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Possible role of prostaglandin E1 on adrenergic neurotransmission in the guinea-pig Taenia coli
European Journal of Pharmacology, 40 (1976) 209--214 © Elsevler]North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
209
POSSIBLE ROLE OF PROSTAGLANDIN E1 ON ADRENERGIC NEUROTRANSMISSION IN THE GUINEA-PIG TAENIA COLI MITSUO SAKATO and YASUO SHIMO *
Department of Pharmacology, Dokkyo University School of Medicine, Mibu, Tochigi 321-02, Japan Reveived 25 September 1975, revised MS received 29 December 1975, accepted 21 July 1976
M. SAKATO and Y. SHIMO, Possible role of prostaglandin E 1 on adrenergic neurotransmission in the guinea-pig taenia coli, European J. Pharmacol. 40 (1976) 209--214. The effects of prostaglandin (PG) El were investigated on the responses to adrenergic and non-adrenergic inhibitory nerve stimulation using the perivascular nerve--taenia preparation of the guinea pig. PGEI caused a rapid and sustained contraction and markedly inhibited the response to adrenergic but not to non-adrenergic inhibitory nerve stimulation. It was also observed that PGE 1 had some desensitizing action to exogenous noradrenaline o n the postjunctional site. Although indomethacin decreased the tone of the preparation, it potentiated the response to adrenergic nerve stimulation without any effects on the response to non-adrenergic inhibitory nerve stimulation. From these observations, it was concluded that endogenous PGE 1 may also play a regulatory role in adrenergic inhibitory neurotransmission, mainly by inhibitory action on noradrenaline release and partly by a similar action on the postjunctional site. Non-adrenergic inhibitory nerve Adrenergic neurotransmission
Prostaglandin E 1
1. Introduction
It has been postulated that prostaglandins (PGs) of the E type operate on adrenergic neurotransmission by a negative feedback mechanism and thereby modulate the effector response to nerve impulse (see Hedqvist, 1973a). The experimental basis for this assumption largely derives from observations made in spleen, heart and vas deferens. These organs, however, all respond to adrenergic nerve stimulation with a contraction. Therefore, in order to generalize this hypothesis, it appeared desirable to investigate the possibility that PGEs play a regulatory role on adrenergic neurotransmission in organs such as stomach and intestine which respond with relaxation to stimulation of the nerve. * Supported by grant 057289 from the Ministry of Education, Japan.
Taenia coli
Indomethacin
The inhibitory effects of PGE on the response to adrenergic nerve stimulation have been reported in the rabbit jejunum (Persson and Hedqvist, 1973; Hedqvist, 1974a), the rabbit ileum (Abdel-Aziz, 1974) and the guinea-pig (Shimo and Sakato, 1974). Recently, a potentiation of PGE on the contractile response of guinea-pig ileum by coaxial stimulation has also been explained by the inhibitory effect of PGE on the noradrenaline release from sympathetic nerve terminals (Kadlec et al., 1974). However, there seems to be no evidence clearly indicating that endogenous PGE regulates adrenergic inhibitory neurotransmission in the gut. In order to get insight into this question, the effects of following drugs on the responses to adrenergic nerve stimulation were examined in refined nerve--muscle preparations such as perivascular nerve--taenia preparation of guinea pig. Indomethacin a PG biosynthesis inhibitor (Vane, 1971; Smith and
210
Willis, 1971; Flower et al., 1972), and exogenous PGs were used. Although the response of the taenia to non-adrenergic inhibitory nerve stimulation was reported not to be affected by PGE2 and indomethacin (Kadlec et al., 1974), the effects of these drugs on the response of the taenia were also investigated in the present experiments. This was done so as to compare the effect with that on the response to adrenergic nerve stimulation and also to confirm the results of Kadlec et al. (1974). A brief report has already been presented (Shimo and Sakato, 1975).
2. Materials and methods Male guinea pigs, 250--350 g, were sacrificed by a blow on the head. The taenia, about 3 cm in length, was dissected together with mesenteric vessels from the caecum for the perivascular nerve--taenia preparation according to Burnstock et al. (1966). Each preparation was suspended in a 50 ml organ bath containing Tyrode solution bubbled with a mixture of 5% CO2 in oxygen and maintained at 37°C. The composition of the solution was as follows (mM): NaCl 137, KC1 2.7, MgC12 1.0, CaC12 1.8, NaH2PO4 0.42, NaHCO3 11.9, glucose 5.6. Tyrode solution also contained atropine (3.5 × 10 .6 M) throughout the experiments. Mechanical responses of the taenia were recorded on a polygraph with isotonic transducer with an applied load of 0.5 g. A pair of stimulating electrodes, made of two platinum wire loops 3 mm apart, were placed around the mesenteric blood vessels and another pair of electrode rings around the taenia for stimulation of the perivascular nerves and transmural stimulation of the taenia respectively. Electronic stimulators were used to deliver square-wave pulses. The pulse duration was 1 msec and the intensity was supramaximal. Perivascular nerve stimulation was given with trains of 30 pulses at a 10 Hz frequency. Transmural stimulation of the taenia was given with single pulse.
M. SAKATO, Y. SHIMO
In the presence of atropine, perivascular nerve stimulation and transmural stimulation were applied alternately on the same preparation. Both types of stimulation caused a relaxation of the taenia. Tetrodotoxin (6.3 × 10 -7 M) completely abolished the responses to both stimulations, indicating that these relaxations were due to nerve excitation. Guanethidine (5 × 10 -6 M) almost completely abolished the response to perivascular nerve stimulation while having no effect on that to transmural stimulation. According to Burnstock et al. (1966), there are two types of inhibitory fibres in the intramural nerves of the taenia; one is adrenergic and the other is nonadrenergic. It was thus thought that the relaxation caused by transmural stimulation with a single pulse was due to excitation only of non-adrenergic inhibitory nerves. Drugs used were: prostaglandin E1 and F2~ (Ono), indomethacin and tetrodotoxin (Sankyo), polyphloretine phosphate (Leo), atropine sulfate and 1-noradrenaline bitartrate (Wako), guanethidine sulfate (Ciba). Indomethacin was dissolved in ethanol. All other drugs were dissolved in physiological saline.
3. Results
3.1. The effects of PGE, and PGF2~ on transmural and perivascular nerve stimulation Addition of PGF2~ (2.8 × 10 -7 M) to the bathing medium, immediately caused a rapid, maximal contraction which gradually declined to a new steady state level. During the sustained contraction, no change in amplitude of both inhibitory responses to perivascular nerve and transmural stimulation was observed (fig. 1). PGE1 also caused a contraction of the taenia, but markedly inhibited the response to perivascular nerve stimulation during the sustained contraction, and had little or no effect on the response to transmural stimulation. Fig. 2 shows dose--effect curves of PGEI on the responses to perivascular nerve and
PROSTAGLANDIN E ~ ON ADRENERGIC NEUROTRANSMISSION
211
PF~ 5x 10-5 g/ml
,
,
PGF2~ 2.8 x10-7M Fig. 1. Effects o f prostaglandin F ~ ( P G F ~ , 2.8 X 10 -'I M) on responses t o perivascular nerve (o; 10 Hz,
30 pulses) and transmural (A; single pulse) stimulation. PGF2~ bad no effect on responses to both types of stimulation. Vertical bar indicates 0.5 ram. Time marker indicates 5 rain.
transmural stimulation. The percent changes in the responses to both types of stimulation at the different PGE~ concentration were calculated relative to the responses obtained before applying PGE~. Threshold inhibition was obtained with approximately 2.8 × 1 0 -9 M, and 70% inhibition was observed with a concentration o f 2.8 × 10 -7 M. On the other hand, there was little or no inhibition of the response to transmural stimulation even at the high concentration of 2.8 × 10 -7 M.
T5
100
{
Z~ o~
Fig. 3. Effect of prostaglandin E 1 (PGE1, 8.5 × 10 -8 M) on the response to perivascular nerve stimulation (o; 10 Hz, 30 pulses) in the presence of polyphloretine phosphate (PPP, 5 X 10 -s g/ml). PGEI reduced the response to perivascular nerve stimulation even when the contractile action of PGE1 was almost completely blocked by PPP. Vertical bar indicates 0.5 ram. Time marker indicates 5 min.
3.2. The effect o f PGE1 in the presence o f polyphloretine phosphate Addition ofpolyphloretine phosphate (PPP, 5 X 10 -s M) to the bathing medium resulted in a gradual decrease of basal tone of the taenia, but did n o t change the amplitude of the response to perivascular nerve stimulation. Approximately 20 min after the addition of PPP to the medium, the contractile effect of PGE~ (8.5 X 10 -8 M) was almost completely blocked, although a small, transient biphasic effect was seen initially. Under these conditions, the inhibitory effect of PGE~ on the response to perivascular nerve stimulation was still comparable in sign to that found in the absence of PPP (fig. 3).
3.3. The effect o f PGEI on the response to noradrenaline
~ 5c oo yv z
2:8
85
28
85
PGE~ (nM)
Fig. 2. Dose--effect curves for the effect of prostaglandin (PG) El on responses to perivascular nerve (PS; 10 Hz, 30 pulses) and transmural (TS; single pulse) stimulation. Ordinate: amplitude of relaxation as a percentage of that obtained before PGE1. Mean -+S.E.M. (n = 8). Abscissa: log concentration of PGE1.
In order to elucidate the mechanism of the inhibitory action of PGEI, the responsiveness of the taenia to exogenous noradrenaline was checked in the presence of PGE1. The concentration of noradrenaline applied was 3 X 10 -7 M, since this caused a relaxation comparable to that due to nerve stimulation. The responses to noradrenaline were reduced to 83.0 + 3.73%, 77.2 + 6.42% and 64.4 + 6.44% (mean -+S.E.M., n = 6) of the control, by PGE1 of 2.8 X 10 -s, 8.5 X 10 -s and 2.8 X
212
M. SAKATO, Y. SHIMO
ND 8.4 x 10-6M
Fig. 4. Effects of indomethacin (IND, 8.4 × 10 -6 M) on responses to perivascular nerve (o; 10 Hz, 30 pulses) and transmural (A; single pulse) stimulation. Indomethacin increased the response to perivascular nerve stimulation, but did not affect the response to transmural stimulation. Vertical bar indicates 0.5 mm. Time marker indicates 5 rain.
10 -7 M respectively. These results indicate that PGE~ has some densensitizing effect to noradrenaline at the postjunctional receptor. 3.4. The effects o f i n d o m e t h a c i n on the responses to perivascular nerve and transmural stimulations
Favourable evidence for endogenous PGs playing a role in adrenergic neuroeffector transmission was obtained using the PG biosynthesis inhibitor, indomethacin. Although indomethacin (8.4 × 10 -6 M) markedly decreased the basal tone for 30 min, the response to perivascular nerve stimulation was still twice as great as that of the control. On the contrary, the response to transmural stimulation was not affected by the presence of the drug (fig. 4). Ethanol (0.5 v/v %), the solvent for the drug, did not influence the response to either type of stimulation.
4. Discussion It was shown, in the perivascular nerve-taenia preparation from the guinea pig, that PGE~ markedly inhibited the response to perivascular nerve stimulation during sustained contraction. Three possible mechanisms
could be considered for the mode of action of PGE1 on the decreased responsiveness to perivascular nerve stimulation: a first possibility is that the inhibitory action of PGE 1 is due to a statistical summation of its contractile effect and of the inhibitory effect of noradrenaline released by the stimulation. In the present experiments, however, this possibility would be excluded by the finding that PGEI still was inhibitory in the presence of PPP which blocked the contractile action of PGE~ (fig. 3), and that PGE~ had no effect on the response to non-adrenergic nerve stimulation. Furthermore, PGF2~ also had no effect on the inhibitory response during the sustained contraction (fig. 1). A second possibility is that the postjunctional adrenergic receptor might be desensitized to noradrenaline by the PGE1, resulting in the decrease of responsiveness to adrenergic nerve stimulation. However the results showed that the responsiveness of the preparation to exogenous noradrenaline was obviously reduced in the presence of PGE1. This indicates that the inhibitory effect of PGE, on nerve stimulation might be partly due to its postjunctional action. The third possible explanation is that PGE~ acts predominantly at the prejunctional level, reducing the release of transmitter, and thus the relaxing effect of nerve stimulation. Although, in the present experiments, there is no direct evidence available for this assumption, in the isolated spleen (Hedqvist, 1970) and the guinea-pig vas deferens (Hedqvist, 1974b), the inhibitory effects of PGE on noradrenaline release from the adrenergic nerve terminals have been demonstrated using 3H-noradrenaline. Furthermore, it was reported that, when the preparation was treated with eicosatetraynoic acid, a biosynthesis inhibitor of PG, there was an increase of the noradrenaline release by nerve stimulation from the cat spleen (Hedqvist et al., 1971) and the vas deferens (Hedqvist, 1973b). From these observations on the effect of endogenous and exogenous PGE, it has been concluded that PGE acts on the nerve terminals to decrease the release of noradrenaline in these organs.
PROSTAGLANDIN E1 ON ADRENERGIC NEUROTRANSMISSION
Hedqvist et al. (1971) proposed a hypothesis for the physiological role of PGE in adrenergic nerve effector transmission. In the series of the events being postulated to control the transmission, it is not clearly established whether endogenous PGE is released from the postjunctional site by the contraction of the smooth muscle or by some action of noradrenaline itself on the cell membrane. If PGE release is associated only with the contraction of the effector organ, the results obtained from the present experiments, in which indomethacin potentiates the inhibitory response to adrenergic nerve stimulation, would n o t be explained by such a mechanism since the response of the taenia to adrenergic nerve stimulation is relaxation. From these considerations, it could be supposed that the release of PGE from the postjunctional site might be n o t due to the configuration change of the smooth muscle cell but to noradrenaline itself stimulating biosynthesis and/or a mechanism for PGE release at the postjunctional site. With regard to transmission of the nonadrenergic inhibitory nerves, it has been reported that PGE has no effect on the inhibitory response to electrical stimulation b u t is responsible for the rebound contraction following stimulation of the nerves (Kadlec et al., 1974; Burnstock et al., 1975). The present experiments confirmed the effect of PGE on the inhibitory response, but gave no information a b o u t the effect of PGE on the rebound contraction as the stimulation used was a single pulse which could n o t cause rebound contraction of the taenia (Burnstock et al., 1966). From the findings obtained so far a b o u t neurotransmission other than adrenergic, the regulatory role of PGE in inhibitory neurotransmission appears to be confined to adrenergic nerve effector transmission.
Acknowledgement We wish to thank Professor S. Katori, Department of Pharmacology, School of Medicine, Kitazato University, for a generous gift of polyphloretine phosphate.
213
References Abdel-Aziz, A., 1974, Blockade by prostaglandins E 2 and F l a of the response of the rabbit ileum to stimulation of sympathetic nerve and its reversal by some antihistamines, dexamphetamine and methylphenidate, European J. Pharmacol. 25, 226. Burnstock, G., G. Campbell and M.J. Rand, 1966, The inhibitory innervation of the taenia of the guinea-pig caecum, J. Physiol., London 182, 504. Burnstock, G., T. Cocks, B. Paddle and J. StaszewskaBarczak, 1975, Evidence that prostaglandin is responsible for the 'rebound contraction' following stimulation of non-adrenergic, non-cholinergic ('purinergic') inhibitory nerves, European J. Pharmacol. 3 1 , 3 6 0 . Flower, R., R. Gryglewski, K. Herbaczyfiska-Cedro and J.R. Vane, 1972, Effects of anti-inflammatory drugs on prostaglandin biosynthesis, Nature New Biol. 2 3 8 , 1 0 4 . Hedqvist, P., 1970, Control by prostaglandin E 2 of sympathetic neurotransmission in the spleen, Life Sci. 9 , 2 6 9 . Hedqvist, P., 1973a, Autonomic neurotransmission, in: The Prostaglandins, Vol. 1, ed. P.W. Ramwell (Plenum Press, New York and London) p. 101. Hedqvist, P., 1973b, Prostaglandin as a tool for local control of transmitter release from sympathetic nerve, Brain Res. 6 2 , 4 8 3 . Hedqvist, P., 1974a, Restriction of transmitter release from adrenergic nerves mediated by prostaglandins and a-adrenoreceptors, Pol. J. Pharmacol. Pharm. 26,119. Hedqvist, P., 1974b, Prostaglandin action on noradrenaline release and mechanical responses in the stimulated guinea pig vas deferens, Acta Physiol. Scand. 90, 86. Hedqvist, P., L. StjSxne and A. Wennmalm, 1971, Facilitation of sympathetic neurotransmission in the cat spleen after inhibition of prostaglandin synthesis, Acta Physiol. Scand. 8 3 , 4 3 0 . Kadlec, O., K. Magek and I. ~eferna, 1974, A modulating role of prostaglandins in contraction of the guinea-pig ileum, Brit. J. Pharmacol. 51,565. Persson, N.-•. and P. Hedqvist, 1973, Reduced intestinal muscular response to adrenergic nerve stimulation after the administration of prostaglandins, Acta Physiol. Scand., Suppl. 396, 108. Shimo, Y. and M. Sakato, 1974, Effects of prostaglandin E on the inhibitory responses of taenia coli produced by electrical stimulation, Jap. J. Pharmacol. 24 Suppl., 32. Shimo, Y. and M. Sakato, 1975, Effects of prostaglandins of the E types and their antagonist on the inhibitory responses of guinea-pig taenia coli, Abstract, in: Sixth International Congress of Pharmacology, p. 157.
214 Smith, J.B. and A.L. Willis, 1971, Aspirine selectively inhibits prostaglandin production in human platelets, Nature New Biol. 231,235.
M. SAKATO, Y. SHIMO Vane, J.R., 1971, Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs, Nature New Biol. 231,232.
209
POSSIBLE ROLE OF PROSTAGLANDIN E1 ON ADRENERGIC NEUROTRANSMISSION IN THE GUINEA-PIG TAENIA COLI MITSUO SAKATO and YASUO SHIMO *
Department of Pharmacology, Dokkyo University School of Medicine, Mibu, Tochigi 321-02, Japan Reveived 25 September 1975, revised MS received 29 December 1975, accepted 21 July 1976
M. SAKATO and Y. SHIMO, Possible role of prostaglandin E 1 on adrenergic neurotransmission in the guinea-pig taenia coli, European J. Pharmacol. 40 (1976) 209--214. The effects of prostaglandin (PG) El were investigated on the responses to adrenergic and non-adrenergic inhibitory nerve stimulation using the perivascular nerve--taenia preparation of the guinea pig. PGEI caused a rapid and sustained contraction and markedly inhibited the response to adrenergic but not to non-adrenergic inhibitory nerve stimulation. It was also observed that PGE 1 had some desensitizing action to exogenous noradrenaline o n the postjunctional site. Although indomethacin decreased the tone of the preparation, it potentiated the response to adrenergic nerve stimulation without any effects on the response to non-adrenergic inhibitory nerve stimulation. From these observations, it was concluded that endogenous PGE 1 may also play a regulatory role in adrenergic inhibitory neurotransmission, mainly by inhibitory action on noradrenaline release and partly by a similar action on the postjunctional site. Non-adrenergic inhibitory nerve Adrenergic neurotransmission
Prostaglandin E 1
1. Introduction
It has been postulated that prostaglandins (PGs) of the E type operate on adrenergic neurotransmission by a negative feedback mechanism and thereby modulate the effector response to nerve impulse (see Hedqvist, 1973a). The experimental basis for this assumption largely derives from observations made in spleen, heart and vas deferens. These organs, however, all respond to adrenergic nerve stimulation with a contraction. Therefore, in order to generalize this hypothesis, it appeared desirable to investigate the possibility that PGEs play a regulatory role on adrenergic neurotransmission in organs such as stomach and intestine which respond with relaxation to stimulation of the nerve. * Supported by grant 057289 from the Ministry of Education, Japan.
Taenia coli
Indomethacin
The inhibitory effects of PGE on the response to adrenergic nerve stimulation have been reported in the rabbit jejunum (Persson and Hedqvist, 1973; Hedqvist, 1974a), the rabbit ileum (Abdel-Aziz, 1974) and the guinea-pig (Shimo and Sakato, 1974). Recently, a potentiation of PGE on the contractile response of guinea-pig ileum by coaxial stimulation has also been explained by the inhibitory effect of PGE on the noradrenaline release from sympathetic nerve terminals (Kadlec et al., 1974). However, there seems to be no evidence clearly indicating that endogenous PGE regulates adrenergic inhibitory neurotransmission in the gut. In order to get insight into this question, the effects of following drugs on the responses to adrenergic nerve stimulation were examined in refined nerve--muscle preparations such as perivascular nerve--taenia preparation of guinea pig. Indomethacin a PG biosynthesis inhibitor (Vane, 1971; Smith and
210
Willis, 1971; Flower et al., 1972), and exogenous PGs were used. Although the response of the taenia to non-adrenergic inhibitory nerve stimulation was reported not to be affected by PGE2 and indomethacin (Kadlec et al., 1974), the effects of these drugs on the response of the taenia were also investigated in the present experiments. This was done so as to compare the effect with that on the response to adrenergic nerve stimulation and also to confirm the results of Kadlec et al. (1974). A brief report has already been presented (Shimo and Sakato, 1975).
2. Materials and methods Male guinea pigs, 250--350 g, were sacrificed by a blow on the head. The taenia, about 3 cm in length, was dissected together with mesenteric vessels from the caecum for the perivascular nerve--taenia preparation according to Burnstock et al. (1966). Each preparation was suspended in a 50 ml organ bath containing Tyrode solution bubbled with a mixture of 5% CO2 in oxygen and maintained at 37°C. The composition of the solution was as follows (mM): NaCl 137, KC1 2.7, MgC12 1.0, CaC12 1.8, NaH2PO4 0.42, NaHCO3 11.9, glucose 5.6. Tyrode solution also contained atropine (3.5 × 10 .6 M) throughout the experiments. Mechanical responses of the taenia were recorded on a polygraph with isotonic transducer with an applied load of 0.5 g. A pair of stimulating electrodes, made of two platinum wire loops 3 mm apart, were placed around the mesenteric blood vessels and another pair of electrode rings around the taenia for stimulation of the perivascular nerves and transmural stimulation of the taenia respectively. Electronic stimulators were used to deliver square-wave pulses. The pulse duration was 1 msec and the intensity was supramaximal. Perivascular nerve stimulation was given with trains of 30 pulses at a 10 Hz frequency. Transmural stimulation of the taenia was given with single pulse.
M. SAKATO, Y. SHIMO
In the presence of atropine, perivascular nerve stimulation and transmural stimulation were applied alternately on the same preparation. Both types of stimulation caused a relaxation of the taenia. Tetrodotoxin (6.3 × 10 -7 M) completely abolished the responses to both stimulations, indicating that these relaxations were due to nerve excitation. Guanethidine (5 × 10 -6 M) almost completely abolished the response to perivascular nerve stimulation while having no effect on that to transmural stimulation. According to Burnstock et al. (1966), there are two types of inhibitory fibres in the intramural nerves of the taenia; one is adrenergic and the other is nonadrenergic. It was thus thought that the relaxation caused by transmural stimulation with a single pulse was due to excitation only of non-adrenergic inhibitory nerves. Drugs used were: prostaglandin E1 and F2~ (Ono), indomethacin and tetrodotoxin (Sankyo), polyphloretine phosphate (Leo), atropine sulfate and 1-noradrenaline bitartrate (Wako), guanethidine sulfate (Ciba). Indomethacin was dissolved in ethanol. All other drugs were dissolved in physiological saline.
3. Results
3.1. The effects of PGE, and PGF2~ on transmural and perivascular nerve stimulation Addition of PGF2~ (2.8 × 10 -7 M) to the bathing medium, immediately caused a rapid, maximal contraction which gradually declined to a new steady state level. During the sustained contraction, no change in amplitude of both inhibitory responses to perivascular nerve and transmural stimulation was observed (fig. 1). PGE1 also caused a contraction of the taenia, but markedly inhibited the response to perivascular nerve stimulation during the sustained contraction, and had little or no effect on the response to transmural stimulation. Fig. 2 shows dose--effect curves of PGEI on the responses to perivascular nerve and
PROSTAGLANDIN E ~ ON ADRENERGIC NEUROTRANSMISSION
211
PF~ 5x 10-5 g/ml
,
,
PGF2~ 2.8 x10-7M Fig. 1. Effects o f prostaglandin F ~ ( P G F ~ , 2.8 X 10 -'I M) on responses t o perivascular nerve (o; 10 Hz,
30 pulses) and transmural (A; single pulse) stimulation. PGF2~ bad no effect on responses to both types of stimulation. Vertical bar indicates 0.5 ram. Time marker indicates 5 rain.
transmural stimulation. The percent changes in the responses to both types of stimulation at the different PGE~ concentration were calculated relative to the responses obtained before applying PGE~. Threshold inhibition was obtained with approximately 2.8 × 1 0 -9 M, and 70% inhibition was observed with a concentration o f 2.8 × 10 -7 M. On the other hand, there was little or no inhibition of the response to transmural stimulation even at the high concentration of 2.8 × 10 -7 M.
T5
100
{
Z~ o~
Fig. 3. Effect of prostaglandin E 1 (PGE1, 8.5 × 10 -8 M) on the response to perivascular nerve stimulation (o; 10 Hz, 30 pulses) in the presence of polyphloretine phosphate (PPP, 5 X 10 -s g/ml). PGEI reduced the response to perivascular nerve stimulation even when the contractile action of PGE1 was almost completely blocked by PPP. Vertical bar indicates 0.5 ram. Time marker indicates 5 min.
3.2. The effect o f PGE1 in the presence o f polyphloretine phosphate Addition ofpolyphloretine phosphate (PPP, 5 X 10 -s M) to the bathing medium resulted in a gradual decrease of basal tone of the taenia, but did n o t change the amplitude of the response to perivascular nerve stimulation. Approximately 20 min after the addition of PPP to the medium, the contractile effect of PGE~ (8.5 X 10 -8 M) was almost completely blocked, although a small, transient biphasic effect was seen initially. Under these conditions, the inhibitory effect of PGE~ on the response to perivascular nerve stimulation was still comparable in sign to that found in the absence of PPP (fig. 3).
3.3. The effect o f PGEI on the response to noradrenaline
~ 5c oo yv z
2:8
85
28
85
PGE~ (nM)
Fig. 2. Dose--effect curves for the effect of prostaglandin (PG) El on responses to perivascular nerve (PS; 10 Hz, 30 pulses) and transmural (TS; single pulse) stimulation. Ordinate: amplitude of relaxation as a percentage of that obtained before PGE1. Mean -+S.E.M. (n = 8). Abscissa: log concentration of PGE1.
In order to elucidate the mechanism of the inhibitory action of PGEI, the responsiveness of the taenia to exogenous noradrenaline was checked in the presence of PGE1. The concentration of noradrenaline applied was 3 X 10 -7 M, since this caused a relaxation comparable to that due to nerve stimulation. The responses to noradrenaline were reduced to 83.0 + 3.73%, 77.2 + 6.42% and 64.4 + 6.44% (mean -+S.E.M., n = 6) of the control, by PGE1 of 2.8 X 10 -s, 8.5 X 10 -s and 2.8 X
212
M. SAKATO, Y. SHIMO
ND 8.4 x 10-6M
Fig. 4. Effects of indomethacin (IND, 8.4 × 10 -6 M) on responses to perivascular nerve (o; 10 Hz, 30 pulses) and transmural (A; single pulse) stimulation. Indomethacin increased the response to perivascular nerve stimulation, but did not affect the response to transmural stimulation. Vertical bar indicates 0.5 mm. Time marker indicates 5 rain.
10 -7 M respectively. These results indicate that PGE~ has some densensitizing effect to noradrenaline at the postjunctional receptor. 3.4. The effects o f i n d o m e t h a c i n on the responses to perivascular nerve and transmural stimulations
Favourable evidence for endogenous PGs playing a role in adrenergic neuroeffector transmission was obtained using the PG biosynthesis inhibitor, indomethacin. Although indomethacin (8.4 × 10 -6 M) markedly decreased the basal tone for 30 min, the response to perivascular nerve stimulation was still twice as great as that of the control. On the contrary, the response to transmural stimulation was not affected by the presence of the drug (fig. 4). Ethanol (0.5 v/v %), the solvent for the drug, did not influence the response to either type of stimulation.
4. Discussion It was shown, in the perivascular nerve-taenia preparation from the guinea pig, that PGE~ markedly inhibited the response to perivascular nerve stimulation during sustained contraction. Three possible mechanisms
could be considered for the mode of action of PGE1 on the decreased responsiveness to perivascular nerve stimulation: a first possibility is that the inhibitory action of PGE 1 is due to a statistical summation of its contractile effect and of the inhibitory effect of noradrenaline released by the stimulation. In the present experiments, however, this possibility would be excluded by the finding that PGEI still was inhibitory in the presence of PPP which blocked the contractile action of PGE~ (fig. 3), and that PGE~ had no effect on the response to non-adrenergic nerve stimulation. Furthermore, PGF2~ also had no effect on the inhibitory response during the sustained contraction (fig. 1). A second possibility is that the postjunctional adrenergic receptor might be desensitized to noradrenaline by the PGE1, resulting in the decrease of responsiveness to adrenergic nerve stimulation. However the results showed that the responsiveness of the preparation to exogenous noradrenaline was obviously reduced in the presence of PGE1. This indicates that the inhibitory effect of PGE, on nerve stimulation might be partly due to its postjunctional action. The third possible explanation is that PGE~ acts predominantly at the prejunctional level, reducing the release of transmitter, and thus the relaxing effect of nerve stimulation. Although, in the present experiments, there is no direct evidence available for this assumption, in the isolated spleen (Hedqvist, 1970) and the guinea-pig vas deferens (Hedqvist, 1974b), the inhibitory effects of PGE on noradrenaline release from the adrenergic nerve terminals have been demonstrated using 3H-noradrenaline. Furthermore, it was reported that, when the preparation was treated with eicosatetraynoic acid, a biosynthesis inhibitor of PG, there was an increase of the noradrenaline release by nerve stimulation from the cat spleen (Hedqvist et al., 1971) and the vas deferens (Hedqvist, 1973b). From these observations on the effect of endogenous and exogenous PGE, it has been concluded that PGE acts on the nerve terminals to decrease the release of noradrenaline in these organs.
PROSTAGLANDIN E1 ON ADRENERGIC NEUROTRANSMISSION
Hedqvist et al. (1971) proposed a hypothesis for the physiological role of PGE in adrenergic nerve effector transmission. In the series of the events being postulated to control the transmission, it is not clearly established whether endogenous PGE is released from the postjunctional site by the contraction of the smooth muscle or by some action of noradrenaline itself on the cell membrane. If PGE release is associated only with the contraction of the effector organ, the results obtained from the present experiments, in which indomethacin potentiates the inhibitory response to adrenergic nerve stimulation, would n o t be explained by such a mechanism since the response of the taenia to adrenergic nerve stimulation is relaxation. From these considerations, it could be supposed that the release of PGE from the postjunctional site might be n o t due to the configuration change of the smooth muscle cell but to noradrenaline itself stimulating biosynthesis and/or a mechanism for PGE release at the postjunctional site. With regard to transmission of the nonadrenergic inhibitory nerves, it has been reported that PGE has no effect on the inhibitory response to electrical stimulation b u t is responsible for the rebound contraction following stimulation of the nerves (Kadlec et al., 1974; Burnstock et al., 1975). The present experiments confirmed the effect of PGE on the inhibitory response, but gave no information a b o u t the effect of PGE on the rebound contraction as the stimulation used was a single pulse which could n o t cause rebound contraction of the taenia (Burnstock et al., 1966). From the findings obtained so far a b o u t neurotransmission other than adrenergic, the regulatory role of PGE in inhibitory neurotransmission appears to be confined to adrenergic nerve effector transmission.
Acknowledgement We wish to thank Professor S. Katori, Department of Pharmacology, School of Medicine, Kitazato University, for a generous gift of polyphloretine phosphate.
213
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