HETEROLOGOUS EXPRESSION OF A P(2)X-PURINOCEPTOR IN RAT CHROMAFFIN CELLS DETECTS VESICULAR ATP RELEASE

A cloned P-2x-purinoceptor was transiently expressed in single isolate d rat adrenal chromaffin cells and evaluated for the detection of rele ased ATP. After cytoplasmic injection of the P-2x complementary RNA (c RNA; 4-24 h), application of ATP produced an inwardly rectifying curre nt 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 a 50% effective concentration of 18.2 mu M. Suramin, a P-2x-antag onist, attenuated the ATP-induced current. Depolarizing voltage pulses to 0 mV or application of histamine, stimuli that trigger exocytosis, resulted in the appearance of suramin-sensitive spontaneous transient inward currents (at -60 mV) that resembled excitatory postsynaptic cu rrents although they were slower in time course. Concurrent detection of catecholamine release with a carbon fiber electrode often showed co incidence of the amperometric current with the synaptic currentlike ev ents suggesting that ATP and catecholamines were released from the sam e vessicle. These data demonstrate that expression of a P-2x-purinocep tor in chromaffin cells produces a functional autoreceptor capable of detecting vesicular release of ATP. In combination with carbon fiber a mperometry, simultaneous vesicular release of two neurotransmitters fr om a single chromaffin cell could be monitored. The P-2x-purinoceptor, however, produced a regenerative effect on release apparently resulti ng from the high Ca2+ permeability of the receptor. Thus modification of the P-2x-purinoceptor would be required before the system could be applied to examining processes involved in stimulus-release coupling.