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• W2002620715 abstract "Thiamine monophosphatase (TMPase, also known as fluoride-resistant acid phosphatase) is a classic histochemical marker of small-diameter dorsal root ganglia neurons. The molecular identity of TMPase is currently unknown. We found that TMPase is identical to the transmembrane isoform of prostatic acid phosphatase (PAP), an enzyme with unknown molecular and physiological functions. We then found that PAP knockout mice have normal acute pain sensitivity but enhanced sensitivity in chronic inflammatory and neuropathic pain models. In gain-of-function studies, intraspinal injection of PAP protein has potent antinociceptive, antihyperalgesic, and antiallodynic effects that last longer than the opioid analgesic morphine. PAP suppresses pain by functioning as an ecto-5′-nucleotidase. Specifically, PAP dephosphorylates extracellular adenosine monophosphate (AMP) to adenosine and activates A1-adenosine receptors in dorsal spinal cord. Our studies reveal molecular and physiological functions for PAP in purine nucleotide metabolism and nociception and suggest a novel use for PAP in the treatment of chronic pain. Thiamine monophosphatase (TMPase, also known as fluoride-resistant acid phosphatase) is a classic histochemical marker of small-diameter dorsal root ganglia neurons. The molecular identity of TMPase is currently unknown. We found that TMPase is identical to the transmembrane isoform of prostatic acid phosphatase (PAP), an enzyme with unknown molecular and physiological functions. We then found that PAP knockout mice have normal acute pain sensitivity but enhanced sensitivity in chronic inflammatory and neuropathic pain models. In gain-of-function studies, intraspinal injection of PAP protein has potent antinociceptive, antihyperalgesic, and antiallodynic effects that last longer than the opioid analgesic morphine. PAP suppresses pain by functioning as an ecto-5′-nucleotidase. Specifically, PAP dephosphorylates extracellular adenosine monophosphate (AMP) to adenosine and activates A1-adenosine receptors in dorsal spinal cord. Our studies reveal molecular and physiological functions for PAP in purine nucleotide metabolism and nociception and suggest a novel use for PAP in the treatment of chronic pain. Painful and tissue-damaging stimuli are sensed by small-diameter nociceptive neurons located in the dorsal root ganglia (DRG) and trigeminal ganglia (Woolf and Ma, 2007Woolf C.J. Ma Q. Nociceptors—noxious stimulus detectors.Neuron. 2007; 55: 353-364Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). For nearly 50 years, it was known that many small-diameter DRG neurons expressed a histochemically identifiable acid phosphatase (Colmant, 1959Colmant H.J. Aktivitatsschwankungen der sauren Phosphatase im Ruckenmark und den Spinalganglien der Ratte nach Durchschneidung des Nervus ischiadicus.Arch. Psychiatr. Nervenkr. 1959; 199: 60-71Crossref Scopus (40) Google Scholar), commonly referred to as fluoride-resistant acid phosphatase (FRAP) or thiamine monophosphatase (TMPase) (Dodd et al., 1983Dodd J. Jahr C.E. Hamilton P.N. Heath M.J. Matthew W.D. Jessell T.M. Cytochemical and physiological properties of sensory and dorsal horn neurons that transmit cutaneous sensation.Cold Spring Harb. Symp. Quant. Biol. 1983; 48: 685-695Crossref PubMed Google Scholar, Knyihar-Csillik et al., 1986Knyihar-Csillik E. Bezzegh A. Boti S. Csillik B. Thiamine monophosphatase: a genuine marker for transganglionic regulation of primary sensory neurons.J. Histochem. Cytochem. 1986; 34: 363-371Crossref PubMed Scopus (64) Google Scholar). TMPase dephosphorylates diverse substrates, including the vitamin B1 derivative thiamine monophosphate (TMP) and 5′-nucleotide monophosphates (Dodd et al., 1983Dodd J. Jahr C.E. Hamilton P.N. Heath M.J. Matthew W.D. Jessell T.M. Cytochemical and physiological properties of sensory and dorsal horn neurons that transmit cutaneous sensation.Cold Spring Harb. Symp. Quant. Biol. 1983; 48: 685-695Crossref PubMed Google Scholar, Sanyal and Rustioni, 1974Sanyal S. Rustioni A. Phosphatases in the substantia gelatinosa and motoneurones: a comparative histochemical study.Brain Res. 1974; 76: 161-166Crossref PubMed Scopus (11) Google Scholar, Silverman and Kruger, 1988aSilverman J.D. Kruger L. Acid phosphatase as a selective marker for a class of small sensory ganglion cells in several mammals: spinal cord distribution, histochemical properties, and relation to fluoride-resistant acid phosphatase (FRAP) of rodents.Somatosens. Res. 1988; 5: 219-246Crossref PubMed Scopus (47) Google Scholar). TMPase was intensively studied in the 1980s in an effort to determine its molecular identity and function. TMPase marks most nonpeptidergic DRG neurons, a subset of peptidergic DRG neurons, and unmyelinated axon terminals in lamina II of the dorsal spinal cord (Carr et al., 1990Carr P.A. Yamamoto T. Nagy J.I. Calcitonin gene-related peptide in primary afferent neurons of rat: co-existence with fluoride-resistant acid phosphatase and depletion by neonatal capsaicin.Neuroscience. 1990; 36: 751-760Crossref PubMed Scopus (46) Google Scholar, Dalsgaard et al., 1984Dalsgaard C.J. Ygge J. Vincent S.R. Ohrling M. Dockray G.J. Elde R. Peripheral projections and neuropeptide coexistence in a subpopulation of fluoride-resistant acid phosphatase reactive spinal primary sensory neurons.Neurosci. Lett. 1984; 51: 139-144Crossref PubMed Scopus (46) Google Scholar, Dodd et al., 1983Dodd J. Jahr C.E. Hamilton P.N. Heath M.J. Matthew W.D. Jessell T.M. Cytochemical and physiological properties of sensory and dorsal horn neurons that transmit cutaneous sensation.Cold Spring Harb. Symp. Quant. Biol. 1983; 48: 685-695Crossref PubMed Google Scholar, Hunt and Rossi, 1985Hunt S.P. Rossi J. Peptide- and non-peptide-containing unmyelinated primary afferents: the parallel processing of nociceptive information.Philos. Trans. R. Soc. Lond. B Biol. Sci. 1985; 308: 283-289Crossref PubMed Scopus (117) Google Scholar, Knyihar-Csillik et al., 1986Knyihar-Csillik E. Bezzegh A. Boti S. Csillik B. Thiamine monophosphatase: a genuine marker for transganglionic regulation of primary sensory neurons.J. Histochem. Cytochem. 1986; 34: 363-371Crossref PubMed Scopus (64) Google Scholar, Nagy and Hunt, 1982Nagy J.I. Hunt S.P. Fluoride-resistant acid phosphatase-containing neurones in dorsal root ganglia are separate from those containing substance P or somatostatin.Neuroscience. 1982; 7: 89-97Crossref PubMed Scopus (211) Google Scholar, Silverman and Kruger, 1988aSilverman J.D. Kruger L. Acid phosphatase as a selective marker for a class of small sensory ganglion cells in several mammals: spinal cord distribution, histochemical properties, and relation to fluoride-resistant acid phosphatase (FRAP) of rodents.Somatosens. Res. 1988; 5: 219-246Crossref PubMed Scopus (47) Google Scholar). Since peptidergic and nonpeptidergic neurons are generally considered to be nociceptive (Woolf and Ma, 2007Woolf C.J. Ma Q. Nociceptors—noxious stimulus detectors.Neuron. 2007; 55: 353-364Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar), these anatomical studies suggested that TMPase might function in nociception. Moreover, TMPase staining in lamina II of spinal cord is reduced or eliminated when peripheral nerves are damaged (Colmant, 1959Colmant H.J. Aktivitatsschwankungen der sauren Phosphatase im Ruckenmark und den Spinalganglien der Ratte nach Durchschneidung des Nervus ischiadicus.Arch. Psychiatr. Nervenkr. 1959; 199: 60-71Crossref Scopus (40) Google Scholar, Csillik and Knyihar-Csillik, 1986Csillik B. Knyihar-Csillik E. The Protean Gate: Structure and Plasticity of the Primary Nociceptive Analyzer. Akademiai Kiado, Budapest1986Google Scholar, Shields et al., 2003Shields S.D. Eckert 3rd, W.A. Basbaum A.I. Spared nerve injury model of neuropathic pain in the mouse: a behavioral and anatomic analysis.J. Pain. 2003; 4: 465-470Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, Tenser, 1985Tenser R.B. Sequential changes of sensory neuron (fluoride-resistant) acid phosphatase in dorsal root ganglion neurons following neurectomy and rhizotomy.Brain Res. 1985; 332: 386-389Crossref PubMed Scopus (23) Google Scholar, Tenser et al., 1991Tenser R.B. Viselli A.L. Savage D.H. Reversible decrease of fluoride resistant acid phosphatase-positive neurons after herpes simplex virus infection.Neurosci. Lett. 1991; 130: 85-88Crossref PubMed Scopus (5) Google Scholar). Ultimately, studies of TMPase waned when it was found that isolectin B4 (IB4) colocalized with TMPase and was an easier-to-use marker of nonpeptidergic neurons (Silverman and Kruger, 1988bSilverman J.D. Kruger L. Lectin and neuropeptide labeling of separate populations of dorsal root ganglion neurons and associated “nociceptor” thin axons in rat testis and cornea whole-mount preparations.Somatosens. Res. 1988; 5: 259-267Crossref PubMed Scopus (171) Google Scholar, Silverman and Kruger, 1990Silverman J.D. Kruger L. Selective neuronal glycoconjugate expression in sensory and autonomic ganglia: relation of lectin reactivity to peptide and enzyme markers.J. Neurocytol. 1990; 19: 789-801Crossref PubMed Scopus (290) Google Scholar). More importantly, the gene encoding TMPase was never identified, making it impossible to study the molecular and physiological function of TMPase in sensory neurons. In an attempt to identify the TMPase gene, Dodd and coworkers partially purified TMPase protein from rat DRG using chromatography (Dodd et al., 1983Dodd J. Jahr C.E. Hamilton P.N. Heath M.J. Matthew W.D. Jessell T.M. Cytochemical and physiological properties of sensory and dorsal horn neurons that transmit cutaneous sensation.Cold Spring Harb. Symp. Quant. Biol. 1983; 48: 685-695Crossref PubMed Google Scholar). The partially purified rat protein was inhibited by the nonselective acid phosphatase inhibitor L(+)-tartrate and was similar in molecular weight to the secretory isoform of human prostatic acid phosphatase (PAP, also known as ACPP), the only known isoform of PAP at the time (Ostrowski and Kuciel, 1994Ostrowski W.S. Kuciel R. Human prostatic acid phosphatase: selected properties and practical applications.Clin. Chim. Acta. 1994; 226: 121-129Crossref PubMed Scopus (3) Google Scholar). These biochemical experiments hinted that TMPase might be secretory PAP (Dodd et al., 1983Dodd J. Jahr C.E. Hamilton P.N. Heath M.J. Matthew W.D. Jessell T.M. Cytochemical and physiological properties of sensory and dorsal horn neurons that transmit cutaneous sensation.Cold Spring Harb. Symp. Quant. Biol. 1983; 48: 685-695Crossref PubMed Google Scholar). However, subsequent studies using anti-PAP antibodies failed to immunostain small-diameter DRG neurons and their axon terminals in lamina II (i.e., the neurons and axons that contain TMPase) (Dodd et al., 1983Dodd J. Jahr C.E. Hamilton P.N. Heath M.J. Matthew W.D. Jessell T.M. Cytochemical and physiological properties of sensory and dorsal horn neurons that transmit cutaneous sensation.Cold Spring Harb. Symp. Quant. Biol. 1983; 48: 685-695Crossref PubMed Google Scholar, Silverman and Kruger, 1988aSilverman J.D. Kruger L. Acid phosphatase as a selective marker for a class of small sensory ganglion cells in several mammals: spinal cord distribution, histochemical properties, and relation to fluoride-resistant acid phosphatase (FRAP) of rodents.Somatosens. Res. 1988; 5: 219-246Crossref PubMed Scopus (47) Google Scholar). As summarized by Silverman and Kruger in 1988, these data made it impossible to determine whether TMPase was PAP or some other enzyme. In light of this unsolved question regarding the molecular nature of TMPase and the historical use of TMPase as a nociceptive neuron marker, we sought to definitively identify the TMPase gene and ascertain its function in nociception. Our experiments revealed that TMPase was a recently discovered transmembrane (TM) isoform of PAP (TM-PAP) (Quintero et al., 2007Quintero I.B. Araujo C.L. Pulkka A.E. Wirkkala R.S. Herrala A.M. Eskelinen E.L. Jokitalo E. Hellstrom P.A. Tuominen H.J. Hirvikoski P.P. Vihko P.T. Prostatic acid phosphatase is not a prostate specific target.Cancer Res. 2007; 67: 6549-6554Crossref PubMed Scopus (65) Google Scholar) and was not the secretory isoform of PAP. This molecular identification then allowed us to use modern molecular and genetic approaches to rigorously study the function of PAP/TMPase in nociceptive circuits. Using our PAP knockout mice, we found that deletion of PAP increased thermal hyperalgesia (increased pain sensitivity) and mechanical allodynia in animal models of chronic pain. Conversely, a single intraspinal injection of PAP protein had antinociceptive, antihyperalgesic, and antiallodynic effects that lasted for up to 3 days, much longer than a single injection of the commonly used opioid analgesic morphine. Mechanistically, we found that PAP is an ectonucleotidase that dephosphorylates extracellular AMP to adenosine and requires A1-adenosine receptors (A1Rs) for antinociception. PAP has been intensively studied for 70 years in the prostate cancer field (Gutman and Gutman, 1938Gutman A.B. Gutman E.B. An “acid” phosphatase occurring in the serum of patients with metastasizing carcinoma of the prostate gland.J. Clin. Invest. 1938; 17: 473-478Crossref PubMed Google Scholar). Despite decades of research, the molecular and physiological functions for PAP remained unknown. Our studies with pain-sensing neurons identify the in vivo substrate, the molecular mechanism, and the physiological function for this medically relevant protein. Moreover, we show that PAP functions in nociception. Considering that TM-PAP is expressed throughout the body (Quintero et al., 2007Quintero I.B. Araujo C.L. Pulkka A.E. Wirkkala R.S. Herrala A.M. Eskelinen E.L. Jokitalo E. Hellstrom P.A. Tuominen H.J. Hirvikoski P.P. Vihko P.T. Prostatic acid phosphatase is not a prostate specific target.Cancer Res. 2007; 67: 6549-6554Crossref PubMed Scopus (65) Google Scholar), PAP could regulate diverse physiological processes that are dependent on adenosine (Jacobson and Gao, 2006Jacobson K.A. Gao Z.G. Adenosine receptors as therapeutic targets.Nat. Rev. Drug Discov. 2006; 5: 247-264Crossref PubMed Scopus (1081) Google Scholar). In rats, mice, and humans, PAP is expressed as a secreted protein or as a type 1 transmembrane (TM) protein, with the catalytic acid phosphatase domain localized extracellularly (Figure 1A) (Quintero et al., 2007Quintero I.B. Araujo C.L. Pulkka A.E. Wirkkala R.S. Herrala A.M. Eskelinen E.L. Jokitalo E. Hellstrom P.A. Tuominen H.J. Hirvikoski P.P. Vihko P.T. Prostatic acid phosphatase is not a prostate specific target.Cancer Res. 2007; 67: 6549-6554Crossref PubMed Scopus (65) Google Scholar, Roiko et al., 1990Roiko K. Janne O.A. Vihko P. Primary structure of rat secretory acid phosphatase and comparison to other acid phosphatases.Gene. 1990; 89: 223-229Crossref PubMed Scopus (39) Google Scholar, Vihko, 1979Vihko P. Human prostatic acid phosphatases: purification of a minor enzyme and comparisons of the enzymes.Invest. Urol. 1979; 16: 349-352PubMed Google Scholar). The secretory isoform has been used as a diagnostic marker for prostate cancer for nearly 70 years, whereas the TM isoform was only recently discovered (Gutman and Gutman, 1938Gutman A.B. Gutman E.B. An “acid” phosphatase occurring in the serum of patients with metastasizing carcinoma of the prostate gland.J. Clin. Invest. 1938; 17: 473-478Crossref PubMed Google Scholar, Quintero et al., 2007Quintero I.B. Araujo C.L. Pulkka A.E. Wirkkala R.S. Herrala A.M. Eskelinen E.L. Jokitalo E. Hellstrom P.A. Tuominen H.J. Hirvikoski P.P. Vihko P.T. Prostatic acid phosphatase is not a prostate specific target.Cancer Res. 2007; 67: 6549-6554Crossref PubMed Scopus (65) Google Scholar). To determine whether either PAP isoform is expressed in DRG, we performed in situ hybridization with isoform-specific antisense riboprobes. These experiments revealed that TM-PAP was expressed in a subset of small-diameter DRG neurons (Figure 1B), while the secretory isoform was expressed at low-to-undetectable levels (Figure 1C). Importantly, TM-PAP is localized to the plasma membrane and vesicular membranes, just like TMPase (Csillik and Knyihar-Csillik, 1986Csillik B. Knyihar-Csillik E. The Protean Gate: Structure and Plasticity of the Primary Nociceptive Analyzer. Akademiai Kiado, Budapest1986Google Scholar, Quintero et al., 2007Quintero I.B. Araujo C.L. Pulkka A.E. Wirkkala R.S. Herrala A.M. Eskelinen E.L. Jokitalo E. Hellstrom P.A. Tuominen H.J. Hirvikoski P.P. Vihko P.T. Prostatic acid phosphatase is not a prostate specific target.Cancer Res. 2007; 67: 6549-6554Crossref PubMed Scopus (65) Google Scholar). We also found that PAP was expressed in human DRG using RT-PCR and intron-spanning primers (data not shown), consistent with localization of TMPase to small-diameter human DRG neurons (Silverman and Kruger, 1988aSilverman J.D. Kruger L. Acid phosphatase as a selective marker for a class of small sensory ganglion cells in several mammals: spinal cord distribution, histochemical properties, and relation to fluoride-resistant acid phosphatase (FRAP) of rodents.Somatosens. Res. 1988; 5: 219-246Crossref PubMed Scopus (47) Google Scholar). To directly test whether PAP had TMPase histochemical activity, we overexpressed mouse TM-PAP in HEK293 cells, then stained these cells using TMP histochemistry. Cells transfected with TM-PAP were heavily stained when the plasma membrane was left intact (Figure 2A), indicating that TM-PAP can dephosphorylate TMP extracellularly. TMPase staining was even greater when the plasma membrane was permeabilized with detergent (Figure S1H available online). In contrast, control cells transfected with empty vector were not stained (Figure 2B). Two additional phosphatases (soluble acid phosphatase 1 [ACP1] and placental alkaline phosphatase) lacked TMPase activity (Figure S1). DRG neurons express at least eight different acid phosphatase genes (M.J.Z, unpublished data), any one of which could be TMPase. To determine whether PAP was the only enzyme in sensory neurons capable of dephosphorylating TMP, we analyzed DRG and spinal cord tissues from PAPΔ3/Δ3 (henceforth referred to as PAP−/−) knockout mice (P.V. et al., abstract from Proceedings of the AACR, 2005, 96th Annual Meeting, Anaheim, CA). In these mice, deletion of exon 3 causes complete loss of secretory and transmembrane PAP catalytic activity (P.V. et al., abstract from Proceedings of the AACR, 2005). Strikingly, TMP histochemical staining of DRG neurons and axon terminals in spinal cord was abolished in PAP−/− mice (Figures 2C–2F). Absence of TMP staining in PAP−/− mice was not due to developmental loss of DRG neurons, as wild-type and PAP−/− mice had equivalent numbers of P2X3-expressing neurons relative to all NeuN+ neurons in lumbar ganglia (43.4% ± 1.9% verses 42.4% ± 1.9% (SEM); 1500 NeuN+ neurons counted per genotype). P2X3 is an ATP-gated ion channel that is colocalized with PAP (see below). Moreover, loss of TMPase staining in the spinal cord was not due to loss of axon terminals in the dorsal horn (Figure S2). These gain- and loss-of-function experiments conclusively prove that TMPase in small-diameter DRG neurons is the transmembrane isoform of PAP. In addition, by combining immunofluorescence and TMP histochemistry, we observed colocalization between PAP and TMPase in DRG neurons (Figures S3A–S3C) and in axon terminals in lamina II of the spinal cord (Figures S3D–S3F). This anti-PAP antibody did not stain DRG or spinal cord sections from PAP−/− mice, confirming antibody specificity. Finally, upon finding that PAP was TMPase, we reanalyzed two published microarray data sets that measured changes in gene expression in DRG following peripheral nerve injury (Costigan et al., 2002Costigan M. Befort K. Karchewski L. Griffin R.S. D'Urso D. Allchorne A. Sitarski J. Mannion J.W. Pratt R.E. Woolf C.J. Replicate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury.BMC Neurosci. 2002; 3: 16Crossref PubMed Scopus (441) Google Scholar, Davis-Taber, 2006Davis-Taber R.A. Transcriptional profiling of dorsal root ganglia in a neuropathic pain model using microarray and laser capture microdissection.Drug Dev. Res. 2006; 67: 308-330Crossref Scopus (10) Google Scholar). In both studies, PAP was one of the most heavily downregulated genes. This is consistent with the fact that TMPase histochemical activity is greatly reduced in DRG and dorsal horn after peripheral nerve injury (Colmant, 1959Colmant H.J. Aktivitatsschwankungen der sauren Phosphatase im Ruckenmark und den Spinalganglien der Ratte nach Durchschneidung des Nervus ischiadicus.Arch. Psychiatr. Nervenkr. 1959; 199: 60-71Crossref Scopus (40) Google Scholar, Csillik and Knyihar-Csillik, 1986Csillik B. Knyihar-Csillik E. The Protean Gate: Structure and Plasticity of the Primary Nociceptive Analyzer. Akademiai Kiado, Budapest1986Google Scholar, Shields et al., 2003Shields S.D. Eckert 3rd, W.A. Basbaum A.I. Spared nerve injury model of neuropathic pain in the mouse: a behavioral and anatomic analysis.J. Pain. 2003; 4: 465-470Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, Tenser, 1985Tenser R.B. Sequential changes of sensory neuron (fluoride-resistant) acid phosphatase in dorsal root ganglion neurons following neurectomy and rhizotomy.Brain Res. 1985; 332: 386-389Crossref PubMed Scopus (23) Google Scholar, Tenser et al., 1991Tenser R.B. Viselli A.L. Savage D.H. Reversible decrease of fluoride resistant acid phosphatase-positive neurons after herpes simplex virus infection.Neurosci. Lett. 1991; 130: 85-88Crossref PubMed Scopus (5) Google Scholar). TMPase was previously localized to nonpeptidergic DRG neurons and a small number of peptidergic neurons (Carr et al., 1990Carr P.A. Yamamoto T. Nagy J.I. Calcitonin gene-related peptide in primary afferent neurons of rat: co-existence with fluoride-resistant acid phosphatase and depletion by neonatal capsaicin.Neuroscience. 1990; 36: 751-760Crossref PubMed Scopus (46) Google Scholar, Dalsgaard et al., 1984Dalsgaard C.J. Ygge J. Vincent S.R. Ohrling M. Dockray G.J. Elde R. Peripheral projections and neuropeptide coexistence in a subpopulation of fluoride-resistant acid phosphatase reactive spinal primary sensory neurons.Neurosci. Lett. 1984; 51: 139-144Crossref PubMed Scopus (46) Google Scholar, Hunt and Mantyh, 2001Hunt S.P. Mantyh P.W. The molecular dynamics of pain control.Nat. Rev. Neurosci. 2001; 2: 83-91Crossref PubMed Scopus (451) Google Scholar, Nagy and Hunt, 1982Nagy J.I. Hunt S.P. Fluoride-resistant acid phosphatase-containing neurones in dorsal root ganglia are separate from those containing substance P or somatostatin.Neuroscience. 1982; 7: 89-97Crossref PubMed Scopus (211) Google Scholar, Silverman and Kruger, 1988bSilverman J.D. Kruger L. Lectin and neuropeptide labeling of separate populations of dorsal root ganglion neurons and associated “nociceptor” thin axons in rat testis and cornea whole-mount preparations.Somatosens. Res. 1988; 5: 259-267Crossref PubMed Scopus (171) Google Scholar). To show that PAP had a similar distribution and to identify additional proteins that were colocalized with PAP, we performed double-label immunofluorescence with our anti-PAP antibody and various sensory neuron markers. Cell counts from confocal images revealed that virtually all nonpeptidergic DRG neurons, as defined by the markers IB4, Mrgprd-EGFPf, and P2X3, coexpressed PAP (Figures 3A–3I and Table S1). Moreover, PAP+ axons terminated in lamina II of spinal cord in association with nonpeptidergic neuron markers (Figures S4A–S4F). In contrast, a smaller percentage (17.1%) of peptidergic CGRP+ neurons (n = 1364 cells counted) expressed PAP (Figures 3J–3L and Table S1), and there was minimal overlap between PAP+ and peptidergic (CGRP+) axon terminals in spinal cord (Figures S4G–S4I). Finally, 19.1% ± 1.3% of PAP+ neurons expressed the capsaicin and noxious heat receptor TRPV1 (Figures 3M–3O). Taken together, these confocal imaging studies revealed that PAP was preferentially expressed in nonpeptidergic, presumably nociceptive, DRG neurons. PAP was generally thought to function only in the prostate (Ostrowski and Kuciel, 1994Ostrowski W.S. Kuciel R. Human prostatic acid phosphatase: selected properties and practical applications.Clin. Chim. Acta. 1994; 226: 121-129Crossref PubMed Scopus (3) Google Scholar). However, our expression data suggested that PAP might also function in nociceptive neurons. To evaluate pain-related functions for PAP, we tested age-matched wild-type C57BL/6 and PAP−/− male mice (backcrossed to C57BL/6 for ten generations) using acute and chronic pain behavioral assays. We found no significant differences between genotypes using a measure of mechanical sensitivity (electronic von Frey) or several different measures of acute noxious thermal sensitivity (Table S2). In contrast, PAP−/− mice showed significantly greater thermal hyperalgesia and mechanical allodynia relative to wild-type mice in the complete Freund's adjuvant (CFA) model of chronic inflammatory pain (Figures 4A and 4B). In addition, PAP−/− mice showed significantly greater thermal hyperalgesia in the spared nerve injury (SNI) model of neuropathic pain (Figures 4C and 4D) (Shields et al., 2003Shields S.D. Eckert 3rd, W.A. Basbaum A.I. Spared nerve injury model of neuropathic pain in the mouse: a behavioral and anatomic analysis.J. Pain. 2003; 4: 465-470Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Since deletion of PAP enhanced sensitivity in two different models of chronic pain, we hypothesized that excess PAP should have the opposite effect and reduce pain. To test this, we took advantage of the fact that secretory PAP protein is commercially available and has the same N-terminal catalytic region as TM-PAP (Figure 1A). We injected wild-type mice intrathecally (i.t.) into the lumbar region of spinal cord with pure human (h)PAP protein (the secretory isoform). Control mice were injected i.t. with an equivalent amount of heat-denatured, and hence phosphatase-inactive, hPAP protein. In all cases, we determined that hPAP was active or inactive using a sensitive fluorometric-based phosphatase assay (see Experimental Procedures). We then measured noxious thermal and mechanical sensitivity before (baseline, BL) and after hPAP injections (Figures 5A and 5B). Six hours after i.t. injection of hPAP, paw-withdrawal latency to a noxious thermal stimulus significantly increased relative to controls and remained elevated for 3 days (Figure 5A). This antinociceptive effect was dose dependent (Figure S5) and required PAP catalytic activity (Figure 5A). Active hPAP did not alter mechanical sensitivity (Figure 5B) nor did it cause paralysis or sedation. This long-lasting antinociceptive effect was species conserved, as a single i.t. injection of bovine (b)PAP also increased thermal withdrawal latency for 2 days but had no effect on mechanical sensitivity (Figure S6). Finally, i.t. injection of an unrelated protein (bovine serum albumin) or large quantities of a different secreted phosphatase (bovine alkaline phosphatase) did not alter thermal or mechanical sensitivity (Figures S6 and S7). We next used the same behavioral assay to compare PAP antinociception to the commonly used opioid analgesic morphine. We found that PAP and morphine antinociception were similar in magnitude following a single i.t. injection (40.8% ± 3.3% versus 62.2% ± 9.9% increase above baseline at the highest doses, respectively) but that PAP antinociception lasted much longer than morphine (3 days verses 5 hr at the highest doses, respectively; Figures S5 and S8). Similarly, Grant and colleagues found that the same high dose of morphine (50 μg, i.t., single injection) lasted 4.6 ± 1.0 hr in mice (Grant et al., 1995Grant G.J. Cascio M. Zakowski M.I. Langerman L. Turndorf H. Intrathecal administration of liposomal morphine in a mouse model.Anesth. Analg. 1995; 81: 514-518PubMed Google Scholar). We next evaluated the extent to which hPAP affected hyperalgesia and allodynia in the CFA model of inflammatory pain and the SNI model of neuropathic pain. For both chronic pain assays, we used the uninjured paw as control. Strikingly, in both chronic pain models, a single i.t. injection of active hPAP was antihyperalgesic and antiallodynic in the inflamed/injured paw (Figures 5C–5F). As before, a single injection was effective for several days, and phosphatase activity was required for these antinociceptive effects. Since PAP−/− mice showed enhanced hyperalgesia and allodynia in the CFA inflammatory pain model (Figures 4A and 4B), we next tested whether hPAP could rescue these enhanced thermal and mechanical phenotypes in PAP−/− mice. We found that i.t. injection of hPAP increased thermal withdrawal latency in the control paw of PAP−/− mice to the same extent as wild-type mice (Figure 6A, blue lines). This demonstrated that PAP−/− mice were competent to respond to acute increases in PAP activity. Strikingly, injection of hPAP rescued the thermal and mechanical inflammatory pain phenotype in the inflamed paw of PAP−/− mice (Figures 6A and 6B, compare red lines where PAP was injected to black lines where inactive PAP was injected). Importantly, these data also suggest that localized, spinal injection of hPAP can rescue the behavioral deficit caused by deletion of PAP throughout the animal. The antinociceptive effects of PAP require catalytic activity. This suggested PAP might generate, via dephosphorylation, a molecule that regulates nociceptive neurotransmission in the spinal cord. PAP and TMPase can dephosphorylate many different substrates (Dziembor-Gryszkiewicz et al., 1978Dziembor-Gryszkiewicz E. Fikus M. Kazimierczuk Z. Os" @default.
• W2002620715 created "2016-06-24" @default.
• W2002620715 creator A5013117593 @default.
• W2002620715 creator A5047547845 @default.
• W2002620715 creator A5068620787 @default.
• W2002620715 creator A5078197372 @default.
• W2002620715 creator A5078210617 @default.
• W2002620715 creator A5086576972 @default.
• W2002620715 creator A5089260333 @default.
• W2002620715 date "2008-10-01" @default.
• W2002620715 modified "2023-10-10" @default.
• W2002620715 title "Prostatic Acid Phosphatase Is an Ectonucleotidase and Suppresses Pain by Generating Adenosine" @default.
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• W2002620715 doi "https://doi.org/10.1016/j.neuron.2008.08.024" @default.
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