Analysis of P2Y ₁₂ receptor responsiveness to cysteinyl leukotrienes

Leukotriene E 4 (LTE 4 ), the most stable of the cysteinyl leukotrienes (cysLT), binds poorly to classical type 1 and 2 cysLT receptors although it may potently induce bronchial constriction, airway hyperresponsiveness and inflammatory cell influx to the lungs of asthmatic individuals. Evidence of the presence of a previously unidentified LTE 4 receptor has also been provided in CysLT 1 - /CysLT 2 - double knock out mice. A recent study has suggested that purinergic receptor P2Y 12 is required for LTE 4 mediated pulmonary inflammation in a mouse model of asthma. The aim of the study was to characterise the responsiveness of human P2Y 12 to cysteinyl leukotrienes. A model of human CysLT 1 , CysLT 2 and P2Y 12 transiently overexpressed in HEK293 cells was used and responsiveness to different agonists was measured using intracellular calcium and cAMP assays. The responsiveness of human P2Y 12 stably overexpressed in CHO cells was also analysed using a β-arrestin recruitment assay. CysLTs induced concentration dependent calcium mobilisation in cells overexpressing CysLT 1 and CysLT 2 but failed to induce any calcium response in cells expressing P2Y 12 or P2Y 12 +G α 16. In contrast, a selective P2Y 12 agonist 2-MeSADP, induced specific calcium flux in cell expressing P2Y 12 +G α 16. Similarly, specific response to 2-MeSADP, but not to cysLTs was also observed in cells expressing P2Y 12 when intracellular cAMP and β-arrestin signalling was analysed. These results suggest that LTE 4 as well as other cysLTs may not activate intracellular signalling acting through human P2Y 12 and another LTE 4 specific receptor has yet to be identified.