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Effects of acetaldehyde on contractile response to nerve stimulation in guinea-pig vas deferens
European Journal of Pharmacology, 186 (1990) 189-196
189
Elsevier EJP 51474
Effects of acetaldehyde on contractile response to nerve stimulation in guinea-pig vas deferens Ryuji Takeda, Yasunori Momose and Akira Haji Department of Pharmacology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan
Received 28 May 1990, revised MS received 22 June 1990, accepted 26 June 1990
Twitch contractions of the isolated guinea-pig vas deferens induced by sympathetic nerve stimulation were augmented by acetaldehyde (0.1-10 mM). With high concentrations (5-10 raM), acetaldehyde produced a biphasic response consisting of an initial brief depression and a subsequent potentiation of the contraction. The late effect was associated with repetitive contractions that were not prevented by tetrodotoxin. A low concentration of phentolamine (27/~M) increased and a high concentration (1.3 mM) suppressed the potentiating action of acetaldehyde. Acetaldehyde did not induce contractions in surgically sympathectomized vasa or vasa pretreated with reserpine. Acetaldehyde caused a dose-dependent increase in noradrenaline release into the bathing fluid. The study shows that acetaldehyde has a dual effect on sympathetic neuroeffector transmission, and that an increase in noradrenaline secretion appears to contribute to the late facilitatory effect in the isolated vas deferens. Acetaldehyde; Adrenergic transmission; Noradrenaline; Contractile responses; Vas deferens
1. Introduction Acetaldehyde is known as an indirectly acting sympatho-mimetic agent (Akabane et al., 1964; Walsh et al., 1969; Egle et al., 1973). Lai and Hudgins (1975) reported that acetaldehyde produced contractions in the isolated segment of the rat vas deferens in a manner resembling that of tyramine. Similar contractile responses were induced by acetaldehyde in isolated blood vessels such as the rat portal vein (Altura et al., 1978) and the canine auricular (ear) artery (Chiba and Tsukada, 1988). These actions of acetaldehyde appeared to be due to endogenous catecholamines released from the sympathetic nerve terminals (Lai
Correspondence to: R. Takeda, Department of Pharmacology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan.
and Hudgins, 1975; Chiba and Tsukada, 1988). However, there is little data about the effects of acetaldehyde on adrenergic neuroeffector transmission. It has been shown that the mechanical and electrical responses of the isolated vas deferens to sympathetic nerve stimulation are biphasic. These responses are thought to be mediated by two different transmitters co-secreted from the sympathetic nerve terminals (Burnstock and Holman, 1964; Furness, 1974; Meldrum and Burnstock, 1983; Sneddon and Westfall, 1984; Stj~irne and Astrand, 1985; Vizi and Burnstock, 1988). With this in mind, we attempted to clarify the actions of acetaldehyde on the mechanical and electrical events occurring during sympathetic nerve stimulation of the isolated guinea-pig vas deferens. We now report on the influence of acetaldehyde on nerve-stimulated contractions, the contractile response to noradrenaline, and the ability of
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
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acetaldehyde to release noradrenaline. In the accompanying article, we describe the effects of acetaldehyde on the electrical characteristics of the vas deferens during sympathetic neuroeffector transmission (Takeda et al., 1990).
(1969). The right vas deferens was left intact to serve as control. The experiments were conducted 4 days after surgery. 'Chemical sympathectomy' was achieved with reserpine. Four other animals were injected i.p. with 5 m g / k g reserpine on 2 successive days, and were used 24 h after the second injection.
2. Materials and methods
2.1. Contraction experiments Experiments were carried out with 22 albino guinea-pigs, weighing between 250 and 450 g. They were stunned and exsanguinated. The vasa deferentia together with branches of the hypogastric nerve were excised. Longitudinal strips of 10 mm length were prepared from the prostatic side and suspended in a 3 ml organ bath. The strips were perfused at a constant rate of 2 m l / m i n with Krebs solution of the following composition (in mM): NaC1 121.9, KC1 4.7, CaC12 2.5, MgC12 1.2, KH2PO 4 1.2, N a H C O 3 15.5 and glucose 11.5. The solution was maintained at 34-36°C and bubbled with 95% 02 and 5% CO 2 (pH 7.2-7.4). Two sets of silver wire electrodes were placed on the strips in parallel; one set was placed on the hypogastric nerve about 15 mm from its entry into the vas deferens, and the other on the prostatic end of the tissue close to the entry of the hypogastric nerve. The former was used for preganglionic nerve stimulation and the latter for transmural postganglionic nerve stimulation (Birmingham and Wilson, 1963; Sj~Sstrand, 1981). A short train of rectangular pulses (20 pulses at 10 Hz) were applied alternately to preganglionic and postganglionic nerve fibers every 40 s; the pulses lasted 0.1-0.5 ms and were of suprathreshold voltage (5-10 V). Contractions of the vasa deferentia were recorded isometrically by means of a mechano-elec~ric transducer (UL-10, Shinko Tsushin). The resting tension was 0.5 g. 2.2. Denervation Four animals were anesthetized with pentobarbital (30-35 m g / k g i.p.) and the left vas deferens was surgically sympathectomized according to the procedures described by Kasuya et al.
2.3. Measurement of noradrenaline output Freshly incised vas deferens with the hypogastric nerve was superfused for 30 min with oxygenated Krebs solution and the effluent was discarded. The tissue was then superfused with 30 ~tM phenoxybenzamine for 4 min, and the effluent (4-min fraction) was collected in a tube containing 1 / 5 volume of chilled 2 N perchloric acid. During the next 4 min sampling period, the preparation was stimulated transmurally with pulses of suprathreshold intensity (15-20 V, 2 ms in pulse width, 5 Hz in frequency). This stimulation induced sustained contractions of nearly maximal amplitudes. This combination of stimulations was carried out in the absence and presence of acetaldehyde. The effluent were kept on ice for 30 rain and centrifuged at 10000 x g for 30 min. Noradrenaline was extracted and concentrated from the supernatant with a column of the resin Duolite C-20 (Wako) as described by Ito et al. (1962). Noradrenaline was determined fluorometrically using the ethylenediamine condensing method (Weil-Malherbe and Bone, 1952). The tissue was blotted with filter paper and weighed. 2.4. Drugs Acetaldehyde (Wako), noradrenaline hydrochloride (Sankyo), h e x a m e t h o n i u m bromide (Yamanouchi), phentolamine methansulfonate (Ciba), phenoxybenzamine hydrochloride (Nakarai), tetrodotoxin (Sankyo) and reserpine (Torii) were used in this study. Acetaldehyde was freshly distilled from a stock solution and dissolved in Krebs solution immediately before use. It was applied to the tissue by changing the inflow to the organ bath from normal Krebs solution to Krebs containing an appropriate concentration of
191 acetaldehyde (pH 7.2-7.3) without altering the flow rate. 2.5. Statistics
D a t a are presented as means + S.E. Statistical analyses were done with Student's t-test (twosided). Differences with P < 0.05 were considered significant.
3. Results 3.1. Effects on nerve stimulated contractions
Twitch contractions of a fairly constant amplitude were obtained by electrical stimulation of b o t h the preganglionic and postgangfionic nerves with a short train of pulses (20 pulses at 10 Hz). Acetaldehyde reversibly and concentration dependently increased the twitch contractions (fig. 1). At concentrations of 5 and 10 mM, acetaldehyde p r o d u c e d a brief depression of the contractions, a response which appeared shortly after drug application and reached a peak after about 2 rain. In addition, these high concentrations of acetaldehyde later induced repetitive contractions of irregular amplitudes that were not related to nerve stimulation (fig. 1B). Increasing the concentration of acetaldehyde increased the amplitude and
frequency of the repetitive contractions whereas the neurally stimulated contractions decreased slightly (fig. 1C). In the continuous presence of the high concentrations of acetaldehyde, the repetitive contractions lasted about 20 min and then declined abruptly. The neurally stimulated contractions were also severely depressed when the repetitive contractions subsided. The twitch contractions re-appeared within 10 min after the washout of the substance (fig. 1C). However, it took about 30 min for the twitch contractions to return to the pre-application level. Relations between the acetaldehyde concentration and the amplitude of the twitch contractions were assessed at two peak times, 2 min and 8-12 min, during a 30 min superfusion with acetaldehyde. Figure 2 shows that acetaldehyde produced a biphasic effect consisting of an early depression and a late facilitation of the neurally induced contractions. The m i n i m u m effective concentration for the facilitatory action was approximately 0.1 mM, and that for the inhibitory effect was between 1 and 5 m M (fig. 2). H e x a m e t h o n i u m (30/~M) eliminated the twitch contractions induced by hypogastric nerve stimulation but not those evoked by transmural stimulation (fig. 3B). However, h e x a m e t h o n i u m did not affect the acetaldehyde (5 m M ) - i n d u c e d transient depression and late augmentation of transmurally stimulated contractions or the repetitive contrac-
A
AcA I mM B
C
AcA 5 m M
AcA lOmM
1 g J
5 rain
Fig. 1. Effects of acetaldehyde (AcA) on the twitch contractions induced by sympathetic nerve stimulation in isolated vas deferens. Circles represent preganglionic nerve stimulation (20 pulses at 10 Hz, 0.2 ms, 5 V); dots indicate transmural postganglionic nerve stimulation (20 pulses at 10 Hz, 0.2 ms, 10 V). These two stimuli were applied alternately every 40 s throughout the experiment, but are indicated only in the early part of the trace. AcA was applied during the period indicated by the horizontal bar. (A) 1 mM; (B) 5 mM; (C) 10 mM. These records were obtained from the same preparation.
192
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hyde a n d suppressed its early i n h i b i t o r y effect on the twitch contractions. P h e n t o l a m i n e (27 >M) also increased the a l d e h y d e - i n d u c e d repetitive c o n t r a c t i o n s (fig. 4B). A higher c o n c e n t r a t i o n of p h e n t o l a m i n e (1.3 m M ) completely abolished the neurally i n d u c e d contractions. Acetaldehyde, when applied in the presence of this high c o n c e n t r a t i o n of p h e n t o l a m i n e , did not cause c o n t r a c t i o n (fig.
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0.5 1 5 10 AeA (mM) Fig. 2. Relations between the concentration of acetaldehyde (AcA) and the contractile response to the nerve stimulation. Amplitudes of the twitch contractions induced by transmural nerve stimulation were measured at two peak times, 2 min (dots) and 8-12 min (circles), after the start of a 30 min superfusion with acetaldehyde. Amplitudes are presented as percent changes from pre-application amplitudes (ordinate). Average amplitudes with S.E. (vertical bar) are shown. The number of experiments is in parentheses.
tions i n d u c e d by acetaldehyde. T e t r o d o t o x i n (3 /~M) blocked all neurally stimulated contractions. However, this agent did n o t prevent the acetaldeh y d e - i n d u c e d repetitive c o n t r a c t i o n s (fig. 3C). A relatively low c o n c e n t r a t i o n of p h e n t o l a m i n e (27 /~M) increased the nerve-stimulated twitch c o n t r a c t i o n s (fig. 4B). This dose of p h e n t o l a m i n e p o t e n t i a t e d the late a u g m e n t i n g effect of acetalde-
N o contractile response was i n d u c e d in surgically denervated vas deferens by t r a n s m u r a l stimulation, even though the stimulus i n t e n s i t y was 10 times greater (up to 100 V) t h a n that used in the control experiments. However, the p r e p a r a t i o n res p o n d e d with distinct c o n t r a c t i o n s to exogenously applied n o r a d r e n a l i n e at c o n c e n t r a t i o n s of 250 a n d 500 n M . Neither c o n c e n t r a t i o n of n o r a d r e n a line had a contractile effect on the control preparations. This m e a n s that d e n e r v a t i o n supersensitivity had occurred ( K a s u y a et al., 1969). A c e t a l d e h y d e in c o n c e n t r a t i o n s up to 10 m M did not induce a contractile response in this preparation (n = 4). Similar supersensitivity was observed in the vasa pretreated with reserpine (n = 4). This prep a r a t i o n showed n o contractile response to electrical s t i m u l a t i o n or to acetaldehyde (fig. 4D). However, after the p r e p a r a t i o n had been i n c u b a t e d
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Fig. 3. Effects of hexamethonium (C6) and tetrodotoxin (TTX) on the twitch contractions and the acetaldehyde-induced response in isolated vas deferens. Circles and dots indicate preganglionic and postganglionic nerve stimulation, respectively, as shown in fig. 1. (A) A control experiment with 5 mM acetaldehyde (AcA) applied during the bar; (B) 30 btM C6 was applied at the time indicated by an arrow; (C) 3 p.M TTX. W: the preparation was washed with a drug-free Krebs solution for about 30 min.
193
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Fig. 4. Effects of phentolamine and pretreatment with reserpine on the twitch contractions and the acetaldehyde-induced contractions. Records A-C were obtained from the same preparation. (A) A control experiment with 5 mM acetaldehyde (AcA) applied during the bar; (B) 27 ~tM phentolamine, applied at the time indicated by a solid triangle (the trace has been shortened); (C) 1.3 mM phentolamine. Records D-E were obtained from a preparation pretreated with reserpine. (D) A control experiment with 10 mM AcA; (E) the response to AcA (10 mM) after the preparation had been incubated with noradrenaline (5 #M for 20 min).
with noradrenaline, a small tonic contraction associated with phasic irregular contractions occurred during application of 10 mM acetaldehyde. Electrical stimulation of the hypogastric nerve or the transmural postganglionic nerve also produced small but distinct contractions (fig. 4E).
after the aldehyde-induced repetitive contractions had subsided (n = 5).
3.3. Effects on contractions induced by noradrenaline
Noradrenaline was released spontaneously into the bathing fluid. Transmural stimulation increased the noradrenaline output. The evoked release was slightly decreased during the subsequent stimulation. The ratio between the amounts of noradrenaline released by consecutive stimulations was 0.81 + 0.15 ( n = 4). However, when acetaldehyde was present during the second stimulation, the amount of noradrenaline released increased remarkably. Figure 5 summarizes the results of these experiments in which the first stimulation was taken as control and the effects of acetaldehyde were examined with the succeeding stimulations. Under the control conditions, the nervestimulated release of noradrenaline was approximately twofold greater than the spontaneous out-
Application of noradrenaline (12.5 and 25/~M) produced a monophasic contraction with a relatively slow time course (30-90 s) (not illustrated). At concentrations of 2.5/tM or lower, noradrenaline had no effect on the resting tension of normal vasa. In the presence of 1 mM acetaldehyde, this low concentration of noradrenaline produced a distinct contraction (n = 5). Acetaldehyde augmented the peak amplitude and time course of the contractions induced by higher concentrations of noradrenaline. In the presence of 10 mM acetaldehyde, the noradrenaline-induced contractions were severely depressed during the period
3.4. Effects on noradrenaline output
194
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lmM 5mM Fig. 5. Effects of acetaldehyde (AcA) on noradrena]ine output into the bathing fluid. White columns show the mean values with S.E. (vertical bars) of noradrenaline released during a 4 min incubation period immediately before nerve stimulation, shaded columns during a 4 min stimulation period. Stimulation pulses (15-20 V, 2 ms, 5 Hz) were applied transmurally for 4 min. * * P < 0.01: significantly different from the corresponding control values (left two columns, n = 8). AcA 1 mM (center, n = 11), AcA 5 mM (right, n = 14).
put. Application of acetaldehyde caused a dosedependent increase in both the nerve stimulated and non-stimulated release of noradrenaline (fig. 5).
4. Discussion
Twitch contractions induced by hypogastric nerve stimulation were eliminated by hexamethonium, tetrodotoxin or a relatively high dose of phentolamine, an a-adrenoceptor blocking agent. Contractions produced by transmural stimulation were not blocked by hexamethonium but were suppressed by the two other agents. Hence, the contractions evoked by nerve stimulation and by transmural stimulation can be considered to be due to excitation of preganglionic and postganglionic sympathetic nerves, respectively (Birmingham and Wilson, 1963; Btilbring et al., 1981; Sj6strand, 1981). Acetaldehyde potentiated the twitch contractions induced by both stimuli. Potentiation of the transmurally stimulated contractions occurred even when the ganglionic transmission was blocked by hexamethonium. In addition, acetaldehyde had no effect on the action
potentials of postganglionic nerve bundles (Takeda et al., 1990). Thus, the effect of acetaldehyde was not dependent on ganglionic transmission or on spike propagation in postganglionic nerves. It has been reported that, during a train of stimuli, endogenously released noradrenaline contributes to the later part of the contraction, which is distinct from the rapid part induced by the non-adrenergic co-transmitter (Wakade and Krusz, 1972; McGrath, 1978; Meldrum and Burnstock, 1983; Brown et al., 1983). However, the twitch contractions observed in our experiments were totally suppressed by a high dose of phentolamine or by pretreatment with reserpine. Treatment of the reserpinized vas with noradrenaline partly restored the twitch contraction. Further, as shown in the accompanying article, reserpine pretreatment did not eliminate nerve stimulated excitatory junction potentials (EJPs) (Takeda et al., 1990). Therefore, it is presumed that the main part of the twitch contraction is due to noradrenaline, and that the non-adrenergic co-transmitter seems to trigger this contraction. Acetaldehyde produced a concentration-dependent increase in the release of noradrenaline during nerve stimulation, The aldehyde also dose-dependently increased the non-stimulated noradrenaline release. A relatively low concentration of phentolamine enhanced both the nerve-stimulated twitch contractions and the acetaldehyde-induced contractions. It has been postulated that blockade of presynaptically located ~-adrenoceptors causes a large increase in noradrenaline output per stimulus (Langer, 1979: Alberts et al., 1981: BiJlbring et al., 1981). Thus, the increased secretion of noradrenaline could be a major mechanism by which both phentolamine and aldehyde increased the nerve stimulated contractions. With higher concentrations of acetaldehyde, the release of endogenous noradrenaline is increased to generate the repetitive contractions (Lai and Hudgins, 1975). Likewise, the transmitter released by acetaldehyde may lead to additive effects on the contractions produced by exogenous noradrenaline. This notion is supported by the finding that surgical or chemical sympathectomy suppressed the potentiating effect of acetaldehyde. Since the acetaldehyde-induced contractions were not pre-
195
vented by tetrodotoxin, the drug-evoked release of noradrenaline is, at least partly, independent of impulse propagation in sympathetic nerve fibers (Lai and Hudgins, 1975; Chiba and Tsukada, 1988). In addition, when the aldehyde-induced contractions declined, the neurally stimulated contractions were also severely depressed. This result suggests that the transmitter may be depleted by higher concentrations of acetaldehyde. However, since the contractions produced by exogenously applied noradrenaline were also depressed during this period, it is more likely that the postjunctional receptor was 'desensitized' to noradrenaline (Harden, 1983). Another possible explanation is that the transmitter released causes a progressively increasing depolarization of the smooth muscle membrane (Takeda et al., 1990) which later leads to inactivation of action potential generation. When acetaldehyde is washed out, the latter mechanism could be affected before receptor desensitization. This could account for the observation that the neurally stimulated contractions were restored within a relatively short time during the washout of acetaldehyde. The present study demonstrated that acetaldehyde produced biphasic or dual effects on neurally induced contractions. These effects of acetaldehyde were dose- and time-dependent. Similar dual effects of acetaldehyde have been reported for various tissues such as the rat cardiac muscle (Egle et al., 1973; Sakurai et al., 1980), rat portal vein (Altura et al., 1978), frog and cat spinal motor neurons (Takeda and Haji, 1985; Haji and Takeda, 1987). These authors postulated that the aldehyde excited these muscle or nerve cells by liberating excitatory transmitters and depressed directly the postsynaptic membrane. Our electrophysiological studies with the vas deferens suggest that the initial inhibitory effect of acetaldehyde may be due to a decrease in the EJPs and hyperpolarization of the smooth muscle membrane (Takeda et al., 1990). The late facilitatory effect appears to be due to endogenous noradrenaline. The effective concentrations of acetaldehyde in the guinea-pig vas deferens were similar to those reported previously for isolated strips of other smooth muscle organs (Lai and Hudgins, 1975; Altura et al., 1978; Takeda and Momose, 1978;
Chiba and Tsukada, 1988). It is known that the blood level of acetaldehyde reaches nearly 100 #M during the consumption of moderate to large amount of ethanol by humans. However, when ethanol is combined with disulfiram, the level increases to several times that of controls (Weiner, 1979). Presumably acetaldehyde is responsible for at least part of the pharmacolcgical consequences of sympathetic neuroeffector transmission during acute ethanol intoxication or during the disulfiram-ethanol reaction.
References Akabane, J., S. Nakanishi, H. Kohei, R. Matsumura and H. Ogata, 1964, Studies on sympathomimetic action of acetaldehyde: experiments with blood pressure and nictitating membrane, Jap. J. Pharmacol. 14, 295. Alberts, P., T. Bartfai and L. Stj~irne, 1981, Site(s) and ionic basis of alpha-autoinhibition and facifitation of [3H]noradrenaline secretion in guinea-pig vas deferens, J. Physiol. (London) 312, 297. Altura, B.M., A. Carella and B.T. Altura, 1978, Acetaldehyde on vascular smooth muscle: possible role in vasodilator action of ethanol, European J. Pharmacol. 52, 73. Birmingham, A.T. and A.B. Wilson, 1963, Preganglionic and postganglionic stimulation of the guinea-pig isolated vas deferens preparation, Br. J. Pharmacol. 21,569. Brown, D.A., J.R. Docherty, A.M. French, A. MacDonald, J.C. McGrath and N.C. Scott, 1983, Separation of adrenergic and non-adrenergic contractions to field stimulation in the rat vas deferens, Br. J. Pharmacol. 79, 379. Biilbring, E., H. Ohashi and T. Tomita, 1981, Adrenergic mechanisms, in: Smooth muscle, eds. E. Biilbring, A.F. Brading, A.W. Jones and T. Tomita (Edward Arnold, London) p. 219. Burnstock, G. and M.E. Holman, 1964, An electrophysiological investigation of the actions of some autonomic blocking drugs on transmission in the guinea-pig vas deferens, Br. J. Pharmacol. 23, 600. Chiba, S. and M. Tsukada, 1988, Possible mechanism of acetaldehyde-induced noradrenaline release from sympathetic nerve terminals in isolated blood vessels, Br. J. Pharmacol. 95, 177. Egle, J.L., P.M. Hudgins and F.M. Lai, 1973, Cardiovascular effects of intravenous acetaldehyde and propionaldehyde in the anesthetized rat, Toxicol. Appl. Pharmacol. 24, 636. Furness, J.B., 1974, Transmission to the longitudinal muscle of the guinea-pig vas deferens: the effect of pretreatment of guanethidine, Br. J. Pharmacol. 50, 63. Haji, A. and R. Takeda, 1987, Effects of acetaldehyde on the monosynaptic reflex pathway in the cat spinal cord, Arch. Int. Pharmacodyn. Ther. 289, 25.
196 Harden, T.K., 1983, Agonist-induced desensitization of the beta-adrenergic receptor linked adenylate cyclase, Pharmacol. Rev. 35, 5. Ito. T., M. Matsuoka, K. Nakazima, K. Tagawa and R. Imaizumi, 1962, An isolation method of catecholamine and effect of reserpine on the enzyme systems related to the formation and inactivation of catecholamines in brain, Jap. J. Pharmacol. 12, 130. Kasuya, Y., K. Goto, H. Hashimoto, H. Watanabe, H. Munakata and M. Watanabe, 1969, Nonspecific denervation supersensitivity in the rat vas deferens 'in vitro', European J. Pharmacol. 8, 177. Lai, F.M. and P.M. Hudgins, 1975, A comparison of the indirect sympathomimetic actions of tyramine and acetaldehyde in isolated smooth muscle preparations, Toxicol. Appl. Pharmacol. 32, 368. Langer, S.Z., 1979, Presynaptic adrenoceptors and regulation of release, in: The Release of Catecholamines from Adrenergic Neurons, ed. D.M. Paton (Pergamon, Oxford) p. 59. McGrath, J.C., 1978, Adrenergic and 'non-adrenergic' components in the contractile response of the vas deferens to a single indirect stimulus, J. Physiol. (London) 283, 23. Meldrum, L.A. and G. Burnstock, 1983, Evidence that ATP acts as a co-transmitter with noradrenaline in sympathetic nerves supplying the guinea-pig vas deferens, European J. Pharmacol. 92, 161. Sakurai, T., O.H.L. Bing, G. Huber and W.H. Abelmann, 1980, Effects of acetaldehyde upon mechanical properties of isolated rat papillary muscle, J. Pharmacol. Exp. Ther. 214, 219. Sj6strand, N.O., 1981, Smooth muscles of vas deferens and other organs in the male reproductive tract, in: Smooth Muscle, eds. E. Biilbring, A.F. Brading, A.W. Jones and T. Tomita (Edward Arnold, London) p. 367. Sneddon, P. and D.P. Westfall, 1984, Pharmacological evi-
dence that adenosine triphosphate and noradrenaline are co-transmitters in the guinea-pig vas deferens, J. Physiol. (London) 347, 561. Stfiirne, L. and P. Astrand, 1985, Relative pre- and postjunctional roles of noradrenaline and adenosine 5'-triphosphate as neurotransmitters of the sympathetic nerves of guinea-pig and mouse vas deferens, Neuroscience 14, 929. Takeda, R. and A. Haft, 1985, Dual effects of acetaldehyde on electrical activity in the isolated frog spinal cord, European J. Pharmacol. 113, 409. Takeda, R. and Y. Momose, 1978, Effects of acetaldehyde on electrical and mechanical activities of guinea-pig taenia coil Arch. int. Pharmacodyn. Ther. 232, 14. Takeda, R., Y. Momose and A. Haft, 1990, Effects of acetaldehyde on electrical activity during neuroeffector transmission in guinea-pig vas deferens, European J. Pharmacol. 186, 197. Vizi, E.S. and G. Burnstock, 1988, Origin of ATP release in the rat vas deferens: concomitant measurement of [3H]noradrenaline and [14C]ATP, European J. Pharmacol. 158, 69. Wakade, A.R. and J. Krusz, 1972, Effect of reserpine, phenoxybenzamine and cocaine on neuromuscular transmission in the vas deferens of the guinea pig, J. Pharmacol. Exp. Ther. 181,310. Walsh, M.J., P.B. Hollander and E.B. Truitt, Jr. 1969, Sympathomimetic effects of acetaldehyde on the electrical and contractile characteristics of isolated left atria of guinea pig, J. Pharmacol. Exp. Ther. 167, 173. Weil-Malherbe, H. and A.D. Bone, 1952, The chemical estimation of adrenaline-like substances in blood, Biochem. J. 51, 311. Weiner, H., 1979, Aldehyde metabolism, in: Biochemistry and Pharmacology of Ethanol, Vol. 1, eds. E. Majchrowicz and E.P. Noble (Plenum, New York, London) p. 125.
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Elsevier EJP 51474
Effects of acetaldehyde on contractile response to nerve stimulation in guinea-pig vas deferens Ryuji Takeda, Yasunori Momose and Akira Haji Department of Pharmacology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan
Received 28 May 1990, revised MS received 22 June 1990, accepted 26 June 1990
Twitch contractions of the isolated guinea-pig vas deferens induced by sympathetic nerve stimulation were augmented by acetaldehyde (0.1-10 mM). With high concentrations (5-10 raM), acetaldehyde produced a biphasic response consisting of an initial brief depression and a subsequent potentiation of the contraction. The late effect was associated with repetitive contractions that were not prevented by tetrodotoxin. A low concentration of phentolamine (27/~M) increased and a high concentration (1.3 mM) suppressed the potentiating action of acetaldehyde. Acetaldehyde did not induce contractions in surgically sympathectomized vasa or vasa pretreated with reserpine. Acetaldehyde caused a dose-dependent increase in noradrenaline release into the bathing fluid. The study shows that acetaldehyde has a dual effect on sympathetic neuroeffector transmission, and that an increase in noradrenaline secretion appears to contribute to the late facilitatory effect in the isolated vas deferens. Acetaldehyde; Adrenergic transmission; Noradrenaline; Contractile responses; Vas deferens
1. Introduction Acetaldehyde is known as an indirectly acting sympatho-mimetic agent (Akabane et al., 1964; Walsh et al., 1969; Egle et al., 1973). Lai and Hudgins (1975) reported that acetaldehyde produced contractions in the isolated segment of the rat vas deferens in a manner resembling that of tyramine. Similar contractile responses were induced by acetaldehyde in isolated blood vessels such as the rat portal vein (Altura et al., 1978) and the canine auricular (ear) artery (Chiba and Tsukada, 1988). These actions of acetaldehyde appeared to be due to endogenous catecholamines released from the sympathetic nerve terminals (Lai
Correspondence to: R. Takeda, Department of Pharmacology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan.
and Hudgins, 1975; Chiba and Tsukada, 1988). However, there is little data about the effects of acetaldehyde on adrenergic neuroeffector transmission. It has been shown that the mechanical and electrical responses of the isolated vas deferens to sympathetic nerve stimulation are biphasic. These responses are thought to be mediated by two different transmitters co-secreted from the sympathetic nerve terminals (Burnstock and Holman, 1964; Furness, 1974; Meldrum and Burnstock, 1983; Sneddon and Westfall, 1984; Stj~irne and Astrand, 1985; Vizi and Burnstock, 1988). With this in mind, we attempted to clarify the actions of acetaldehyde on the mechanical and electrical events occurring during sympathetic nerve stimulation of the isolated guinea-pig vas deferens. We now report on the influence of acetaldehyde on nerve-stimulated contractions, the contractile response to noradrenaline, and the ability of
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
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acetaldehyde to release noradrenaline. In the accompanying article, we describe the effects of acetaldehyde on the electrical characteristics of the vas deferens during sympathetic neuroeffector transmission (Takeda et al., 1990).
(1969). The right vas deferens was left intact to serve as control. The experiments were conducted 4 days after surgery. 'Chemical sympathectomy' was achieved with reserpine. Four other animals were injected i.p. with 5 m g / k g reserpine on 2 successive days, and were used 24 h after the second injection.
2. Materials and methods
2.1. Contraction experiments Experiments were carried out with 22 albino guinea-pigs, weighing between 250 and 450 g. They were stunned and exsanguinated. The vasa deferentia together with branches of the hypogastric nerve were excised. Longitudinal strips of 10 mm length were prepared from the prostatic side and suspended in a 3 ml organ bath. The strips were perfused at a constant rate of 2 m l / m i n with Krebs solution of the following composition (in mM): NaC1 121.9, KC1 4.7, CaC12 2.5, MgC12 1.2, KH2PO 4 1.2, N a H C O 3 15.5 and glucose 11.5. The solution was maintained at 34-36°C and bubbled with 95% 02 and 5% CO 2 (pH 7.2-7.4). Two sets of silver wire electrodes were placed on the strips in parallel; one set was placed on the hypogastric nerve about 15 mm from its entry into the vas deferens, and the other on the prostatic end of the tissue close to the entry of the hypogastric nerve. The former was used for preganglionic nerve stimulation and the latter for transmural postganglionic nerve stimulation (Birmingham and Wilson, 1963; Sj~Sstrand, 1981). A short train of rectangular pulses (20 pulses at 10 Hz) were applied alternately to preganglionic and postganglionic nerve fibers every 40 s; the pulses lasted 0.1-0.5 ms and were of suprathreshold voltage (5-10 V). Contractions of the vasa deferentia were recorded isometrically by means of a mechano-elec~ric transducer (UL-10, Shinko Tsushin). The resting tension was 0.5 g. 2.2. Denervation Four animals were anesthetized with pentobarbital (30-35 m g / k g i.p.) and the left vas deferens was surgically sympathectomized according to the procedures described by Kasuya et al.
2.3. Measurement of noradrenaline output Freshly incised vas deferens with the hypogastric nerve was superfused for 30 min with oxygenated Krebs solution and the effluent was discarded. The tissue was then superfused with 30 ~tM phenoxybenzamine for 4 min, and the effluent (4-min fraction) was collected in a tube containing 1 / 5 volume of chilled 2 N perchloric acid. During the next 4 min sampling period, the preparation was stimulated transmurally with pulses of suprathreshold intensity (15-20 V, 2 ms in pulse width, 5 Hz in frequency). This stimulation induced sustained contractions of nearly maximal amplitudes. This combination of stimulations was carried out in the absence and presence of acetaldehyde. The effluent were kept on ice for 30 rain and centrifuged at 10000 x g for 30 min. Noradrenaline was extracted and concentrated from the supernatant with a column of the resin Duolite C-20 (Wako) as described by Ito et al. (1962). Noradrenaline was determined fluorometrically using the ethylenediamine condensing method (Weil-Malherbe and Bone, 1952). The tissue was blotted with filter paper and weighed. 2.4. Drugs Acetaldehyde (Wako), noradrenaline hydrochloride (Sankyo), h e x a m e t h o n i u m bromide (Yamanouchi), phentolamine methansulfonate (Ciba), phenoxybenzamine hydrochloride (Nakarai), tetrodotoxin (Sankyo) and reserpine (Torii) were used in this study. Acetaldehyde was freshly distilled from a stock solution and dissolved in Krebs solution immediately before use. It was applied to the tissue by changing the inflow to the organ bath from normal Krebs solution to Krebs containing an appropriate concentration of
191 acetaldehyde (pH 7.2-7.3) without altering the flow rate. 2.5. Statistics
D a t a are presented as means + S.E. Statistical analyses were done with Student's t-test (twosided). Differences with P < 0.05 were considered significant.
3. Results 3.1. Effects on nerve stimulated contractions
Twitch contractions of a fairly constant amplitude were obtained by electrical stimulation of b o t h the preganglionic and postgangfionic nerves with a short train of pulses (20 pulses at 10 Hz). Acetaldehyde reversibly and concentration dependently increased the twitch contractions (fig. 1). At concentrations of 5 and 10 mM, acetaldehyde p r o d u c e d a brief depression of the contractions, a response which appeared shortly after drug application and reached a peak after about 2 rain. In addition, these high concentrations of acetaldehyde later induced repetitive contractions of irregular amplitudes that were not related to nerve stimulation (fig. 1B). Increasing the concentration of acetaldehyde increased the amplitude and
frequency of the repetitive contractions whereas the neurally stimulated contractions decreased slightly (fig. 1C). In the continuous presence of the high concentrations of acetaldehyde, the repetitive contractions lasted about 20 min and then declined abruptly. The neurally stimulated contractions were also severely depressed when the repetitive contractions subsided. The twitch contractions re-appeared within 10 min after the washout of the substance (fig. 1C). However, it took about 30 min for the twitch contractions to return to the pre-application level. Relations between the acetaldehyde concentration and the amplitude of the twitch contractions were assessed at two peak times, 2 min and 8-12 min, during a 30 min superfusion with acetaldehyde. Figure 2 shows that acetaldehyde produced a biphasic effect consisting of an early depression and a late facilitation of the neurally induced contractions. The m i n i m u m effective concentration for the facilitatory action was approximately 0.1 mM, and that for the inhibitory effect was between 1 and 5 m M (fig. 2). H e x a m e t h o n i u m (30/~M) eliminated the twitch contractions induced by hypogastric nerve stimulation but not those evoked by transmural stimulation (fig. 3B). However, h e x a m e t h o n i u m did not affect the acetaldehyde (5 m M ) - i n d u c e d transient depression and late augmentation of transmurally stimulated contractions or the repetitive contrac-
A
AcA I mM B
C
AcA 5 m M
AcA lOmM
1 g J
5 rain
Fig. 1. Effects of acetaldehyde (AcA) on the twitch contractions induced by sympathetic nerve stimulation in isolated vas deferens. Circles represent preganglionic nerve stimulation (20 pulses at 10 Hz, 0.2 ms, 5 V); dots indicate transmural postganglionic nerve stimulation (20 pulses at 10 Hz, 0.2 ms, 10 V). These two stimuli were applied alternately every 40 s throughout the experiment, but are indicated only in the early part of the trace. AcA was applied during the period indicated by the horizontal bar. (A) 1 mM; (B) 5 mM; (C) 10 mM. These records were obtained from the same preparation.
192
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hyde a n d suppressed its early i n h i b i t o r y effect on the twitch contractions. P h e n t o l a m i n e (27 >M) also increased the a l d e h y d e - i n d u c e d repetitive c o n t r a c t i o n s (fig. 4B). A higher c o n c e n t r a t i o n of p h e n t o l a m i n e (1.3 m M ) completely abolished the neurally i n d u c e d contractions. Acetaldehyde, when applied in the presence of this high c o n c e n t r a t i o n of p h e n t o l a m i n e , did not cause c o n t r a c t i o n (fig.
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0.5 1 5 10 AeA (mM) Fig. 2. Relations between the concentration of acetaldehyde (AcA) and the contractile response to the nerve stimulation. Amplitudes of the twitch contractions induced by transmural nerve stimulation were measured at two peak times, 2 min (dots) and 8-12 min (circles), after the start of a 30 min superfusion with acetaldehyde. Amplitudes are presented as percent changes from pre-application amplitudes (ordinate). Average amplitudes with S.E. (vertical bar) are shown. The number of experiments is in parentheses.
tions i n d u c e d by acetaldehyde. T e t r o d o t o x i n (3 /~M) blocked all neurally stimulated contractions. However, this agent did n o t prevent the acetaldeh y d e - i n d u c e d repetitive c o n t r a c t i o n s (fig. 3C). A relatively low c o n c e n t r a t i o n of p h e n t o l a m i n e (27 /~M) increased the nerve-stimulated twitch c o n t r a c t i o n s (fig. 4B). This dose of p h e n t o l a m i n e p o t e n t i a t e d the late a u g m e n t i n g effect of acetalde-
N o contractile response was i n d u c e d in surgically denervated vas deferens by t r a n s m u r a l stimulation, even though the stimulus i n t e n s i t y was 10 times greater (up to 100 V) t h a n that used in the control experiments. However, the p r e p a r a t i o n res p o n d e d with distinct c o n t r a c t i o n s to exogenously applied n o r a d r e n a l i n e at c o n c e n t r a t i o n s of 250 a n d 500 n M . Neither c o n c e n t r a t i o n of n o r a d r e n a line had a contractile effect on the control preparations. This m e a n s that d e n e r v a t i o n supersensitivity had occurred ( K a s u y a et al., 1969). A c e t a l d e h y d e in c o n c e n t r a t i o n s up to 10 m M did not induce a contractile response in this preparation (n = 4). Similar supersensitivity was observed in the vasa pretreated with reserpine (n = 4). This prep a r a t i o n showed n o contractile response to electrical s t i m u l a t i o n or to acetaldehyde (fig. 4D). However, after the p r e p a r a t i o n had been i n c u b a t e d
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Fig. 3. Effects of hexamethonium (C6) and tetrodotoxin (TTX) on the twitch contractions and the acetaldehyde-induced response in isolated vas deferens. Circles and dots indicate preganglionic and postganglionic nerve stimulation, respectively, as shown in fig. 1. (A) A control experiment with 5 mM acetaldehyde (AcA) applied during the bar; (B) 30 btM C6 was applied at the time indicated by an arrow; (C) 3 p.M TTX. W: the preparation was washed with a drug-free Krebs solution for about 30 min.
193
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Fig. 4. Effects of phentolamine and pretreatment with reserpine on the twitch contractions and the acetaldehyde-induced contractions. Records A-C were obtained from the same preparation. (A) A control experiment with 5 mM acetaldehyde (AcA) applied during the bar; (B) 27 ~tM phentolamine, applied at the time indicated by a solid triangle (the trace has been shortened); (C) 1.3 mM phentolamine. Records D-E were obtained from a preparation pretreated with reserpine. (D) A control experiment with 10 mM AcA; (E) the response to AcA (10 mM) after the preparation had been incubated with noradrenaline (5 #M for 20 min).
with noradrenaline, a small tonic contraction associated with phasic irregular contractions occurred during application of 10 mM acetaldehyde. Electrical stimulation of the hypogastric nerve or the transmural postganglionic nerve also produced small but distinct contractions (fig. 4E).
after the aldehyde-induced repetitive contractions had subsided (n = 5).
3.3. Effects on contractions induced by noradrenaline
Noradrenaline was released spontaneously into the bathing fluid. Transmural stimulation increased the noradrenaline output. The evoked release was slightly decreased during the subsequent stimulation. The ratio between the amounts of noradrenaline released by consecutive stimulations was 0.81 + 0.15 ( n = 4). However, when acetaldehyde was present during the second stimulation, the amount of noradrenaline released increased remarkably. Figure 5 summarizes the results of these experiments in which the first stimulation was taken as control and the effects of acetaldehyde were examined with the succeeding stimulations. Under the control conditions, the nervestimulated release of noradrenaline was approximately twofold greater than the spontaneous out-
Application of noradrenaline (12.5 and 25/~M) produced a monophasic contraction with a relatively slow time course (30-90 s) (not illustrated). At concentrations of 2.5/tM or lower, noradrenaline had no effect on the resting tension of normal vasa. In the presence of 1 mM acetaldehyde, this low concentration of noradrenaline produced a distinct contraction (n = 5). Acetaldehyde augmented the peak amplitude and time course of the contractions induced by higher concentrations of noradrenaline. In the presence of 10 mM acetaldehyde, the noradrenaline-induced contractions were severely depressed during the period
3.4. Effects on noradrenaline output
194
100(3
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Mr
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o
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Control
AcA
AcA
lmM 5mM Fig. 5. Effects of acetaldehyde (AcA) on noradrena]ine output into the bathing fluid. White columns show the mean values with S.E. (vertical bars) of noradrenaline released during a 4 min incubation period immediately before nerve stimulation, shaded columns during a 4 min stimulation period. Stimulation pulses (15-20 V, 2 ms, 5 Hz) were applied transmurally for 4 min. * * P < 0.01: significantly different from the corresponding control values (left two columns, n = 8). AcA 1 mM (center, n = 11), AcA 5 mM (right, n = 14).
put. Application of acetaldehyde caused a dosedependent increase in both the nerve stimulated and non-stimulated release of noradrenaline (fig. 5).
4. Discussion
Twitch contractions induced by hypogastric nerve stimulation were eliminated by hexamethonium, tetrodotoxin or a relatively high dose of phentolamine, an a-adrenoceptor blocking agent. Contractions produced by transmural stimulation were not blocked by hexamethonium but were suppressed by the two other agents. Hence, the contractions evoked by nerve stimulation and by transmural stimulation can be considered to be due to excitation of preganglionic and postganglionic sympathetic nerves, respectively (Birmingham and Wilson, 1963; Btilbring et al., 1981; Sj6strand, 1981). Acetaldehyde potentiated the twitch contractions induced by both stimuli. Potentiation of the transmurally stimulated contractions occurred even when the ganglionic transmission was blocked by hexamethonium. In addition, acetaldehyde had no effect on the action
potentials of postganglionic nerve bundles (Takeda et al., 1990). Thus, the effect of acetaldehyde was not dependent on ganglionic transmission or on spike propagation in postganglionic nerves. It has been reported that, during a train of stimuli, endogenously released noradrenaline contributes to the later part of the contraction, which is distinct from the rapid part induced by the non-adrenergic co-transmitter (Wakade and Krusz, 1972; McGrath, 1978; Meldrum and Burnstock, 1983; Brown et al., 1983). However, the twitch contractions observed in our experiments were totally suppressed by a high dose of phentolamine or by pretreatment with reserpine. Treatment of the reserpinized vas with noradrenaline partly restored the twitch contraction. Further, as shown in the accompanying article, reserpine pretreatment did not eliminate nerve stimulated excitatory junction potentials (EJPs) (Takeda et al., 1990). Therefore, it is presumed that the main part of the twitch contraction is due to noradrenaline, and that the non-adrenergic co-transmitter seems to trigger this contraction. Acetaldehyde produced a concentration-dependent increase in the release of noradrenaline during nerve stimulation, The aldehyde also dose-dependently increased the non-stimulated noradrenaline release. A relatively low concentration of phentolamine enhanced both the nerve-stimulated twitch contractions and the acetaldehyde-induced contractions. It has been postulated that blockade of presynaptically located ~-adrenoceptors causes a large increase in noradrenaline output per stimulus (Langer, 1979: Alberts et al., 1981: BiJlbring et al., 1981). Thus, the increased secretion of noradrenaline could be a major mechanism by which both phentolamine and aldehyde increased the nerve stimulated contractions. With higher concentrations of acetaldehyde, the release of endogenous noradrenaline is increased to generate the repetitive contractions (Lai and Hudgins, 1975). Likewise, the transmitter released by acetaldehyde may lead to additive effects on the contractions produced by exogenous noradrenaline. This notion is supported by the finding that surgical or chemical sympathectomy suppressed the potentiating effect of acetaldehyde. Since the acetaldehyde-induced contractions were not pre-
195
vented by tetrodotoxin, the drug-evoked release of noradrenaline is, at least partly, independent of impulse propagation in sympathetic nerve fibers (Lai and Hudgins, 1975; Chiba and Tsukada, 1988). In addition, when the aldehyde-induced contractions declined, the neurally stimulated contractions were also severely depressed. This result suggests that the transmitter may be depleted by higher concentrations of acetaldehyde. However, since the contractions produced by exogenously applied noradrenaline were also depressed during this period, it is more likely that the postjunctional receptor was 'desensitized' to noradrenaline (Harden, 1983). Another possible explanation is that the transmitter released causes a progressively increasing depolarization of the smooth muscle membrane (Takeda et al., 1990) which later leads to inactivation of action potential generation. When acetaldehyde is washed out, the latter mechanism could be affected before receptor desensitization. This could account for the observation that the neurally stimulated contractions were restored within a relatively short time during the washout of acetaldehyde. The present study demonstrated that acetaldehyde produced biphasic or dual effects on neurally induced contractions. These effects of acetaldehyde were dose- and time-dependent. Similar dual effects of acetaldehyde have been reported for various tissues such as the rat cardiac muscle (Egle et al., 1973; Sakurai et al., 1980), rat portal vein (Altura et al., 1978), frog and cat spinal motor neurons (Takeda and Haji, 1985; Haji and Takeda, 1987). These authors postulated that the aldehyde excited these muscle or nerve cells by liberating excitatory transmitters and depressed directly the postsynaptic membrane. Our electrophysiological studies with the vas deferens suggest that the initial inhibitory effect of acetaldehyde may be due to a decrease in the EJPs and hyperpolarization of the smooth muscle membrane (Takeda et al., 1990). The late facilitatory effect appears to be due to endogenous noradrenaline. The effective concentrations of acetaldehyde in the guinea-pig vas deferens were similar to those reported previously for isolated strips of other smooth muscle organs (Lai and Hudgins, 1975; Altura et al., 1978; Takeda and Momose, 1978;
Chiba and Tsukada, 1988). It is known that the blood level of acetaldehyde reaches nearly 100 #M during the consumption of moderate to large amount of ethanol by humans. However, when ethanol is combined with disulfiram, the level increases to several times that of controls (Weiner, 1979). Presumably acetaldehyde is responsible for at least part of the pharmacolcgical consequences of sympathetic neuroeffector transmission during acute ethanol intoxication or during the disulfiram-ethanol reaction.
References Akabane, J., S. Nakanishi, H. Kohei, R. Matsumura and H. Ogata, 1964, Studies on sympathomimetic action of acetaldehyde: experiments with blood pressure and nictitating membrane, Jap. J. Pharmacol. 14, 295. Alberts, P., T. Bartfai and L. Stj~irne, 1981, Site(s) and ionic basis of alpha-autoinhibition and facifitation of [3H]noradrenaline secretion in guinea-pig vas deferens, J. Physiol. (London) 312, 297. Altura, B.M., A. Carella and B.T. Altura, 1978, Acetaldehyde on vascular smooth muscle: possible role in vasodilator action of ethanol, European J. Pharmacol. 52, 73. Birmingham, A.T. and A.B. Wilson, 1963, Preganglionic and postganglionic stimulation of the guinea-pig isolated vas deferens preparation, Br. J. Pharmacol. 21,569. Brown, D.A., J.R. Docherty, A.M. French, A. MacDonald, J.C. McGrath and N.C. Scott, 1983, Separation of adrenergic and non-adrenergic contractions to field stimulation in the rat vas deferens, Br. J. Pharmacol. 79, 379. Biilbring, E., H. Ohashi and T. Tomita, 1981, Adrenergic mechanisms, in: Smooth muscle, eds. E. Biilbring, A.F. Brading, A.W. Jones and T. Tomita (Edward Arnold, London) p. 219. Burnstock, G. and M.E. Holman, 1964, An electrophysiological investigation of the actions of some autonomic blocking drugs on transmission in the guinea-pig vas deferens, Br. J. Pharmacol. 23, 600. Chiba, S. and M. Tsukada, 1988, Possible mechanism of acetaldehyde-induced noradrenaline release from sympathetic nerve terminals in isolated blood vessels, Br. J. Pharmacol. 95, 177. Egle, J.L., P.M. Hudgins and F.M. Lai, 1973, Cardiovascular effects of intravenous acetaldehyde and propionaldehyde in the anesthetized rat, Toxicol. Appl. Pharmacol. 24, 636. Furness, J.B., 1974, Transmission to the longitudinal muscle of the guinea-pig vas deferens: the effect of pretreatment of guanethidine, Br. J. Pharmacol. 50, 63. Haji, A. and R. Takeda, 1987, Effects of acetaldehyde on the monosynaptic reflex pathway in the cat spinal cord, Arch. Int. Pharmacodyn. Ther. 289, 25.
196 Harden, T.K., 1983, Agonist-induced desensitization of the beta-adrenergic receptor linked adenylate cyclase, Pharmacol. Rev. 35, 5. Ito. T., M. Matsuoka, K. Nakazima, K. Tagawa and R. Imaizumi, 1962, An isolation method of catecholamine and effect of reserpine on the enzyme systems related to the formation and inactivation of catecholamines in brain, Jap. J. Pharmacol. 12, 130. Kasuya, Y., K. Goto, H. Hashimoto, H. Watanabe, H. Munakata and M. Watanabe, 1969, Nonspecific denervation supersensitivity in the rat vas deferens 'in vitro', European J. Pharmacol. 8, 177. Lai, F.M. and P.M. Hudgins, 1975, A comparison of the indirect sympathomimetic actions of tyramine and acetaldehyde in isolated smooth muscle preparations, Toxicol. Appl. Pharmacol. 32, 368. Langer, S.Z., 1979, Presynaptic adrenoceptors and regulation of release, in: The Release of Catecholamines from Adrenergic Neurons, ed. D.M. Paton (Pergamon, Oxford) p. 59. McGrath, J.C., 1978, Adrenergic and 'non-adrenergic' components in the contractile response of the vas deferens to a single indirect stimulus, J. Physiol. (London) 283, 23. Meldrum, L.A. and G. Burnstock, 1983, Evidence that ATP acts as a co-transmitter with noradrenaline in sympathetic nerves supplying the guinea-pig vas deferens, European J. Pharmacol. 92, 161. Sakurai, T., O.H.L. Bing, G. Huber and W.H. Abelmann, 1980, Effects of acetaldehyde upon mechanical properties of isolated rat papillary muscle, J. Pharmacol. Exp. Ther. 214, 219. Sj6strand, N.O., 1981, Smooth muscles of vas deferens and other organs in the male reproductive tract, in: Smooth Muscle, eds. E. Biilbring, A.F. Brading, A.W. Jones and T. Tomita (Edward Arnold, London) p. 367. Sneddon, P. and D.P. Westfall, 1984, Pharmacological evi-
dence that adenosine triphosphate and noradrenaline are co-transmitters in the guinea-pig vas deferens, J. Physiol. (London) 347, 561. Stfiirne, L. and P. Astrand, 1985, Relative pre- and postjunctional roles of noradrenaline and adenosine 5'-triphosphate as neurotransmitters of the sympathetic nerves of guinea-pig and mouse vas deferens, Neuroscience 14, 929. Takeda, R. and A. Haft, 1985, Dual effects of acetaldehyde on electrical activity in the isolated frog spinal cord, European J. Pharmacol. 113, 409. Takeda, R. and Y. Momose, 1978, Effects of acetaldehyde on electrical and mechanical activities of guinea-pig taenia coil Arch. int. Pharmacodyn. Ther. 232, 14. Takeda, R., Y. Momose and A. Haft, 1990, Effects of acetaldehyde on electrical activity during neuroeffector transmission in guinea-pig vas deferens, European J. Pharmacol. 186, 197. Vizi, E.S. and G. Burnstock, 1988, Origin of ATP release in the rat vas deferens: concomitant measurement of [3H]noradrenaline and [14C]ATP, European J. Pharmacol. 158, 69. Wakade, A.R. and J. Krusz, 1972, Effect of reserpine, phenoxybenzamine and cocaine on neuromuscular transmission in the vas deferens of the guinea pig, J. Pharmacol. Exp. Ther. 181,310. Walsh, M.J., P.B. Hollander and E.B. Truitt, Jr. 1969, Sympathomimetic effects of acetaldehyde on the electrical and contractile characteristics of isolated left atria of guinea pig, J. Pharmacol. Exp. Ther. 167, 173. Weil-Malherbe, H. and A.D. Bone, 1952, The chemical estimation of adrenaline-like substances in blood, Biochem. J. 51, 311. Weiner, H., 1979, Aldehyde metabolism, in: Biochemistry and Pharmacology of Ethanol, Vol. 1, eds. E. Majchrowicz and E.P. Noble (Plenum, New York, London) p. 125.