Figure 1.
Purification of recombinant mPAP.
(A) A thrombin cleavage site (Tr) followed by hexahistidine tag (H6) and stop codon (*) were added to the C-terminus of the secretory isoform of mPAP. SP = signal peptide of mPAP. Map is not drawn to scale. (B) Amino acid sequence at the junction between the catalytic domain and Tr-H6 tag. Arrow marks thrombin cleavage site. Asterisk marks stop codon. (C) GelCode blue-stained SDS-PAGE gel and (D) western blot of purified recombinant mPAP protein (1 µg and 5 µg, respectively). The western blot was probed with an anti-hexahistidine antibody.
Figure 2.
Inhibition of mPAP by L-(+)-tartrate.
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.
Figure 3.
mPAP dephosphorylates purine nucleotides in a pH-dependent manner.
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.
Figure 4.
TM-PAP dephosphorylates extracellular purine nucleotides in a pH-dependent manner.
HEK 293 cells were transfected with expression vectors containing (A–F) mouse TM-PAP or (G–L) the fluorescent protein Venus as a control. Cells were then histochemically stained using AMP, ADP or ATP (each 6 mM) as substrate at pH 7.0 or pH 5.6. Cells were not permeabilized with detergent. Scale bar (bottom right panel), 50 µm for all panels.
Figure 5.
Dose-dependent antinociceptive effects of intrathecal mPAP.
(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; P<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 (** P<0.005; *** P<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.
Figure 6.
The antinociceptive effects of mPAP can be transiently inhibited with a selective A1R antagonist.
Wild-type mice were tested for (A) noxious thermal and (B) mechanical sensitivity before (baseline, BL) and following injection of CFA (CFA-arrow) into one hindpaw. The non-inflamed hindpaw served as control. All mice were injected with active mPAP (mPAP-arrow; 2 U, i.t.). Two days later, half the mice were injected with vehicle (CPX/V-arrow, circles; intraperitoneal (i.p.); 1 hr before behavioral measurements) while the other half were injected with 8-cyclopentyl-1, 3-dipropylxanthine (CPX/V-arrow, squares; 1 mg/kg i.p.; 1 hr before behavioral measurements). There were significant differences over time between mice injected with vehicle and mice injected with CPX (Repeated measure two-way ANOVA; P<0.01). Post-hoc paired t-tests were used to compare responses at each time point between vehicle (n = 10) and CPX-injected mice (n = 10); same paw comparisons. *** P<0.0005. All data are presented as means±s.e.m.