Prostanoid and purinergic signalling are essential for the proinflammatory component of acute and chronic kidney disease. Nucleotides are released early in response to cell perturbation, whereas the prostaglandin system is activated downstream. Interestingly, a direct effect of the COX-2 product prostaglandin E2-1-glyceryl ester (PGE2-G) on the UDP-sensitive P2Y6 receptor expressed in the proximal tubule is suggested. Thus, we hypothesized that PGE2-G amplifies renal inflammation directly via P2Y6-receptor activation. To verify the PGE2-G mediated P2Y6 activation, we used 132-1N1 astrocytoma cells devoid of P2 receptors for specific expression of the P2Y6 receptor. We monitored [Ca2+]i signalling by both life-cell microscopy and plate reader by detection of Fluo-4 fluorescence in human P2Y6 expressing 132-1N1 cells (hP2Y6 cells). Wild type 132-1N1 cells (WT cells) were used as negative control. The hP2Y6 cells were activated by UDP in a concentration-dependent manner. WT cells did not react to UDP but to carbachol. Surprisingly, stimulation of hP2Y6 cells with PGE2-G from several batches in concentrations from 10-11 to 10-6 M did not inflict any change in [Ca2+]i. We observed a PGE2-G-induced [Ca2+]i increase compared to vehicle control at concentrations of 10-4 M dissolved in DMSO. However, we detected a completely similar response in WT cells. PGE2-G in our preparations was confirmed by HPLC. Thus, our data cannot confirm a direct effect of PGE2-G on P2Y6 receptors and thus, does not support that this type of signalling is relevant during renal inflammation
