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. 2010 Dec;62(6):500-5.
doi: 10.1016/j.phrs.2010.08.003. Epub 2010 Sep 8.

P2Y(13) receptor is responsible for ADP-mediated degranulation in RBL-2H3 rat mast cells

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

P2Y(13) receptor is responsible for ADP-mediated degranulation in RBL-2H3 rat mast cells

Zhan-Guo Gao et al. Pharmacol Res. 2010 Dec.

Abstract

Extracellular ADP is known to play many important physiological roles. In this study, we identified the P2Y(13) receptor in a rat mast cell line (RBL-2H3) and explored the functional role of ADP, its endogenous agonist. ADP induced both intracellular calcium mobilization and release of hexosaminidase (Hex). In an assay of intracellular calcium, ADP was 100-fold less potent than and equally efficacious as the P2Y(1) receptor-selective agonist MRS2365. However, ADP was more potent and efficacious than MRS2365 in inducing Hex release and in enhancing antigen-induced Hex release. ADP-induced intracellular calcium mobilization was blocked by phospholipase C inhibitor U73122 and by P2Y(1) receptor-selective antagonist MRS2500, but not by pertussis toxin (PTX), suggesting a mechanism mediated by the G(q)-coupled P2Y(1) receptor, but not P2Y(13) (G(i)-coupled) or P2X receptors. ADP-induced Hex release was blocked by PTX and a selective P2Y(13) receptor antagonist MRS2211, but not by MRS2500 or P2Y(1) receptor-specific siRNA, suggesting a G(i)-coupled P2Y(13) receptor-related mechanism. Measurement of gene expression confirmed high expression of both P2Y(1) and P2Y(13) receptors (in comparison to a previously reported P2Y(14) receptor) in RBL-2H3 cells. Thus, we demonstrated that ADP-mediated intracellular calcium mobilization and Hex release in RBL-2H3 cells are via P2Y(1) and P2Y(13) receptors, respectively. Selective antagonists of the P2Y(13) receptor might be novel therapeutic agents for various allergic conditions.

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Figures

Figure 1
Figure 1
P2Y1 receptor agonist-induced intracellular calcium mobilization in RBL-2H3 cells. Results are expressed as mean ± S.E. from a triplicate determination representative of three independent experiments performed in duplicate or triplicate. The calcium mobilization was measured 20 s after addition of agonists and lasted for 120 s. The protocol used was similar to previously described [29]. The mean EC50 values of agonists are listed in the text.
Figure 2
Figure 2
Effects of various inhibitors on agonist-induced intracellular calcium mobilization in RBL-2H3 cells. A. Effects of pretreatment with PTX, a phospholipase C inhibitor U73122 and various concentrations of the P2Y1 receptor antagonist MRS2500. Cells were pretreated with PTX (100 ng/ml) for 24 h or with U73122 (10 μM) and MRS2500 for 20 min before the measurements. B. Schild analysis of antagonism by MRS2500 of the agonist effect of ADP. C. Effect of MRS2500 on the selective P2Y1 receptor agonist MRS2365-induced calcium release. D. Schild analysis of antagonism by MRS2500 of the agonist effect of MRS2365. The calcium mobilization was measured 20 s after addition of agonists and lasted for 120 s. The protocol used for measurement of calcium mobilization was similar to previously described [29]. The EC50 values (nM) of ADP and MRS2365 were 278 ± 42 and 4.5 ± 0.8 nM, respectively.
Figure 3
Figure 3
Hex release from RBL-2H3 cell induced by antigen (10 ng/ml) in the presence and absence of P2Y receptor agonists. A. Initial testing of the effect of antigen (10 ng/ml) in the absence and presence of ADP (100 μM). B. Enhancement by various agonists of antigen-mediated Hex release. Cells were primed with anti-DNP-BSA antibody for 24 h before testing for the release of Hex. Results are expressed as mean ± S.E. and were from three separate experiments performed in duplicate. #P < 0.05; ##P < 0.01, compared with control.
Figure 4
Figure 4
Effects of the phospholipase C inhibitor, U73122 (A, 10 μM) and pertussis toxin (PTX, 100 ng/ml) (B) and a calcium chelator, BAPTA-AM (10 μM) on antigen-mediated Hex release. Results are expressed as mean ± SE and are from three independent experiments performed in duplicate. The basal value of percentage Hex release was about 5%. U73122 and BAPTA-AM inhibited both antigen-induced and ADP-enhanced Hex release, whereas PTX only blocked ADP-enhanced Hex release.
Figure 5
Figure 5
A. Effects of P2Y receptor antagonists on ADP-induced enhancement of antigen-mediated Hex release in RBL-2H3 cells. MRS2500 and MRS2211 are antagonists for the P2Y1 and P2Y13 receptors, respectively. Cells were primed with anti-DNP-BSA antibody for 24 h before testing for the release of Hex. Results are expressed as mean ± S.E. and were from 3-4 separate experiments performed in duplicate. B. Effect of P2Y1 receptor-specific siRNA on ADP-induced enhancement of antigen-induced Hex release. The experimental procedures were as described previously [6].
Figure 5
Figure 5
A. Effects of P2Y receptor antagonists on ADP-induced enhancement of antigen-mediated Hex release in RBL-2H3 cells. MRS2500 and MRS2211 are antagonists for the P2Y1 and P2Y13 receptors, respectively. Cells were primed with anti-DNP-BSA antibody for 24 h before testing for the release of Hex. Results are expressed as mean ± S.E. and were from 3-4 separate experiments performed in duplicate. B. Effect of P2Y1 receptor-specific siRNA on ADP-induced enhancement of antigen-induced Hex release. The experimental procedures were as described previously [6].
Figure 6
Figure 6
ADP-induced Hex release in the absence of antigen DNP-BSA. Results were from 3-4 experiments performed in duplicate. *Significantly different from basal Hex release, P < 0.05.
Figure 7
Figure 7
Expression of P2Y1, P2Y12 and P2Y13 receptor genes determined using qRT-PCR in RBL-2H3 cells. Results are expressed as mean ± SE from three separate experiments performed in triplicate. The GAPDH was used as an endogenous positive control. The expression level of the known P2Y14 receptor [ref. 6] was expressed as 1. The gene expression levels of P2Y1, P2Y12 and P2Y13 receptors were expressed as their relative values to that of the P2Y14. The gene expression of the P2Y12 receptor was essentially undetectable in RBL-2H3 cells. The P2Y11 gene is absent in rodents.
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