Pharmacological characterization of atp receptors in ampulla from
frog semicircular canal.
Butlen, Daniel, Christian Bernard and Evelyne Ferrary .
1 Institut National de la Sant[acute]e et de la Recherche
M[acute]edicale, Unit[acute]e 426, Facult[acute]e de M[acute]edecine
Xavier Bichat, 75870 Paris Cedex 18, France; and 2 Laboratoire de
Neurophysiologie Sensorielle, Universit[acute]e de Rouen, 76821 Mont
-Saint-Aignan Cedex, France
APStracts 5:0123R, 1998.
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]-ADP- -S}
to a few ampullas microdissected from frog semicircular canals. When
determined at 4 C in absence of divalent cations, [35S]-ADP--S
binding was saturable with incubation time and reversible after
elimination of free radioligand. The dissociation kinetics were
biphasic and comprised a major- component which was rapidly
reversible and a minor component which dissociated slowly. [35S]-ADP
-[beta]-S binding was competitively inhibited by unlabeled with an
apparent dissociation constant KB = 0.48 +/- 0.09 M and a Hill
coefficient n = 0.70 +/- 0.06 and Scatchard analysis revealed a minor
class of high affinity binding sites (RT1 = 52 +/- 11 fmol [35S]-ADP
-[beta]-S bound/ampulla and KD1 = 0.15 +/- 0.04 [mu]M) and a major
class of low affinity binding sites (RT2 = 436 +/- 79 fmol [35S]
-ADP--S bound/ampulla and KD2 = 2.0 +/- 0.8 [mu]M). The pattern of
stereospecificity for recognition of unlabeled structural ATP
analogues was: ADP-[beta]-S >/= ([alpha], [beta])-Me-ATP = ADP = ATP
-[gamma]-S > ATP = Ap4A = AMP > Bz-ATP >/= 2-MeS-ATP > dTTP =
GTP = ITP = XTP = CTP = UTP = UDP, 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 heterogenous 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.
Received 2 July 1997; accepted in final form 25 March 1998.
APS Manuscript Number R418-7.
Article publication pending Am. J. Physiol. (Regulatory Integrative
Comp. Physiology).
ISSN 1080-4757 Copyright 1998 The American Physiological Society.
Published in APStracts on 24 April 1998