Heterologous expression of a P2x-purinoceptor in rat chromaffin cells detects vesicular ATP release.
A cloned P2x-purinoceptor was transiently expressed in single isolated rat adrenal chromaffin cells and evaluated for the detection of released ATP. After cytoplasmic injection of the P2x complementary RNA (cRNA; 4-24 h), 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 a 50% effective concentration of 18. 2 microM. Suramin, a P2x-antagonist, 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 currents although they were slower in time course. Concurrent detection of catecholamine release with a carbon fiber electrode often showed coincidence of the amperometric current with the synaptic currentlike 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.
Hollins
B
B
Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912-2300, USA.
Ikeda
S R
SR
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002395:Catecholamines / Q000378:metabolism; D019439:Chromaffin Cells / Q000378:metabolism*; D003001:Cloning, Molecular; D066298:In Vitro Techniques; D008297:Male; D018408:Patch-Clamp Techniques; D018075:RNA, Complementary / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011983:Receptors, Purinergic / Q000235:genetics*; D013572:Synaptic Vesicles / Q000378:metabolism*
D002395:Catecholamines; D018075:RNA, Complementary; D011983:Receptors, Purinergic; D000255:Adenosine Triphosphate