine 5'-mono- Authors' contributions NAS carried out the behavioral studies and drafted figure legends, MKV carried phosphate disodium salt (Fluka, 01930) as substrate. out molecular and biochemical studies and drafted portions of the manuscript, Reactions were terminated by adding 950 μL of the mala- MJZ designed the mNT5E expression construct, conceived of and designed chite green color reagent [0.03% (w/v) malachite green the study and wrote the manuscript. All authors read and approved the final manuscript. oxalate, 0.2% (w/v) sodium molybdate, 0.05% (v/v) Triton X-100, dissolved in 0.7 M HCl] followed by incubation at Acknowledgements room temperature for 30 minutes. Inorganic phosphate We thank Yvette Chuang for excellent technical assistance, Jost Vielmetter, Inderjit Nangiana and Michael Anaya at the Caltech Protein Expression Center was quantified by measuring OD and comparing to an for purifying mNT5E protein. This work was supported by grants to MJZ from inorganic phosphate (KH PO ) standard curve [47]. Unit 2 4 The Searle Scholars Program, The Klingenstein Foundation, The Whitehall Foundation, Rita Allen Foundation and NINDS (R01NS060725, R01NS067688). (U) definition: 1 U hydrolyzes 1 nmol of AMP per minute NAS was supported by NINDS (F30NS063507) and a MSTP grant at 37°C at pH 7.0. α,β-me-ADP was purchased from ( T32GM008719). MJZ is a Rita Allen Foundation Milton E. Cassel Scholar. Sigma (M3763). Author Details Behavior Department of Cell and Molecular Physiology, UNC Neuroscience Center, University of North Carolina, CB #7545, Chapel Hill, North Carolina 27599, USA All behavioral experiments involving vertebrate animals were approved by the Institutional Animal Care and Use Received: 14 February 2010 Accepted: 14 April 2010 Published: 14 April 2010 Committee at the University of North Carolina at Chapel T © T M h h o 2 i is s l0 e arti i1 c su 0 an la S cle o r P O w i p a s ain e ava et n 2010, A aicce llable ; lic ss arti 6 e f n :20 r so ee m cl B :e h i d o ttp:/ M ist ed C ri/bu ww e te n w d t.m r u aln o Lt d le e d. c r th u larpai e term n.co s o m f th /co en C te re n a tti /6 ve /1 C /2 o0 mmons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hill. 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Adv Drug Deliv Rev 2003, 55(8):1007-1041. doi: 10.1186/1744-8069-6-20 Cite this article as: Sowa et al., Recombinant ecto-5'-nucleotidase (CD73) has long lasting antinociceptive effects that are dependent on adenosine A1 receptor activation Molecular Pain 2010, 6:20

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Molecular Pain – Springer Journals

Published: Apr 14, 2010

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