Characterization of Ectonucleotidases in Nociceptive Circuits

Pain is one of the most common medical complaints in the United States, affecting almost a quarter of American adults. There are numerous treatments for acute and chronic pain, but none of them are completely effective and many have intolerable side effects. New treatments are needed that are safer, more efficacious, and more cost-effective. We have focused on trying to understand better the mechanisms involved in the regulation of pain (nociceptive) signaling in order to develop novel therapies. Two important compounds involved in pain signaling are adenosine triphosphate (ATP) and adenosine. ATP has pro-nociceptive properties, while adenosine is antinociceptive. ATP can be converted to adenosine through a step-wise process catalyzed by enzymes on the surface of cells called ectonucleotidases. These enzymes could play a pivotal role in regulation of nociception by degrading pro-nociceptive ATP while simultaneously producing antinociceptive adenosine. Prior to this work, the exact ectonucleotidases present in nociceptive circuits were unknown. Here, we identify and characterize the first two known AMP-degrading ectonucleotidases involved in nociception, prostatic acid phosphatase (PAP) and Ecto-5'-nucleotidase (NT5E). Genetic deletion of these enzymes does not affect acute nociception, but leads to enhanced pain sensitivity in chronic inflammatory and neuropathic pain models. Conversely, intraspinal injection of PAP or NT5E protein has antinociceptive, antihyperalgesic, and antiallodynic effects that last longer than the opioid analgesic morphine. Both PAP and NT5E suppress pain by the production of adenosine from endogenous AMP and subsequent activation of the A1-adenosine receptor (A1R). Further, chronic activation of A1R by PAP leads to depletion of cellular levels of the signaling molecule PIP2. Depletion of PIP2 before or after chemical or physical injury (through injection of PAP) reduces pain hypersensitivity, highlighting an important role for PIP2 levels in the modulation of nociceptive signaling. We are the first to show this important role for PIP2 in setting the dynamic pain threshold in nociceptors. These studies not only identify two potentially new targets for the development of chronic pain therapy, but also highlight a new model for the dynamic modulation of pain sensitivity through the regulation of neuronal PIP2 levels

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

Predictors of depression recovery in HIV-infected individuals managed through measurement-based care in infectious disease clinics

Treatment of comorbid chronic disease, such as depression, in people living with HIV/AIDS (PLWHA) increasingly falls to HIV treatment providers. Guidance in who will best respond to depression treatment and which patient-centered symptoms are best to target is limited

PAP and NT5E inhibit nociceptive neurotransmission by rapidly hydrolyzing nucleotides to adenosine

Peer reviewe

Prostatic Acid Phosphatase Is an Ectonucleotidase and Suppresses Pain by Generating Adenosine

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 anti-nociceptive, anti-hyperalgesic and anti-allodynic 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

Recombinant Mouse PAP Has pH-Dependent Ectonucleotidase Activity and Acts through A1-Adenosine Receptors to Mediate Antinociception

Prostatic acid phosphatase (PAP) is expressed in nociceptive neurons and functions as an ectonucleotidase. When injected intraspinally, the secretory isoforms of human and bovine PAP protein have potent and long-lasting antinociceptive effects that are dependent on A1-adenosine receptor (A1R) activation. In this study, we purified the secretory isoform of mouse (m)PAP using the baculovirus expression system to determine if recombinant mPAP also had antinociceptive properties. We found that mPAP dephosphorylated AMP, and to a much lesser extent, ADP at neutral pH (pH 7.0). In contrast, mPAP dephosphorylated all purine nucleotides (AMP, ADP, ATP) at an acidic pH (pH 5.6). The transmembrane isoform of mPAP had similar pH-dependent ectonucleotidase activity. A single intraspinal injection of mPAP protein had long-lasting (three day) antinociceptive properties, including antihyperalgesic and antiallodynic effects in the Complete Freund's Adjuvant (CFA) inflammatory pain model. These antinociceptive effects were transiently blocked by the A1R antagonist 8-cyclopentyl-1, 3-dipropylxanthine (CPX), suggesting mPAP dephosphorylates nucleotides to adenosine to mediate antinociception just like human and bovine PAP. Our studies indicate that PAP has species-conserved antinociceptive effects and has pH-dependent ectonucleotidase activity. The ability to metabolize nucleotides in a pH-dependent manner could be relevant to conditions like inflammation where tissue acidosis and nucleotide release occur. Lastly, our studies demonstrate that recombinant PAP protein can be used to treat chronic pain in animal models

Regulator of G-Protein Signaling 14 (RGS14) Is a Selective H-Ras Effector

Background: Regulator of G-protein signaling (RGS) proteins have been well-described as accelerators of Ga-mediated GTP hydrolysis (‘‘GTPase-accelerating proteins’’ or GAPs). However, RGS proteins with complex domain architectures are now known to regulate much more than Ga GTPase activity. RGS14 contains tandem Ras-binding domains that have been reported to bind to Rap- but not Ras GTPases in vitro, leading to the suggestion that RGS14 is a Rap-specific effector. However, more recent data from mammals and Drosophila imply that, in vivo, RGS14 may instead be an effector of Ras.Methodology/Principal Findings: Full-length and truncated forms of purified RGS14 protein were found to bind indiscriminately in vitro to both Rap- and Ras-family GTPases, consistent with prior literature reports. In stark contrast, however, we found that in a cellular context RGS14 selectively binds to activated H-Ras and not to Rap isoforms. Co- transfection / co-immunoprecipitation experiments demonstrated the ability of full-length RGS14 to assemble a multiprotein complex with components of the ERK MAPK pathway in a manner dependent on activated H-Ras. Small interfering RNA-mediated knockdown of RGS14 inhibited both nerve growth factor- and basic fibrobast growth factor- mediated neuronal differentiation of PC12 cells, a process which is known to be dependent on Ras-ERK signaling.Conclusions/Significance: In cells, RGS14 facilitates the formation of a selective Ras?GTP-Raf-MEK-ERK multiprotein complex to promote sustained ERK activation and regulate H-Ras-dependent neuritogenesis. This cellular function for RGS14 is similar but distinct from that recently described for its closely-related paralogue, RGS12, which shares the tandem Ras- binding domain architecture with RGS14

mPAP dephosphorylates purine nucleotides in a pH-dependent manner.

<p>Plot of initial velocity at the indicated concentrations of AMP, ADP and ATP at (A) pH 7.0 and (B) pH 5.6. Reactions (n = 3 per point) were stopped after 3 min. Inorganic phosphate was measured using malachite green. All data are presented as means±s.e.m. Error bars are obscured due to their small size.</p

Dose-dependent antinociceptive effects of intrathecal mPAP.

<p>(A) Effects of increasing amounts of mPAP on paw withdrawal latency to a radiant heat source. (B) Paw withdrawal threshold to a semi-flexible tip mounted on an electronic von Frey apparatus. (A, B) BL = Baseline. Injection (i.t.) volume was 5 µL. n = 8 wild-type mice were used per dose. There were significant differences over time between mice injected with heat-inactivated (0 U) mPAP and mice injected with active (1 U or 2 U) mPAP (Repeated measure two-way ANOVA; <i>P</i><0.0001 for each dose). Post-hoc paired t-tests were used to compare responses at each time point between mice injected with active mPAP to mice injected with heat-inactivated mPAP (** <i>P</i><0.005; *** <i>P</i><0.0005). For the heat-inactivated mPAP control, the protein concentration was equivalent to the maximum 2 U dose of mPAP (1.1 mg/mL). All data are presented as means±s.e.m.</p

Inhibition of mPAP by L-(+)-tartrate.

<p>The indicated concentrations of L-(+)-tartrate were added to reactions (n = 3 per concentration) containing mPAP (1 U/mL), 100 mM sodium acetate, pH 5.6 and the fluorescent acid phosphatase substrate DiFMUP. Relative fluorescence units (RFU). All data are presented as means±s.e.m. Prism 5.0 (GraphPad Software, Inc) was used to generate curve.</p