Pharmacological characterization of ATP receptors in ampulla from frog semicircular canal

Abstract

Phosphoinositidase C activities sensitive to purine and pyrimidine nucleotides have been identified earlier in ampulla from Rana ridibunda semicircular canal. The aim of this study was to characterize the pharmacological properties of other P2 receptors borne by this structure. A microassay was developed to measure the binding of [35S]adenosine 5′- O-(2-thiodiphosphate) ([35S]ADPβS) to a few ampullas microdissected from frog semicircular canals. When determined at 4°C in the absence of divalent cations, [35S]ADPβS binding was saturable with incubation time and reversible after elimination of free radioligand. The dissociation kinetics were biphasic and comprised a major component that was rapidly reversible and a minor component that dissociated slowly. [35S]ADPβS binding was competitively inhibited by unlabeled ADPβS with an apparent dissociation constant of 0.48 ± 0.09 μM and a Hill coefficient of 0.70 ± 0.06, and Scatchard analysis revealed a minor class of high-affinity binding sites (RT1 = 52 ± 11 fmol [35S]ADPβS bound/ampulla and K d1 = 0.15 ± 0.04 μM) and a major class of low-affinity binding sites (RT2 = 436 ± 79 fmol [35S]ADPβS bound/ampulla and K d2 = 2.0 ± 0.8 μM). The pattern of stereospecificity for recognition of unlabeled structural ATP analogs was ADPβS ≥ α,β-methyleneadenosine 5′-triphosphate = ADP = adenosine 5′- O-(3-thiotriphosphate) > ATP = diadenosine tetraphosphate = AMP > 2′- and 3′- O-(4-benzoylbenzoyl)-adenosine 5′-triphosphate ≥ 2-methylthioadenosine 5′-triphosphate > 2-desoxythymidine 5′-triphosphate = guanosine 5′-triphosphate = inosine-5′-triphosphate = xanthosine 5′-triphosphate = cytosine 5′-triphosphate = uridine 5′-triphosphate = uridine-5′-diphosphate, whereas cAMP and adenosine were devoid of activity. For antagonists, suramin revealed competitive inhibitor potencies, whereas reactive blue 2 and DIDS acted as pure noncompetitive inhibitors. Results suggest that the population of labeled receptors is heterogeneous and contains a low number of P2Y-like receptors and a large number of P2X-like receptors whose molecular subtypes and functions in endolymph homeostasis remain to be defined.