Evidence that ATP, released from the postsynaptic site by noradrenaline, is involved in mechanical responses of guinea-pig vas deferens: Cascade transmission

Abstract

The release of endogenous ATP and [ 3H]noradrenaline and the mechanical response of the guinea-pig vas deferens to field stimulation of its motor nerves were examined using a perfusion system. The release of ATP at rest was 0.83 ± 0.13pmol/g per min and ATP released by field stimulation (8 Hz, 480 shocks) was 5.47 ± 1.23pmol/g. The evoked release was completely inhibited when Ca 2+ was removed and 1 mM EGTA was added, or by 1 μM tetrodotoxin. The release of ATP and [ 3H]noradrenaline in response to field stimulation was constant with an S 2/S 1 ratio of 1.10 ± 0.11 for ATP and 0.92 ± 0.03 for [ 3H]noradrenaline, respectively (where S 1 and S 2 are stimulation periods). Prazosin (1 μM), a potent α 1-adrenoceptor antagonist, significantly reduced the stimulation-evoked release of ATP by 75% and significantly reduced both mechanical twitch and tonic responses, but enhanced the release of [ 3H]noradrenaline. This finding indicates that there is an α 1-adrenoceptor-mediated release of endogenous ATP. However, the prazosin-insensitive portion of ATP release (25%) is considered to be of presynaptic origin. The stimulation of α 1-adrenoceptors by 1-noradrenaline or methoxamine in concentrations ranging from 10 to 100 μM resulted in a concentration-dependent release of ATP and a biphasic contraction of the vas deferens: a twitch response was followed by a tonic contraction. Prazosin (1 μM) completely prevented the effect of 1-noradrenaline or methoxamine on both ATP release and mechanical response. When Ca 2+ was omitted and EGTA (1 mM) was added, 1-noradrenaline was still able to release ATP but failed to produce contraction. Nifedipine, a Ca-channel and ATP receptor antagonist, reduced the twitch contraction and enhanced the release of ATP from muscle in response to noradrenaline administration. This finding indicates that the release of ATP from the muscle is not linked to mechanical contraction. When the vas deferens was made deficient in noradrenaline by 6-hydroxydopamine pretreatment (100 + 250rmmg/kg, i.p.), electrical field stimulation failed to release [ 3H]noradrenaline and ATP. Under these conditions, exogenous 1-noradrenaline was much more effective in releasing ATP from the smooth muscle and producing twitch responses, followed by a tonic contraction. After reserpine pretreatment (2 × 5mg/kg, i.p.), the field stimulation-evoked release of ATP and both phases of contraction were markedly reduced. These findings provide neurochemical evidence, from measurement of the release of ATP by the luciferin-luciferase method, that ATP is released from the smooth muscle of guinea-pig vas deferens in response to α 1-adrenoceptor stimulation by noradrenaline released from the sympathetic axon terminals. ATP released from the postsynaptic site in response to a chemical signal of presynaptic origin (noradrenaline) acts in loco via stimulation of P 2x receptors present on the smooth muscle outer surface. It produces a twitch response and contributes to the tonic contraction produced by noradrenaline. This leads to the proposal of a new type of transmission in smooth muscle—cascade transmission—where a transmitter, noradrenaline releases a secondary transmitter, ATP, from the postsynaptic site. This ATP, in addition to the ATP of presynaptic origin, becomes involved in the signal transmission via stimulation of postsynaptic P 2x receptors.