Bernd Driessen

Functional consequences of inhibition of nucleotide breakdown in rat vas deferens: a study with Evans blue

The effect of Evans blue on nucleotide breakdown, nucleotide-evoked contractions and electrically evoked contractions, overflow of ATP and overflow of tritium (after labelling with [3H]-noradrenaline) was studied in rat vas deferens. Pieces of vas deferens degraded 83 to 85% of added ATP, ADP and 2-methylthio ATP (all 100 μM) over 30 min. Evans blue (100 μM) reduced this degradation to 22 to 26%. Nucleotides elicited contraction with potency declining in the order α, \-methylene ATP > 2-methylthio ATP > ATP > ADP. Evans blue (100 μM) shifted the concentration-response curve of α, \-methylene ATP to the right and increased the maximum. Concentration-response curves of ATP, ADP and 2-methylthio ATP, in contrast, were shifted to the left and responses were much potentiated. In the presence of Evans blue, the rank order of potency was ATP > 2-methylthio ATP > α, \-methylene ATP > ADP. Electrical field stimulation (100 pulses at 10 Hz) elicited contraction and an overflow of tritium and ATP. Evans blue (100 μM) did not alter the contraction and the evoked overflow of tritium but increased 24-fold the evoked overflow of ATP. The results indicate that Evans blue may serve as an — albeit impure — ecto-nucleotidase inhibitor in functional experiments. Such experiments demonstrate that the low potency of ATP (and also ADP and 2-methylthio ATP) in eliciting contraction, and the small size of the overflow of ATP upon sympathetic nerve stimulation, are due to rapid breakdown.

Release of ATP in rat vas deferens: origin and role of calcium

Release of endogenous ATP elicited by electrical (neural) stimulation and exogenous agonists was studied in the rat isolated vas deferens. The aims were to dissect neural and postjunctional contributions to the nerve activity-evoked overflow of ATP and to clarify the role of transmitter receptors and calcium in postjunctional ATP release.In tissues preincubated with [3H]-noradrenaline, electrical stimulation (100 pulses/10 Hz) elicited contraction and an overflow of tritium and ATP. Contractions as well as ATP overflow were reduced by prazosin 0.3 μM and even more so by prazosin 0.3 μM combined with suramin 300 μM. They were also reduced by nifedipine 10 μM and even more so by nifedipine 10 μM combined with ryanodine 20 μM (the additional effect of ryanodine on ATP overflow was not significant). In tissues not pretreated with [3H]-noradrenaline, exogenous noradrenaline 10 μM and α,β-methylene ATP 10 μM elicited contraction and an overflow of ATP. Responses to noradrenaline were blocked by prazosin 0.3 μM but not suramin 300 μM and were greatly reduced by nifedipine 10 μM and in Ca2+-free medium. Responses to α,β-methylene ATP were blocked by suramin 300 μM but not prazosin 0.3 μM were reduced by nifedipine 10 μM (effect on ATP overflow not significant) and were reduced even more in Ca2+-free medium. Neuropeptide Y 0.3 μM caused only very small contraction and ATP overflow. The electrically as well as the agonist-evoked ATP overflow correlated well with the contraction responses except in experiments with suramin which retarded the removal, by vas deferens tissue, of ATP from the medium.Itsis concluded that the overflow of ATP from rat vas deferens elicited by electrical (neural) stimulation is at least 90% postjunctional, presumably smooth muscle, in origin. ATP is released from postjunctional cells as a consequence of both α1-adrenoceptor and P2-purinoceptor activation. Ca2+ is a second messenger in the postjunctional ATP release response; its major part enters through L-type channels. A “purely neural” overflow of ATP was not isolated under the conditions of the experiments.

Depression of C fiber-evoked activity by intrathecally administered reactive red 2 in rat thalamic neurons

To investigate the possible role of spinal purinoceptors in nociception, the potent P2-purinoceptor antagonist reactive red 2 was studied in rats under urethane anesthesia in which nociceptive activity was elicited by electrical stimulation of afferent C fibers in the sural nerve and recorded from single neurons in the ventrobasal complex of the thalamus. Intrathecal (i.t.) application of reactive red 2 (6-200 micrograms) caused a dose-dependent reduction of the evoked activity in thalamic neurons. The estimated ED50 was 30 micrograms, and the maximum depression of nociceptive activity amounted to about 70% of the control activity at a dose of 100 micrograms. Morphine, administered i.t. at a maximally effective dose (80 micrograms), inhibited the evoked nociceptive activity by only up to 55% of the control activity. An i.t. co-injection of reactive red 2 (100 micrograms) and morphine (80 micrograms) caused a maximum reduction of the evoked thalamic activity by up to 85% of the control activity, thus, exceeding significantly the effect elicited by either drug alone. Similarly, i.t. co-injection of almost equipotent dosages of reactive red 2 (30 micrograms) and morphine (30 micrograms) caused a maximum reduction of the evoked activity by up to 72% of the control activity, which again exceeded significantly the effect of either drug alone. The results suggest that in rats reactive red 2 exerts antinociception by blockade of P2-purinoceptors in the spinal cord and, hence, support the idea that ATP may play an important role in spinal transmission of nociceptive signals. An activation of the spinal opioid system does not seem to contribute to the effect of reactive red 2 but might act additive or even synergistically with its antinociceptive action.