Evidence that 2-methylthioATP and 2-methylthioADP are both agonists at the rat hepatocyte P2Y(1) receptor

Abstract

In the absence of selective antagonists, pharmacological characterization of P2Y receptor subtypes has relied heavily upon their distinct agonist profiles. 2-methylthioADP (2-MeSADP) is a selective agonist for the P2Y(1) receptor. The agonist action of 2-MeSATP at the P2Y(1) receptor has recently been questioned. The effects of both 2-MeSADP and 2-MeSATP have been studied on rat hepatocytes injected with the bioluminescent Ca(2+) indicator, aequorin. Single hepatocytes generate series of repetitive transients in cytosolic free calcium concentration ([Ca(2+)](i)) when stimulated with agonists acting through the phosphoinositide signalling pathway. The transients induced by 2-MeSADP and 2-MeSATP in the same cell were indistinguishable, indicating that they act at a common receptor. In contrast the transients evoked by ATP and UTP had very different profiles. Treatment of 2-MeSATP with an ATP-regenerating system to remove contaminating 2-MeSADP did not abolish its agonist activity. Application of the P2Y(1) antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P) inhibited the transients induced by both 2-MeSADP and 2-MeSATP. In contrast the transients induced by ATP and UTP were enhanced by the addition of A3P5P. These results indicate that both 2-MeSADP and 2-MeSATP are agonists at the rat hepatocyte P2Y(1) receptor.

Figure 1

Similarities in the [Ca²⁺]_i response of a single rat hepatocyte to 2-MeSADP and 2-MeSATP, and the contrasting effects of ATP and UTP. Transients were recorded from a single aequorin-injected hepatocyte in response to stimulation with agonists at the concentrations indicated. The short duration transients induced by 10 μM 2-MeSADP were found to be indistinguishable from those induced by 10 μM 2-MeSATP. In contrast stimulation with either 1.5 μM ATP or 1.5 μM UTP induced transients of much longer duration in the same hepatocyte. The break in the x-axis represents a period of 15 min when the cell was superfused with WME alone. This result is typical of 20 experiments.

Figure 2

(a) Purification of 2-MeSATP to remove contaminating 2-MeSADP does not alter its ability to evoke [Ca²⁺]_i transients in rat hepatocytes. The 2-MeSATP stock was treated with an ATP-regenerating (RS) for 3 h and applied to an aequorin-injected hepatocyte. Stimulation with the same concentration of treated or untreated nucleotide evoked transients which were indistinguishable. The break in the x axis represents a period of 10 min when the cell was superfused with WME alone. This trace is representative of nine experiments. (b) HPLC profile of unpurified 2-MeSATP showing 2-MeSADP contamination. Peaks: (1) 2-MeSADP; (2) 2-MeSATP. (c) HPLC profile of 2-MeSATP after incubation with 20 units ml⁻¹ CPK and 20 mM PC for 3 h. 2-MeSADP contamination has been effectively removed (1). This sample was taken from the stock used to stimulate the cell depicted in (a).

Figure 3

Inhibitory effects of A3P5P on [Ca²⁺]_i transients induced by 2-MeSADP. Application of 10 μM A3P5P to a single cell responding to 2-MeSADP resulted in a decrease in frequency of transients. Increasing the concentration of A3P5P to 50 μM led to abolition of transients. This result is typical of five experiments. Transients induced by 2-MeSATP were affected similarly by addition of A3P5P (n=5).

Figure 4

Enhancement of UTP-induced [Ca²⁺]_i transients by the addition of A3P5P. Application of 10 μM A3P5P to a cell responding to UTP, led to an increase in duration of individual [Ca²⁺]_i transients. This result is typical of three experiments. ATP-induced transients were affected similarly by the addition of A3P5P (n=3).