Svetlana T. Mihaylova-Todorova

Evidence for the differential release of the cotransmitters ATP and noradrenaline from sympathetic nerves of the guinea‐pig vas deferens.

1. Experiments were carried out to quantify the stimulation‐evoked overflow of catecholamines and purines (ATP, ADP, AMP and adenosine) from an in vitro sympathetic nerve‐smooth muscle preparation of the guinea‐pig vas deferens and from isolated bovine adrenal chromaffin cells. The superfused preparations were stimulated for 60 s with electrical field stimulation (EFS; vas deferens), dimethylphenylpiperazinium (chromaffin cells) or KCl (both preparations). 2. Samples of superfusate were taken at 10 s intervals during the 60 s stimulation period for analysis of purines by HPLC‐fluorescence detection and catecholamines by HPLC‐electrochemical detection. 3. The evoked overflow of catecholamines and purines from chromaffin cells occurred with the same time course and in a constant ratio of approximately 4:1 (catecholamine to purine). These findings are compatible with the release of catecholamines and purines from a homogeneous population of exocytotic vesicles in the chromaffin cells. 4. The evoked overflow of purines and noradrenaline (NA) from the vas deferens preparation differed from the pattern of overflow from chromaffin cells and there was also some temporal disparity in the overflow of the two cotransmitters. The evoked overflow of ATP exceeded that of NA. In addition, the overflow of NA was tonic while the overflow of ATP and the other purines was phasic. 5. The EFS‐evoked overflow of NA and the purines from the guniea‐pig vas deferens preparation was examined after treatment with the neuronal amine‐uptake inhibitors desipramine and cocaine, the alpha 1‐adrenoceptor agonist methoxamine, the alpha 1‐adrenoceptor antagonist prazosin, the alpha 2‐adrenoceptor antagonists idazoxan and yohimbine, the noradrenaline‐depleting drug reserpine and the adrenergic neuron‐blocking agent guanethidine. The results of these studies, together with an analysis of the metabolic degradation of extracellular ATP, indicated that the temporal disparity in the overflow of NA and ATP is unlikely to be due to differences in the clearance of the cotransmitters or to the release of purines from non‐neuronal sites. These results indicate that evoked overflow of the cotransmitters accurately reflects release from nerves. This pattern of release from nerves suggests that the two cotransmitters are released from two separate populations of exocytotic vesicles. 6. Superfusion of the vas deferens with exogenous epsilon‐ATP, a fluorescent derivative of ATP, revealed that there was essentially no metabolism of the nucleotide over 60 s unless the tissue was subjected to EFS. Upon EFS, there was a rapid and nearly complete degradation of ATP with a corresponding increase in ADP, AMP and adenosine. This indicates the presence of a nerve stimulation‐dependent metabolism of ATP.

Chapter 2 Modulation of purinergic neurotransmission

Publisher Summary This chapter highlights some recent developments in the understanding of ATP as a cotransmitter. Four main topics of purinergic research are emphasized in the chapter: the storage and release of ATP and its regulation, the structure and classification of P2-receptor subtypes, the postjunctional effector mechanisms by which ATP mediates its neurotransmitter actions, and the mechanism of inactivation of the neurotransmitter actions of ATP by ATPases. Recent studies indicate that there are more than one population of storage vesicles in the nerves, as the release of various cotransmitters varies over time and can be differentially modulated by drugs. The subclassification of P2 receptors has advanced in the past few years because of the use of molecular biology methods allowing the cloning and expression of 14 different subclasses of P2 receptors, seven P2X, and seven P2Y. Determination of the functional significance of various receptor subtypes would be helped by the development of selective agonists and antagonists.

Release of soluble nucleotidases: a novel mechanism for neurotransmitter inactivation?

The support of the NIH and American Heart Foundation to Prof. D. P. Westfall, Astra Charnwood and the Wellcome Trust (C. K., P. S.), the Caledonian Research Foundation (C. K.) and Carnegie Trust (P. S.) is gratefully acknowledged. We also thank the Scottish Hospital Endowments Research Trust for refurbishing the laboratories in which some of these experiments were performed.