A selective high-affinity antagonist of the P2Y14 receptor inhibits UDP-glucose-stimulated chemotaxis of human neutrophils

Abstract

The nucleotide-sugar-activated P2Y14 receptor (P2Y14-R) is highly expressed in hematopoietic cells. Although the physiologic functions of this receptor remain undefined, it has been strongly implicated recently in immune and inflammatory responses. Lack of availability of receptor-selective high-affinity antagonists has impeded progress in studies of this and most of the eight nucleotide-activated P2Y receptors. A series of molecules recently were identified by Gauthier et al. (Gauthier et al., 2011) that exhibited antagonist activity at the P2Y14-R. We synthesized one of these molecules, a 4,7-disubstituted 2-naphthoic acid derivative (PPTN), and studied its pharmacological properties in detail. The concentration-effect curve of UDP-glucose for promoting inhibition of adenylyl cyclase in C6 glioma cells stably expressing the P2Y14-R was shifted to the right in a concentration-dependent manner by PPTN. Schild analyses revealed that PPTN-mediated inhibition followed competitive kinetics, with a KB of 434 pM observed. In contrast, 1 μM PPTN exhibited no agonist or antagonist effect at the P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, or P2Y13 receptors. UDP-glucose-promoted chemotaxis of differentiated HL-60 human promyelocytic leukemia cells was blocked by PPTN with a concentration dependence consistent with the KB determined with recombinant P2Y14-R. In contrast, the chemotactic response evoked by the chemoattractant peptide fMetLeuPhe was unaffected by PPTN. UDP-glucose-promoted chemotaxis of freshly isolated human neutrophils also was blocked by PPTN. In summary, this work establishes PPTN as a highly selective high-affinity antagonist of the P2Y14-R that is useful for interrogating the action of this receptor in physiologic systems.

Fig. 1.

Structure and synthetic route of PPTN. The synthetic procedure is briefly described in Materials and Methods and is based on Boger et al. (1996) and Belly et al. (2009). Additional details of the procedures also are provided (Supplemental Experimental Procedures).

Fig. 2.

PPTN blocks P2Y₁₄-R–dependent elevation of cyclic AMP levels in P2Y₁₄-C6 cells. (A) Cyclic AMP accumulation was quantified in P2Y₁₄-C6 cells in the presence of 30 μM forskolin and the indicated concentrations of UDP-glucose (UDPG) as described in Materials and Methods. The results are the mean of triplicate determinations and are representative of results of at least three separate experiments. (B) Basal and forskolin (30 μM)–stimulated cyclic AMP accumulation was quantified in the absence or presence of 1 μM PPTN in wild-type (WT) C6 glioma cells. The results are the mean of triplicate determinations and are representative of results of three separate experiments. (C) Basal and forskolin (30 μM)–stimulated cyclic AMP accumulation was quantified in the absence or presence of 100 nM PPTN in P2Y₁₄-C6 glioma cells. The results are the mean of triplicate determinations and are representative of results of three separate experiments. NS, not significant; (*) and (#), significantly different from vehicle and forskolin, respectively; P < 0.05 by unpaired t test.

Fig. 3.

Concentration-dependent effect of PPTN on cyclic AMP levels in P2Y₁₄-C6 cells. Basal and forskolin (30 μM)–stimulated cyclic AMP accumulation was quantified in the presence of the indicated concentrations of PPTN in P2Y₁₄-C6 glioma cells. The results are the mean of triplicate determinations and are representative of results of at least three separate experiments.

Fig. 4.

High-affinity competitive antagonism of the human P2Y₁₄-R by PPTN. (A) Concentration effect curves for UDP-glucose (UDPG) for inhibition of forskolin (30 μM)–stimulated cyclic AMP accumulation were generated in P2Y₁₄-C6 glioma cells in the presence of the indicated concentrations of PPTN. The data are the mean of triplicate determinations, and the results are similar to those obtained in four separate experiments. Typical values for basal and forskolin-stimulated [³H]cyclic AMP accumulation were approximately 1000 and 9000 cpm, respectively. (B) A Schild plot was generated by determining the dose ratio (concentration of agonist necessary to produce a chosen level of effect in the presence of the indicated concentration of PPTN divided by the concentration of agonist that produces the same effect in the absence of antagonist) for each concentration of antagonist and then plotting log (dose ratio -1) on the Y-axis versus concentration of antagonist presented on the X-axis. The K_B determined from the intercept of the regressed line on the X- axis was 1150 pM, and a slope of 0.91 was observed. The results are similar to those obtained in four separate experiments.

Fig. 5.

PPTN blocks the agonist action of UDP and a receptor-selective synthetic analog at the P2Y₁₄-R. Forskolin (30 μM)–stimulated cyclic AMP accumulation was quantified in P2Y₁₄-C6 cells in the presence of 1 μM UDP-glucose (UDPG), UDP, or MRS2906 with or without 300 nM PPTN. The results are from triplicate determinations and are representative of results from three different experiments.

Fig. 6.

Lack of effect of PPTN at the P2Y₁-R, P2Y₂-R, P2Y₄-R, P2Y₆-R, and P2Y₁₁-R. [³H]Inositol phosphate accumulation was determined in P2Y₁-1321N1 (red), P2Y₂-1321N1 (blue), P2Y₄-1321N1 (green), P2Y₆-1321N1 (purple), and P2Y₁₁-1321N1 (black) cells in the absence of agonist (open symbols) or in the presence of a concentration of the cognate agonist (closed symbols) for each receptor that produced approximately 70–80% of the maximal effect observed with that agonist. The agonists and their concentrations were 2MeSADP (1 μM), UTP (300 nM), UTP (1 μM), UDP (1 μM), and ATP (100 μM) for the P2Y₁-R, P2Y₂-R, P2Y₄-R, P2Y₆-R, and P2Y₁₁-R, respectively. The results are the mean of triplicate determinations and are representative of results of at least three different experiments for each receptor. Typical values for basal and agonist-stimulated [³H]inositol phosphate accumulation were approximately 1000 and 6000 cpm, respectively, for P2Y₁-1321N1 cells, approximately 3000 and 10,000 cpm, respectively, for P2Y₂-1321N1 cells, approximately 3000 and 14,000 cpm, respectively, for P2Y₄-1321N1 cells, approximately 3000 and 25,000 cpm, respectively, for P2Y₆-1321N1 cells, and approximately 1000 and 8000 cpm, respectively, for P2Y₁₁-1321N1 cells.

Fig. 7.

Lack of effect of PPTN at the P2Y₁₂-R and P2Y₁₃-R. (A) Forskolin (30 μM)–stimulated cyclic AMP accumulation was quantified in P2Y₁₂-CHO cells in the presence of the indicated concentrations of PPTN in the absence (open red circle) or presence (closed red circle) of 1 μM 2MeSADP. The results are the mean of triplicate determinations and are representative of results of at least three different experiments for each receptor. Typical values for basal and forskolin-stimulated [³H]cyclic AMP accumulation in P2Y₁₂-CHO cells were approximately 700 and 4000 cpm, respectively. Data from Fig. 4 were replotted (dotted blue line) to provide a comparative illustration of the concentration-dependent effects of PPTN for antagonizing the effect of 316 nM UDP-glucose at the P2Y₁₄-R. (B) Agonist-stimulated [³H]inositol phosphate accumulation was quantified in the presence of the indicated concentrations of PPTN in the absence (open symbols) or presence (closed symbols) of agonists in COS-7 cells transfected with expression vectors for Gα_q/i and the human P2Y₁₃-R (green) or P2Y₁₄-R (blue). The agonists used were 3 μM ADP and 1 μM UDP-glucose for the P2Y₁₃-R and P2Y₁₄-R, respectively. The results are the mean of triplicate determinations and are representative of results of at least three different experiments for each receptor.

Fig. 8.

PPTN inhibits UDP-glucose–promoted chemotaxis in differentiated HL-60 human promyelocytic leukemia cells. (A) Chemotaxis of dHL-60 cells was quantified in the presence of 1, 10, and 100 μM UDP-glucose (UDPG). The agonist was added to the lower compartment of a Boyden chamber. After 2 hours of incubation at 37°C, the fluorescence of the bottom compartment was read and expressed as chemotaxis index (see Materials and Methods). The results are the mean ± S.E.M. of three independent experiments, each one performed in quadruplicate. (B) dHL-60 cells were added to the upper compartment in the presence of the indicated concentrations of PPTN, and chemotaxis index was quantified as above in the presence of either 10 μM or 100 μM UDP-glucose added to the lower compartment. The data are the mean ± S.E.M. of results from three independent experiments, each performed in quadruplicate. (C) The effect of PPTN on fMLP-induced chemotaxis was quantified. Vehicle or 100 nM fMLP was added to the lower compartment, and dHL-60 cells were added with or without 100 nM PPTN to the upper compartment. The results are the mean ± S.E.M. from three separate experiments, each performed in quadruplicate. NS, not significantly different from fMLP alone.

Fig. 9.

PPTN inhibits UDP-glucose–promoted chemotaxis in human neutrophils. Neutrophil migration was quantified in response to vehicle or 100 μM UDP-glucose (UDPG) added to the lower compartment of a Boyden chamber. Neutrophils were loaded in the upper chamber with or without 10 nM PPTN. The chemotaxis (Ctx) index was quantified after 2 hours, as described in Materials and Methods. The results are the mean ± S.E.M. from three separate experiments, each performed in quadruplicate; (*) and (#), significantly different from vehicle and UDP-glucose, respectively; P < 0.05 by 2-way analysis of variance.