Abstract

Many cells express both P2X cation channels and P2Y G-protein-coupled receptors that are costimulated by nucleotides released during physiologic or pathophysiologic responses. For example, during hemostasis and thrombosis, ATP-gated P2X1 channels and ADP-stimulated P2Y1 and P2Y12 G-protein coupled receptors play important roles in platelet activation. It has previously been reported that P2X1 receptors amplify P2Y1-evoked Ca²⁺ responses in platelets, but the underlying mechanism and influence on function is unknown. In human platelets, we show that maximally activated P2X1 receptors failed to stimulate significant aggregation but could amplify the aggregation response to a submaximal concentration of ADP. Costimulation of P2X1 and P2Y1 receptors generated a superadditive Ca²⁺ increase in both human platelets and human embryonic kidney 293 (HEK293) cells via a mechanism dependent on Ca²⁺ influx rather than Na⁺ influx or membrane depolarization. The potentiation, due to an enhanced P2Y1 response, was observed if ADP was added up to 60 seconds after P2X1 activation. P2X1 receptors also enhanced Ca²⁺ responses when costimulated with type 1 protease activated and M1 muscarinic acetylcholine receptors. The P2X1-dependent amplification of G_q-coupled [Ca²⁺]_i increase was mimicked by ionomycin and was not affected by inhibition of protein kinase C, Rho-kinase, or extracellular signal-regulated protein kinase 1/2, which suggests that it results from potentiation of inositol 1,4,5-trisphosphate receptors and/or phospholipase C. We conclude that Ca²⁺ influx through P2X1 receptors amplifies Ca²⁺ signaling through P2Y1 and other G_q-coupled receptors. This represents a general form of co-incidence detection of ATP and coreleased agonists, such as ADP at sites of vascular injury or synaptic transmitters acting at metabotropic G_q-coupled receptors.