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The role of P2Y14 and other P2Y receptors in degranulation of human LAD2 mast cells

Abstract

Mast cell degranulation affects many conditions, e.g., asthma and urticaria. We explored the potential role of the P2Y14 receptor (P2Y14R) and other P2Y subtypes in degranulation of human LAD2 mast cells. All eight P2YRs were expressed at variable levels in LAD2 cells (quantitative real-time RT-PCR). Gene expression levels of ADP receptors, P2Y1R, P2Y12R, and P2Y13R, were similar, and P2Y11R and P2Y4R were highly expressed at 5.8- and 3.8-fold of P2Y1R, respectively. Least expressed P2Y2R was 40-fold lower than P2Y1R, and P2Y6R and P2Y14R were ≤50 % of P2Y1R. None of the native P2YR agonists alone induced β-hexosaminidase (β-Hex) release, but some nucleotides significantly enhanced β-Hex release induced by C3a or antigen, with a rank efficacy order of ATP > UDPG ≥ ADP >> UDP, UTP. Although P2Y11R and P2Y4R are highly expressed, they did not seem to play a major role in degranulation as neither P2Y4R agonist UTP nor P2Y11R agonists ATPγS and NF546 had a substantial effect. P2Y1R-selective agonist MRS2365 enhanced degranulation, but ~1,000-fold weaker compared to its P2Y1R potency, and the effect of P2Y6R agonist 3-phenacyl-UDP was negligible. The enhancement by ADP and ATP appears mediated via multiple receptors. Both UDPG and a synthetic agonist of the P2Y14R, MRS2690, enhanced C3a-induced β-Hex release, which was inhibited by a P2Y14R antagonist, specific P2Y14R siRNA and pertussis toxin, suggesting a role of P2Y14R activation in promoting human mast cell degranulation.

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Abbreviations

ADP:

Adenosine 5′-diphosphate

AR-C66096:

(2-(Propylthio)adenosine-5′-O-(β,γ-difluoromethylene)triphosphate tetrasodium salt)

ATP:

Adenosine 5′-triphosphate

BSA:

Bovine serum albumin

(FcεRI):

Fc region of immunoglobulin E (IgE)

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

GPCR:

G-protein-coupled receptor

β-Hex:

β-Hexosaminidase

MRS2211:

2-[(2-Chloro-5-nitrophenyl)azo]-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-4-pyridinecarboxaldehyde

MRS2365:

[[(1R,2R,3S,4R,5S)-4-[6-amino-2-(methylthio)-9H-purin-9-yl]-2,3-dihydroxybicyclo[3.1.0]hex-1-yl]methyl]diphosphoric acid mono ester trisodium salt

MRS2500:

(1R,2S,4S,5S)-4-[2-iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy)bicyclo[3.1.0]hexane-1-methanol dihydrogen phosphate ester tetraammonium salt

MRS2690:

2-Thiouridine-5′-diphosphoglucose

NF340:

4,4′-(Carbonylbis(imino-3,1-(4-methy l-phenylene)carbonylimino))bis(naphthalene-2,6-disulfonic acid) tetrasodium salt)

NF546:

4,4′-(Carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)carbonylimino))-bis(1,3-xylene-alpha,alpha′-diphosphonic acid tetrasodium salt)

NP-BSA:

4-Hydroxy-3-nitrophenylacetyl hapten amide conjugated to bovine serum albumin

PTX:

Pertussis toxin

qRT-PCR:

Quantitative real time polymerase chain reaction

RBL:

Rat basophilic leukemia

UDP:

Uridine 5′-diphosphate

UTP:

Uridine 5′-triphosphate

UDPG:

Uridine-5′-diphosphoglucose (UDP-glucose)

XAC:

8-[4-[[[[(2-Aminoethyl)amino]carbonyl]methyl)oxy]phenyl]-1,3-dipropylxanthine

PPTN:

4-(4-(Piperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic acid (mesylate salt form)

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Acknowledgments

This work is supported by the NIDDK Intramural Research Program, National Institutes of Health. We thank Drs. Arnold Kirshenbaum and Dean Metcalfe (NIAID, NIH, Bethesda, MD, USA) for providing LAD2 cells. We thank Prof. Mortimer M. Civan (University of Pennsylvania) for helpful discussions.

Author information

Correspondence to Zhan-Guo Gao or Kenneth A. Jacobson.

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Gao, Z., Wei, Q., Jayasekara, M.P.S. et al. The role of P2Y14 and other P2Y receptors in degranulation of human LAD2 mast cells. Purinergic Signalling 9, 31–40 (2013). https://doi.org/10.1007/s11302-012-9325-4

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Keywords

  • P2Y
  • Mast cells
  • Uracil nucleotide
  • Degranulation
  • GPCR
  • G protein-coupled receptors