Subcellular localization and organ distribution of adenotin 1 in rat tissues

Abstract

The release of endogenous ATP, measured by the luciferin-luciferase assay, and the release of [3H]acetylcholine from the isolated superior cervical ganglion of the rat loaded with [3H]choline were studied simultaneously. Electrical field stimulation enhanced the release of endogenous ATP and acetylcholine in a [Ca2+]o-dependent manner. The Na+ channel blocker, tetrodotoxin (1 μM) inhibited the stimulation-evoked release of endogenous ATP and of [3H]acetylcholine, but did not change the resting release. The release of ATP was dependent on the frequency of stimulation between 2 and 10 Hz, when the number of shocks was kept constant (360 shocks), while acetylcholine was not released in a frequency-dependent fashion. Ten days after cutting of the preganglionic nerve of the superior cervical ganglion the stimulation-evoked release of acetylcholine and ATP was abolished and the uptake of [3H]choline was significantly reduced but not inhibited. Hexamethonium, (100 μM) a nicotinic acetylcholine receptor antagonist, significantly reduced the release of both acetylcholine and ATP, indicating a positive feedback modulation of ACh and ATP release. 8-Cyclopentyl-1,3-dipropylxanthine (10 nM), the selective A1-adenosine receptor antagonist exhibited similar effect on the release of ATP and acetylcholine: both of them were augmented, showing that the stimulation-evoked release of ATP and acetylcholine are under the inhibitory control of A1-adenosine receptors. When the temperature was reduced to 7°C to inhibit carrier-mediated processes, the resting and stimulated release of acetylcholine was not changed. Conversely, the release of ATP in response to stimulation was reduced by 79.9±5.6%, and the basal release was also almost completely blocked. Carbamylcholine by itself was able to release ATP, but not acetylcholine, in a hexamethonium-inhibitable manner, even from ganglia whose preganglionic nerve had been cut 10 days prior to experiments, suggesting that ATP release can occur in response to nicotinic receptor stimulation of postsynaptic cells. The breakdown of ATP or AMP by superior cervical ganglion was measured by high performance liquid chromatography combined with UV detection. ATP and AMP, added to the tissues, were readily decomposed: the Km (apparent Michaelis constant) and Vmax (apparent maximal velocity) were 475±24 μM and 3.50±0.18 nmol/min per mg for ectoATPase and 1550±120 μM and 14.5±0.9 nmol/min per mg tissue for 5′-nucleotidase. In addition, by using electron microscopic enzyme histochemistry, the presence of ectoATPase was also shown in the superior cervical ganglion. It is concluded that endogenous ATP and acetylcholine are released simultaneously in response to stimulation of preganglionic nerve terminals in the superior cervical ganglion in a [Ca2+]o-dependent, tetrodotoxin-sensitive manner and is metabolized by ectoenzymes present in the tissue.The dissociation of the release of ATP and acetylcholine at different stimulation frequencies and temperatures shows that the release-ratio of acetylcholine and ATP can vary upon the condition of stimulation: this can reflect either the different composition of synaptic vesicles in the preganglionic nerve terminals or a significant contribution of non-exocytotic, carrier-mediated type of release of ATP to the bulk release.