ATP overflow from the isolated mouse vas deferens
The present work led to the conclusion that the release of endogenous ATP from field-stimulated adrenergic nerves innervating the mouse vas deferens can be systematically and quantitatively detected when combining "overflow" techniques and the refined version of the highly specific luciferase-catalysed reaction, presented here. The refined version of the luciferin-luciferase assay was quantitative over the range used and was reproducible within 10%. It was, thus, possible to compare ATP overflow following various number of pulses, various frequencies and to study prejunctional modulation of ATP overflow in the presence of P1-purinoceptors agonists. The temporal pattern of ATP overflow following various stimuli was characterized. Following stimulation, levels of ATP overflow significantly increased over basal, non stimulated ATP before returning to basal levels. Stimulated-ATP levels were variable between experiments. Such variation was not influenced by temperature, number of pulses or the position of the stimulation into the minute aliquot. It is possible that the variation was a consequence of either ectoATPase activity or uptake blockade since untreated tissues were used. An attempt was made to block ATP uptake blockade using suramin but the effects of the compound were incompatible with uptake blockade. Stimulated-ATP behaved in a physiological way. Increasing the number of pulses and the frequency of stimulation significantly increased stimulated-ATP. The non-progressive increase of the per pulse stimulated-ATP when increasing the frequency was explained on the basis that tissues were untreated and thus, it is possible that at low frequency of stimulation the ectoATPase activity was not saturated and, thus, any increase in stimulated-ATP would be degraded. But at a high frequency of stimulation the ectoATPase activity would be saturated, enabling to observed an increase in the per pulse stimulated-ATP. Only a minor part of the stimulated-ATP (around 25%) was due to activation of postsynaptic adrenoceptors (a1-type) supporting previous reports that postjunctional ATP release occurs and indicating that most of the stimulated-ATP was of neuronal origin. The effects of tetrodotoxin on ATP overflow were not expected. TTX could either abolish stimulated-ATP and the muscle contraction, providing further evidence for neurally-released ATP or abolish muscle contraction while a massive release of ATP, unrelated to stimulation was observed. Most probably, the massive release was caused by damaged smooth muscle cells which liberate their internal content of ATP, however, others suggestions have been made. Taken together, the effects of P1-agonists and the effects of activation of presynaptic adrenoceptors led to the conclusion that the modulation of ATP does not ressemble the modulation of noradrenaline indicating a possible differential release for noradrenaline and ATP. If differencial release occurs such system would be better described as multiple-transmission involving noradrenaline and ATP rather than the previous concept of "co-transmission" in which stoichiometric release was expected. A multiple-transmission system would be better prepared for survival and for coding invaluable information which is contained within the action potential. Further work is needed to test the hypothesis that purinergic and noradrenergic transmission may be under separate control mechanisms. Such work requires the simultaneous detection of noradrenaline and ATP overflow, a technique which has been presented in here.