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Functional expression of the P2Y14 receptor in murine spleen derived T-lymphocytes Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis has previously shown that the P2Y14 receptor is expressed in peripheral immune cells such as lymphocytes and neutrophils (Moore et al., 2003). Furthermore, although in transfected cells the P2Y14 receptor couples to pertussis toxin-sensitive Gi/o protein(s) the functional coupling of endogenously expressed P2Y14 receptors to the inhibition of adenylyl cyclase activity has not been reported (Chambers et al., 2000). Therefore, the primary aim of this study was to determine whether the P2Y14 receptor is functionally expressed in enriched populations of murine spleen-derived T and B-lymphocytes. Balb/c mice were sacrificed by cervical dislocation and spleens removed under aseptic conditions. Total RNA was isolated from whole spleen and enriched populations of spleen-derived T and B-lymphocytes using RNA WIZTM . cDNA synthesis was performed using random primers and M-MLV reverse transcriptase and followed by PCR analysis using P2Y14 receptor specific primers: forward 5´-TTCTGGGTCGTGTTTCTTCTG-3´ and reverse 5´-CGAGAGTAGCAGAGTGAATTC-3´. Measurements of [3H]cAMP accumulation were performed using cells pre-labelled with [3H]-adenine as described previously (Germack & Dickenson, 2004). T-cell proliferation was monitored by measuring [3H]-thymidine incorporation.
RT-PCR analysis detected the expression of P2Y14 receptor mRNA in whole murine spleen and enriched populations of T-and B-lymphocytes. Importantly, PCR reactions using GAPDH primers performed prior to cDNA synthesis indicated that the mRNA was not contaminated with genomic DNA. In T-cells UDP-glucose (p[IC50 ]= 6.5 ± 0.6; n=5) induced a small but significant inhibition (22 ± 4%; n=5; P < 0.05) of 5 µM forskolin-stimulated cAMP accumulation suggesting functional coupling of endogenously expressed P2Y14 receptors to the inhibition of adenylyl cyclase activity. In B-cells UDP-glucose (100 µM) had no significant effect on forskolin-stimulated cAMP accumulation. Treatment of T-lymphocytes with pertussis toxin (100 ng ml-1, 16 h; Gi/o blocker) abolished the inhibitory effects of UDP-glucose on forskolin-stimulated cAMP accumulation. Finally, UDP-glucose (100 µM) significantly reduced interleukin-2 (5 ng/ml) and anti CD3 monoclonal antibody (1 µg/ml) induced T-cell proliferation by 48 ± 13% (n=5; P < 0.05) and 45 ± 16% (n=5; P < 0.05), respectively.
In conclusion, we have shown for the first time that the P2Y14 receptor is functionally expressed in murine spleen-derived T-lymphocytes. These observations suggest that UDP-glucose presumably via the P2Y14 receptor may play an important role in modulating immune function.
Chambers, J.K. et al. (2000) J. Biol. Chem. 275, 10767-10771.
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