Effect of palmitoyl Co A on P2Y1 receptor-mediated calcium responses in HEK cells

Eman Alefishat, Vera Ralevic, Stephen Alexander. University of Nottingham, Nottingham, United Kingdom.

Palmitoyl CoA (PaCoA) is an antagonist at recombinant P2Y1 purine receptors, with a four-fold greater potency than CoA (Coddou et al., 2003). We have previously reported to the Society that PaCoA was approximately 100-fold more potent than CoA as an antagonist at P2Y1 receptors in the rat isolated aorta (Alexander et al., 2008). In the present study, we have investigated PaCoA as an antagonist of human P2Y1 receptors expressed endogenously in HEK cells.

HEK cells were cultured for 48 hours to confluency, measuring intracellular calcium levels using Fluo-4 in a Flexstation-96 (Kasorn et al., 2006). Cells were pre-incubated in the absence and presence of three concentrations of PaCoA (10-7-10-5 M) for 30 min, prior to addition of ADP or ATP (10-7-10-4 M).

ADP and ATP evoked calcium responses with pEC50 values of 6.7± 0.1 and 5.5 ± 0.1. Schild analysis of the effects of PaCoA allowed calculation of apparent pA2 values of 7.2 ± 0.2 and 7.0 ± 0.3 using ADP and ATP as agonist, with slopes of 1.4 ± 0.2 and 1.6 ± 0.2, respectively.

These results show that PaCoA is a reasonably potent antagonist at human native P2Y1 receptors in HEK cells. In comparison with our previous investigations of the effects of PaCoA in the rat isolated aorta (pA2 6.3, slope 2.9), there is a small increase in affinity, with a reduction in slope, which may result from the change from a large tissue mass to a monolayer of cells. These data raise the possibility of an endogenous regulation of purinergic signalling involving inhibition of P2Y1 receptors via CoA compounds.

Alexander SPH et al. (2009). Proc BPS 6: 150P.
Coddou C et al. (2003). FEBS Lett 536: 145-150.
Kasorn A et al. (2006). pA2 online 3: 24P.

We gratefully acknowledge and appreciate the sponsorship of the Jordanian government.