Potentiation by 2,2'-pyridylisatogen tosylate of ATP-responses at a recombinant P-2Y1 purinoceptor

1 2,2'-Pyridylisatogen tosylate (PIT) has been reported to be an irreversible antagonist of responses to adenosine 5'-triphosphate (ATP) at metabotropic purinoceptors (of the P-2Y family) in some smooth muscles. When a recombinant P-2Y1 purinoceptor (derived from chick brain) is expressed in Xenopus oocytes, ATP and 2-methylthioATP (2-MeSATP) evoke calcium-activated chloride currents (I-Cl,I-Ca) in a concentration-dependent manner. The effects of PIT on these agonist responses were examined at this cloned P-2Y purinoceptor. 2 PIT (0.1-100 mu M) failed to stimulate P-2Y1 purinoceptors directly but, over a narrow concentration range (0.1-3 mu M), caused a time-dependent potentiation (2-5 fold) of responses to ATP. The potentiation of ATP-responses by PIT was not caused by inhibition of oocyte ecto-ATPase. At high concentrations (3-100 mu M), PIT irreversibly inhibited responses to ATP with a IC50 value of 13 +/- 9 mu M (pK(B) = 4.88 +/- 0.22; n = 3). PIT failed to potentiate inward currents evoked by 2-MeSATP and only inhibited the responses to this agonist in an irreversible manner. 3 Known P-2 purinoceptor antagonists were tested for their ability to potentiate ATP-responses at the chick P-2Y1 purinoceptor. Suramin (IC50 = 230 +/- 80 nM; n = 5) and Reactive blue-2 (IC50 = 580 +/- 130 nM; n = 6) reversibly inhibited but did not potentiate ATP-responses. Coomassie brilliant blue-G (0.1- 3 mu M) potentiated ATP-responses in three experiments, while higher concentrations (3-100 mu M) irreversibly inhibited ATP-responses. The results indicated that potentiation and receptor antagonism were dissociable and not a feature common to all known P-2 purinoceptor antagonists. 4 In radioligand binding assays, PIT showed a low affinity (pK(i) < 5) for a range of membrane receptors, including: alpha(1), alpha(2)-adrenoceptors, 5-HT1A, 5-HT1B, 5-HT2, 5-HT3, D-1, D-2, muscarinic, central benzodiazepine, H-1, mu-opioid, dihydropyridine and batrachotoxin receptors. PIT showed some affinity (PKi = 5.3) for an adenosine (A(1)) receptor. 5 In guinea-pig isolated taenia caeci, PIT (12.5-50 mu M) irreversibly antagonized relaxations to ATP (3-1000 CIM); PIT also directly relaxed the smooth muscle and histamine was used to restore tone. Relaxations to nicotine (10-100 mu M), evoked by stimulating intrinsic NANC nerves of taenia caeci preparations in the presence of hyoscine (0.3 mu M) and guanethidine (17 mu M), were not affected by PIT (50 mu M, for 25-60 min). 6 These experiments indicate that PIT causes an irreversible antagonism of ATP receptors but, for recombinant chick P-2Y1 purinoceptors, this effect is preceded by potentiation of ATP agonism. The initial potentiation by PIT (and by Coomassie brilliant blue-G) of ATP-responses raises the possibility of designing a new class of modulatory drugs to enhance purinergic transmission at metabotropic purinoceptors.