2,2'-Pyridylisatogen tosylate antagonizes P2Y1 receptor signaling without affecting nucleotide binding

Abstract

The effect of 2,2'-pyridylisatogen tosylate (PIT) on the human P2Y(1) receptor and on other recombinant P2Y receptors has been studied. We first examined the modulation by PIT of the agonist-induced accumulation of inositol phosphates. PIT blocked 2-methylthio-ADP (2-MeSADP)-induced accumulation of inositol phosphates in 1321N1 astrocytoma cells stably expressing human P2Y(1) receptors in a non-competitive and concentration-dependent manner. The IC(50) for reduction of the maximal agonist effect was 0.14microM. In contrast, MRS2179, a competitive P2Y(1) receptor antagonist, parallel-shifted the agonist concentration-response curve to the right. PIT also concentration-dependently blocked the P2Y(1) receptor signaling induced by the endogenous agonists, ADP and ATP. A simple structural analogue of PIT was synthesized and found to be inactive as a P2Y(1) receptor antagonist, suggesting that the nitroxyl group of PIT is a necessary structural component for P2Y(1) receptor antagonism. We next examined the possible modulation of the binding of the newly available antagonist radioligand for the P2Y(1) receptor, [3H] MRS2279. It was found that PIT (0.01-10microM) did not inhibit [3H] MRS2279 binding to the human P2Y(1) receptor. PIT (10microM) had no effect on the competition for [3H] MRS2279 binding by agonists, ADP and ATP, suggesting that its antagonism of the P2Y(1) receptor may be allosteric. PIT had no significant effect on agonist activation of other P2Y receptors, including P2Y(2), P2Y(4), P2Y(6), P2Y(11) and P2Y(12) receptors. Thus, PIT selectively and non-competitively blocked P2Y(1) receptor signaling without affecting nucleotide binding.

Fig. 1

Effects of PIT and MRS2179, a competitive P2Y₁ receptor antagonist, on agonist-induced accumulation of IP, as a measure of inositol phosphates in 1321N1 astrocytoma cells stably expressing the human P2Y₁ receptor. Data were from one experiment performed in duplicate, which represents at least three independent experiments of similar results. The EC₅₀ value of 2-MeSADP listed in the text was calculated from three independent experiments performed in duplicate.

Fig. 2

Chemical structures of pyridylisatogen and MRS3461.

Fig. 3

(A) Effects of PIT and a structural analogue MRS3461 on agonist 2-MeSADP (100 nM)-induced accumulation of IP, as a measure of inositol phosphates in 1321N1 astrocytoma cells stably expressing P2Y₁ receptors (n = 3). (B) Effect of PIT on agonist ADP (1 μM)-induced accumulation of inositol phosphates.

Fig. 4

Effects of PIT on activation of human P2Y₂, P2Y₄, P2Y₆, P2Y₁₁ and P2Y₁₂ receptors and rat P2Y₆ receptors. Inhibition by 2-MeSADP of forskolin (10 μM)-stimulated activation of adenylate cyclase was the indicator of activity at the P2Y₁₂ receptor (receptor stably expressed in CHO cells). At all of the other receptors, stimulation of PLC was measured (receptors expressed in 1321N1 astrocytoma cells), using as agonists: UTP (P2Y₂, P2Y₄), UDP (P2Y₆), and ATP (P2Y₁₁). Data were from one experiment performed in duplicate, which represents three independent experiments of similar results.

Fig. 5

Effect of PIT and MRS2179 on the binding of [³H] MRS2279 to membranes from astrocytoma cells stably expressing the human P2Y₁ receptor. Membranes (40 μg protein per tube) from 1321N1 astrocytoma cells stably expressing the human P2Y₁ receptor were incubated with [³H] MRS2279 (8 nM) for 30 min at 4 °C. Results were expressed as mean ± S.E.M. from three independent experiments performed in duplicate. The potencies of PIT and MRS2179 are listed in Table 1.

Fig. 6

Effect of PIT on the competition curves of ADP and ATP for binding of the antagonist radioligand [³H] MRS2279. Membranes (40 μg protein per tube) from 1321N1 astrocytoma cells stably expressing the human P2Y₁ receptor were incubated with [³H] MRS2279 (8 nM) for 30 min at 4 °C. Data were expressed as mean ± S.E.M. from three independent experiments performed in duplicate.

Fig. 7

(A) ADP- and ATP-induced accumulation of IP, as a measure of inositol phosphates in 1321N1 astrocytoma cells stably expressing the human P2Y₁ receptor. (B) Effect of PIT on ATP-induced accumulation of inositol phosphates.

Fig. 8

Effect of PIT on the dissociation of the antagonist radioligand [³H] MRS2279 from P2Y₁ receptors. Membranes (40 μg protein per tube) from 1321N1 astrocytoma cells stably expressing the human P2Y₁ receptor were incubated with [³H] MRS2279 (8 nM) for 30 min at 4 °C. The dissociation was initiated by addition of 10 μM MRS2179 in the absence and presence of PIT. Data were from a representative experiment performed in duplicate. The k₋₁ values listed in the text were calculated from three independent experiments performed in duplicate.