Heterologous expression of a P2x-purinoceptor in rat chromaffin cells
detects vesicular ATP release.
Bettye Hollins and Stephen R. Ikeda.
Department of Pharmacology and Toxicology, Medical College of Georgia,
Augusta, GA 30912-2300.
APStracts 4:171N, 1997.
ABSTRACT
A cloned P2x-purinoceptor was transiently expressed in single isolated rat
adrenal chromaffin cells and evaluated for the detection of released ATP.
Following cytoplasmic injection of the P2x cRNA (4-24 hr), application of ATP
produced an inwardly rectifying current over the voltage range -130 to -10 mV
as measured by the whole-cell patch-clamp technique. The dose-response curve
for ATP was sigmoidal with an EC50 of 18.2 æM. Suramin, a P2x-antagonist,
attenuated the ATP-induced current. Depolarizing voltage pulses to 0 mV or
application of histamine, stimuli which trigger exocytosis, resulted in the
appearance of suramin-sensitive spontaneous transient inward currents (at -60
mV) that resembled excitatory postsynaptic currents although slower in time
course. Concurrent detection of catecholamine release with a carbon fiber
electrode often showed coincidence of the amperometric current with the
synaptic current-like events suggesting that ATP and catecholamines were
released from the same vessicle. These data demonstrate that expression of a
P2x-purinoceptor in chromaffin cells produces a functional autoreceptor
capable of detecting vesicular release of ATP. In combination with carbon
fiber amperometry, simultaneous vesicular release of two neurotransmitters
from a single chromaffin cell could be monitored. The P2x-purinoceptor,
however, produced a regenerative effect on release apparently resulting from
the high Ca2+ permeability of the receptor. Thus, modification of the P2x-
purinoceptor would be required before the system could be applied to examining
processes involved in stimulus-release coupling.
Received 27 December 1996; accepted in final form 24 July 1997.
APS Manuscript Number J1016-6.
Article publication pending J. Neurophysiol.
ISSN 1080-4757 Copyright 1997 The American Physiological Society.
Published in APStracts on 28 August 1997