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. 2010 Mar 15;79(6):873-9.
doi: 10.1016/j.bcp.2009.10.024. Epub 2009 Nov 5.

UDP-glucose acting at P2Y14 receptors is a mediator of mast cell degranulation

Zhan-Guo Gao  1 , Yi Ding, Kenneth A Jacobson
Affiliations

UDP-glucose acting at P2Y14 receptors is a mediator of mast cell degranulation

Zhan-Guo Gao et al. Biochem Pharmacol. .

Abstract

UDP-glucose (UDPG), a glycosyl donor in the biosynthesis of carbohydrates, is an endogenous agonist of the G protein-coupled P2Y(14) receptor. RBL-2H3 mast cells endogenously express a P2Y(14) receptor at which UDPG mediates degranulation as indicated by beta-hexosaminidase (HEX) release. Both UDPG and a more potent, selective 2-thio-modified UDPG analog, MRS2690 (diphosphoric acid 1-alpha-d-glucopyranosyl ester 2-[(2-thio)uridin-5''-yl] ester), caused a substantial calcium transient in RBL-2H3 cells, which was blocked by pertussis toxin, indicating the presence of the G(i)-coupled P2Y(14) receptor, supported also by quantitative detection of abundant mRNA. Expression of the closely related P2Y(6) receptor was over 100 times lower than the P2Y(14) receptor, and the P2Y(6) agonist 3-phenacyl-UDP was inactive in RBL-2H3 cells. P2Y(14) receptor agonists also induced [(35)S]GTPgammaS binding to RBL-2H3 cell membranes, and phosphorylation of ERK1/2, P38 and JNK. UDPG and MRS2690 concentration-dependently enhanced HEX release with EC(50) values of 1150+/-320 and 103+/-18nM, respectively. The enhancement was completely blocked by pertussis toxin and significantly diminished by P2Y(14) receptor-specific siRNA. Thus, mast cells express an endogenous P2Y(14) receptor, which mediates G(i)-dependent degranulation and is therefore a potential novel therapeutic target for allergic conditions.

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Figures

Figure 1
Figure 1
Agonist-induced intracellular calcium mobilization in RBL-2H3 cells. Results are expressed as mean ± S.E. from a triplicate determination representative of 3–4 separate experiments performed in duplicate or triplicate. The mean EC50 values of agonists are listed in the text.
Figure 2
Figure 2
Effects of pretreatment with PTX and a phospholipase C inhibitor U73122 on agonist-induced calcium mobilization in RBL-2H3 cells. Cells were pretreated with PTX (100 ng/ml) for 24 h and with U73122 (10 μM) for 20 min before the measurements. Data are from one experiment performed in triplicate representative of three separate experiments performed in triplicate. Results are expressed as mean ± S.E.
Figure 3
Figure 3
mRNA expression of P2Y2, P2Y4, P2Y6 and P2Y14 receptors in RBL-2H3 mast cells measured by real-time RT-PCR. mRNA expression of P2Y receptors was normalized to the GAPDH level. Results are expressed as fold in comparison with that of the P2Y6 receptor (which showed the lowest expression and was set as 1) and are expressed as mean ± S.E. from 3 independent experiments. The expression levels of P2Y2, P2Y4 and P2Y14 receptors (fold in comparion with that of the P2Y6) are 6.13 ± 0.54, 1.58 ± 0.47 and 175 ± 52, respectively (n=3). #Significantly different from the expression level of the P2Y6 receptor (P<0.01).
Figure 4
Figure 4
UDPG and MRS2690 induced [35S]GTPγS binding to membranes prepared from RBL-2H3 cells. Results are expressed as mean ± SE and are from 3 independent experiments performed in duplicate.
Figure 5
Figure 5
Effect of UDPG (10 μM) and MRS2690 (1 μM) on phosphorylation of ERK1/2 (A), P38 (B,D) and JNK (C) in RBL-2H3 cells. In Figure 5A, cells were pretreated with PTX (100 ng/ml) for 24 h. Results are expressed as mean ± S.E. (n=3).
Figure 6
Figure 6
HEX release in RBL-2H3 cells. Cells were primed with anti-DNP-BSA antibody for 24 h before testing for the release of HEX. (A) Comparison of HEX release induced by UDPG (10 μM), MRS2690 (1 μM) and antigen (10 nM). (B) UDPG and MRS2690 enhanced HEX release in the presence of 10 nM antigen. (C) Effect of PTX (100 ng/ml) pretreatment for 24 h. (D) effect of pretreatment of a PLC inhibitor U73122 (10 μM) for 20 min.
Figure 7
Figure 7
HEX release from RBL-2H3 cell in the presence and absence of P2Y14 receptor siRNA. Results are expressed as mean ± S.E. and were from 3–4 separate experiments performed in duplicate. #p<0.05, compared with control.
Figure 8
Figure 8
A. Effects of PTX (100 ng/ml) and U73122 (10 µM) on HEX release induced by antigen or antigen plus MRS2690. a. Control; b. PTX. c. Antigen (1 µM). d. Antigen+PTX. e. Antigen+U73122. f. Antigen+MRS2690 (1 μM). g. Antigen+MRS2690+PTX. h. Antigen+MRS2690+U73122. j. Antigen+MRS2690+ LY294002 (10 µM). *P<0.01 compared with group (f). #P<0.05 compared with groups (d) and (h). B. Cooperative effect in HEX release between antigen and agonist (either UDPG or MRS2690) in the absence of antibody priming. Cells were grown in the absence of anti-DNP-BSA antibody for 24 h before testing for the release of HEX. I. Control. II. UDPG (10 μM). III. MRS2690 (1 μM). IV. Antigen (1 nM). V. Antigen (1 μM). VI. Antigen (1 nM) + UDPG. VI. Antigen (1 nM) + MRS2690.
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