- Email: [email protected]
The role of prostaglandins in cholinergic neurotransmission in the guinea pig
European Journal of Pharmacology, 34 (1975) 39--47 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
39
T H E R O L E O F P R O S T A G L A N D I N S IN C H O L I N E R G I C N E U R O T R A N S M I S S I O N IN T H E G U I N E A P I G WILLIAM J. HALL, PADRAIC O'NEILL and JOHN D. SHEEHAN Department of Physiology, University College, Cork, Ireland Received 25 March 1975, revised MS received 11 June 1975, accepted 24 June 1975
W.J. HALL, P. O'NEILL and J.D. SHEEHAN, The role of prostaglandins in cholinergic neurotransmission in the guinea pig, European J. Pharmacol. 34 (1975) 39--47. Prostaglandins have contrasting effects on neurotransmission at different cholinergic nerve endings. This is a report on the role of prostaglandins in a number of cholinergic preparations from the guinea pig. In the isolated ileum PGE1 (2 × 10-l° to 5 × 10-s M) potentiated the response to electrical stimulation of the cholinergic nerves. PGE1 (10 -~ M) caused an increase in tone followed by a period of transient inhibition of twitch height. Responses to stimulation of the ileum with drugs were not potentiated by PGE 1. Responses of atropinized or plexus-free muscle to electrical stimulation were also not potentiated by PGEI. Acetylsalicylic acid (2.5 × 10 -a M) diminished the twitch response and the output of acetylcholine from the ileum. Both effects were reversed by PGEI. Qualitatively similar observations were made on the trachea. It is concluded that prostaglandins facilitate acetylcholine release in the ilel~m and trachea. PGE1 diminished the effect of vagal stimulation on the heart rate. The response to stimulation of the phrenic nerve was not affected. Cholinergic neurotransmission
Acetylsalicylic acid
1. I n t r o d u c t i o n Prostaglandins regulate the response to s y m p a t h e t i c nerve s t i m u l a t i o n in a variety o f tissues (Hedqvist, 1 9 7 3 ; H o r t o n , 1 9 7 3 ; B r o d y and K a d o w i t z , 1 9 7 4 ) by altering t r a n s m i t t e r release at the s y m p a t h e t i c nerve terminals. This ability o f p r o s t a g l a n d i n s t o alter transm i t t e r release is n o t restricted t o adrenergic nerves. In the rabbit, p r o s t a g l a n d i n E1 inhibits the b r a d y c a r d i a caused b y vagal stimulation w i t h o u t altering t h a t p r o d u c e d b y an infusion of a c e t y l c h o l i n e ( W e n n m a l m and Hedqvist, 1971). The release o f prostaglandins f r o m the heart, following vagal stimulation, suggests a negative f e e d b a c k c o n t r o l b y t h e m on a c e t y l c h o l i n e release ( W e n n m a l m and Junstad, 1973). The few o t h e r studies on the e f f e c t o f prostaglandins on cholinergic n e u r o t r a n s m i s sion include the r e p o r t b y H a r r y ( 1 9 6 8 ) t h a t
Acetylcholine release
Prostaglandin Et
p r o s t a g l a n d i n E1 increases the t w i t c h response of the guinea-pig ileum to t r a n s m u r a l electrical stimuli. B o t t i n g and S a l z m a n n ( 1 9 7 4 ) investigated the role o f e n d o g e n o u s prostaglandins in the same tissue. T h e y r e p o r t t h a t i n d o m e t h a c i n caused only a small transient r e d u c t i o n in the t w i t c h response t o field stimuli and did n o t cause a significant reduction in the o u t p u t o f acetylcholine. Ehrenpreis et al. ( 1 9 7 3 ) f o u n d t h a t high doses o f i n d o m e t h a c i n did inhibit the c o n t r a c t i o n s o f the electrically s t i m u l a t e d guinea-pig ileum and c o n c l u d e t h a t e n d o g e n o u s prostaglandins facilitate cholinergic n e u r o t r a n s m i s s i o n in the ileum. In the p r e s e n t s t u d y evidence was s o u g h t for this h y p o t h e s i s , using acetylsalicylic acid as a n i n h i b i t o r o f p r o s t a g l a n d i n biosynthesis (Vane, 1 9 7 1 ; Ferreira et al., 1 9 7 1 ; S m i t h and Willis, 1 9 7 1 ; F l o w e r et al., 1972), rather than i n d o m e t h a c i n w h i c h has been s h o w n to have
40 other actions (Northover, 1971, 1972; Flores and Sharp, 1972). In addition, an attempt was made to evaluate the contribution of muscle and neural elements to the potentiation of the twitch response by added prostaglandin E l . Harry (1968) suggested both factors were involved but his investigations do not provide a definitive answer. Since the report of the inhibitory action of prostaglandin E~ on transmitter release from vagal fibres came from a study on rabbit heart (Wennmalm and Hedqvist, 1971) it was also decided to investigate the role of prostaglandins at other cholinergic sites in the guinea pig.
2. Materials and methods Guinea pigs of either sex, weighing 400-600 g, were used.
2.1. Gut segment preparation Guinea pigs were stunned by a blow on the head and bled out. A length of ileum was excised at least 12 cm above the ileo-caecal valve. This was m o u n t e d for electrical stimulation as described by Paton (1955) in a 10 ml organ bath containing Tyrode solution, gassed with 100% O2 and maintained at 37°C. The Tyrode solution had the following composition (mM): NaC1 137, KC1 2.7, CaC12 2, MgC12 0.5, NaH2PO4 0.4, NaHCO3 11.9 and glucose 5.6. Rectangular-wave stimuli were applied transmurally through platinum electrodes from a Grass $4 stimulator at a frequency of 0.1 Hz and a pulse duration of 0.2 msec. The twitch responses were recorded with an isometric transducer linked to an amplifier and pen-recorder. Activation of the muscle was also achieved by drugs or by adequate electrical stimulation of the atropinized preparation.
2. 2. Plexus-free gut strip preparation Plexus-free strips of longitudinal muscle from the ileum were prepared according to
W.J. HALL ET AL. the methods of Rang (1964) and Paton and Zar (1968). In addition to visual examination the absence of a nerve plexus was confirmed by pharmacological means at the end of each experiment. Field stimuli (4--12 V, 50 Hz for 10 sec) at 1 min intervals were used to produce submaximal twitch contractions which were recorded on the isometric system above.
2.3. Measurement of acetylcholine output from gut segment To determine acetylcholine release the methods of Paton and Zar (1968) and Paton and Vizi (1969) were followed in outline. Physostigmine sulphate (6 X 10 -6 M) was added to the Tyrode solution and the ileum was left for 2 hr to reach equilibrium, during which time it was washed continuously by the physostigmine-containing solution. The period allowed for the accumulation of acetylcholine was usually 10 min. The bath fluid was then withdrawn and assayed on guinea-pig ileum in Tyrode solution containing morphine sulphate (2.6 X 10 -s M) and physostigmine sulphate (1.5 X 10 -8 M). To increase specificity, methysergide maleate (2 X 10 -7 M) and mepyramine maleate (3 X 10 -s M) were also included in the bathing fluid. Each segment of gut served as its own control and the a m o u n t of activity released in each test period was expressed as a percentage of the control by a four-point assay. The results were analysed by the one-sample t-test.
2. 4. Heart preparation Guinea pigs were anaesthetised by i.p. administration of sodium pentobarbitone (35 mg/kg). The right carotid artery was cannulated and a fine polythene cannula was pushed towards the aortic valve. In two animals the tip of the cannula was found by post mortem examination at the level of the aortic valve and in two others it was found in the left ventricle. Prostaglandin E~, 5 pg/kg, was infused over a 30 sec period. Control infusions with saline were also made. The periph-
PROSTAGLANDINS AND CHOLINI~RGICTRANSMISSION eral end of the cut right vagus was stimulated electrically (0.1 msec, 20 Hz, 20--40 V) and the heart rate was measured from the simultaneously recorded electrocardiogram. 2. 5. Tracheal preparation The trachea was m o u n t e d according to the method of Jamieson (1962) in Krebs solution gassed with 5% CO 2 in O 2. The bath temperature was 37 °C. The composition of the Krebs solution was as follows (mM): NaC1 118, KC1 4.7, CaC12 2, NaHCO3 25, KH2PO4 1.2, MgSO4 1.2 and glucose 5.6. Propranolol hydrochloride (1.7 × 10 -6 M) was added to the reservoir. The fluid level in the graduated capillary was adjusted to a suitable level above the fluid in the organ bath. The upper end of the fluid column in the capillary consisted of Brodie manometric solution. This facilitated both the reading of the meniscus and the movements of the fluid. Changes of 0.5 pl in tracheal volume could be observed. Rectangular-wave stimuli (0.2 msec, 20 Hz, 40 V for 15 sec) from a Grass $4 stimulator were applied transmurally through platinum electrodes to excite the tissue. 2. 6. Phrenic nerve--diaphragm preparation The preparation was m o u n t e d in Krebs solution, maintained at 37°C and aerated with 5% CO2 in O2. Rectangular-wave stimuli (0.1 msec, 0.1 Hz and 20--40 V) were applied to the nerve and the twitch responses were recorded using an isometric transducer. The preparations were also stimulated at 50 Hz to produce tetanus. 2. 7. Drugs used Drugs were added to the fluid reservoir when lengthy application was required and to the bath fluid for briefer periods of application. The molar concentrations of drugs refer to the active component. The replacement of the bath fluid at least three times was standard procedure to wash out drugs. The fol-
41
lowing drugs were used: acetylcholine chloride, acetylsalicylic acid, atropine sulphate, physostigmine sulphate (Sigma); histamine phosphate (Koch--Light); morphine sulphate (Cahill & Co.); methysergide maleate (Sandoz); mepyramine maleate, prostaglandin E1 (May & Baker); propranolol hydrochloride (I.C.I.); procaine hydrochloride (Antigen). 3. Results 3.1. Effect of PGE1 on the response o f the ileum to nerve stimulation Transmural electrical stimulation of the ileum was found to give more satisfactory results than field stimulation. To ensure that nerves and not muscle were being stimulated a pulse width of 0.2 msec was employed (Harry, 1962). The responses could be blocked by atropine (1.2 > 10 -6 M) and thus it was concluded that cholinergic nerves were being stimulated. In doses ranging from 2 )< 10 -l 0 to 5 X 10 -s M, prostaglandin E1 potentiated the twitch response to transmural electrical stimuli in a dose dependent manner (fig. 1A). While potentiation was demonstrable in all six preparations used, the threshold dose and the degree of potentiation were variable. The maximum increase in twitch height ranged from 30 to 110% with a mean value of 71.1 + 9.3%. At high dose levels, 10 -7 M prostaglandin E i caused an increase in tone followed by a period of transient inhibition of twitch-height (fig. 1B). This inhibition was more evident if the calcium in the bathing fluid was reduced from 2 to 1 mM. A similar increase in tone, due to added histamine, was w i t h o u t effect on twitch height. 3.2. Effect o f PGE~ on the response of the ileum to drugs Responses to stimulation of the ileum by drugs were n o t potentiated by prostaglandin E l . In four gut segment preparations, responses to acetylcholine and histamine which
42
W.J. HALL ET AL.
t w o atropinized preparations, the responses to activation by electrical stimuli (12 V, 50 Hz, 10 sec) were n o t p o t e n t i a t e d by prostaglandin El. I n six p l e x u s - f r e e s t r i p s o f i l e a l l o n g i t u d i n a l muscle the twitch responses to electric field stimuli were not significantly affected by p r o s t a g l a n d i n E l (2 × 1 0 -9 t o 1 0 -7 M). T h e responses of the plexus-free strips to acetylc h o l i n e w e r e also u n a f f e c t e d b y a p p l i e d p r o s t a g l a n d i n E l o v e r a s i m i l a r d o s e r a n g e (fig. 2).
20
3g
.
50
.
.
.
.
.
.
.
A
l O0
~'~ii"~i~ii'~iii')~il~~!i!!~i~ii~~,~i!)! Fig. 1. Effects of PGE1 on the transmurally stimulated (0.2 msec, 0.1 Hz) gut segment. In panel A the twitch responses are potentiated. In panel B an increase m tone is followed by transient inhibition of twitch height. Time: 1 min. Doses of PGEI: x 10 -9 M.
B
20 l O0
Ig m a t c h e d in height the twitch responses to transmural electrical stimuli, were not affected b y p r o s t a g l a n d i n E1 (2 × 10 -9 t o 1 0 -7
M). 3.3. Effect o f PGEI on the response o f atropinized or plexus-free ileal muscle to electrical stimulation I n t w o g u t s e g m e n t s t h e r e s p o n s e s t o rectangular-wave electrical pulses of 0.2 msec duration were first abolished by atropine (1.2 X 10 -6 M). S u b s e q u e n t l y , in six t r i a l s o n t h e s e
x l O'9M ACh Fig. 2. Effect of PGE l on a plexus-free longitudinal muscle strip of guinea-pig ileum stimulated electrically (4 V, 50 Hz for 10 sec) at I min intervals (panel A) and by acetylcholine (panel B). The response to acetylcholine was unaffected and the change in twitch height was < 10%.
PROSTAGLANDINS AND CHOLINERGIC TRANSMISSION Unlike the gut segments, the strips were n o t c o n t r a c t e d b y p r o s t a g l a n d i n El 10 -7 M. In each o f the strips, a t r o p i n e (1.2 X 10 -6 M) or p r o c a i n e (7 × 10 -s M) did n o t alter the t w i t c h responses t o electric field stimuli. This was regarded as c o n f i r m i n g t o t a l r e m o v a l o f the cholinergic nerves.
3.4. Effect o f acetylsalicylic acid on the response of the ileum to nerve stimulation The e f f e c t o f the p r o s t a g l a n d i n s y n t h e t a s e inhibitor, acetylsalicylic acid (2.5 X 10 -4 M), was tested o n the t w i t c h response t o transmural electrical stimuli in six e x p e r i m e n t s . It caused an 84.9 + 6.5% r e d u c t i o n in the responses (p < 0.001). This e f f e c t o f acetylsalicylic acid on the t w i t c h response c o u l d be reversed by p r o s t a g l a n d i n E1 application (fig. 3). Prostaglandin Ej (10 -s M) restored the responses to 74.8 _+ 11.4% o f n o r m a l (p < 0.01). The response o f the ileum t o a c e t y l c h o line was u n a f f e c t e d by acetylsalicylic acid.
43
3.5. Effects o f acetylsalicylic acid and PGE~ acetylcholine o u t p u t from the ileum
on
The e f f e c t o f acetylsalicylic acid (2.5 X 10 .4 M) on the o u t p u t o f a c e t y l c h o l i n e f r o m the t r a n s m u r a l l y s t i m u l a t e d ileum (0.2 msec, 0.1 Hz, 40 V) was m e a s u r e d in six experiments. The o u t p u t o f a c e t y l c h o l i n e was significantly r e d u c e d in the presence o f acetylsalicylic acid t o 6 4 . 9 + 6.0% o f n o r m a l {p < 0.01). Prostaglandin El (10 -s M) restored acet y l c h o l i n e o u t p u t t o 99.0 -+ 5.1% o f n o r m a l (fig. 4). This dose of prostaglandin E1 also caused a significant reversal o f the e f f e c t o f acetylsalicylic acid on the t w i t c h response (fig. 3).
3. 6. Effect o f PGE~ on vagal bradycardia The over a rate in fusions
infusion o f p r o s t a g l a n d i n E l , 5 pg/kg, 30 sec p e r i o d h a d no e f f e c t on h e a r t f o u r preparations. H o w e v e r similar ino f p r o s t a g l a n d i n E] significantly re-
5-
100 - 100 P <0,01 'g
+
80
o
S" o
8O
6O
60 N
o
n= 5 P <0.01
II
4O
40 ::=
n=6 P
2o
/1 A
I
A A+E 1
Fig. 3. Effect of acetylsalicylic acid (A) on the twitch response of the gut segment to transmural electrical stimuli (0.2 msec, 0.1 Hz). The twitch response is expressed as a percentage of the control response, each gut serving as its own control. Acetylsalicylic acid (2.5 × 10 -4 M) caused a highly significant decrease in the response (p < 0.001). Prostaglandin El (10 -8 M) caused a significant reversal of the inhibition.
1
A+E 1
Fig. 4. Effect of acetylsalicylic acid (A) on acetylcholine output from the transmurally stimulated (0.2 msec, 0.1 Hz) gut segment. The output of acetylcholine is expressed as a percentage of the control output, each gut serving as its own control. Acetylsalicylic acid (2.5 × 10 -4 M) caused a significant decrease in acetylcholine output (p < 0.01). Prostaglandin E1 (10 -8 M) restored the output to 99.0 ± 5.1% of normal, p = probability level (t-test). Bars represent ± S.E.M.
44
W.J. H A L L ET AL. 90
n=5
-
I| tl
fl
70 -
<
50 -
'ic
30
n =6 P O,Ol
i-
2C
II
-
2
u
2O
x I0"9M PGE1 10
#= -2
-
~
n=2
-4C S
Fig. 5. E f f e c t o f PGE1 5 ug/kg, infused over a 30 see period on the negative e h r o n o t r o p i c e f f e c t o f stimulating the peripheral end of t h e cut right vagus nerve in the a n a e s t h e t i z e d guinea pig. The vagus was stimulated 30 see after the infusion o f saline or PGE1. S = c o n t r o l infusion of saline. Ej = PGE1 infusion. V.S. = vagal stimulation, p = p r o b a b i l i t y level (Ftest). Bars r e p r e s e n t + S.E.M.
d u c e d the negative c h r o n o t r o p i c effects o f vagal s t i m u l a t i o n in six trials f r o m an 82.9 +6.2% decrease in h e a r t rate in c o n t r o l conditions t o 40.6 :t 6.7% after the infusion o f prostaglandin E, (fig. 5). T h e tests were carried o u t 30 sec after the infusion o f saline or prostaglandin E,.
3. 7. Effects o f PGE, and acetylsalicylic acid on the response of the trachea to nerve stimulation P r o p r a n o l o l h y d r o c h l o r i d e was included in the Krebs solution so t h a t t r a n s m u r a l stimuli caused only c o n s t r i c t o r responses. T h e s e c o u l d be b l o c k e d b y a t r o p i n e (3.46 X 10 `7 M) and thus it was c o n c l u d e d t h a t the constriction was due to s t i m u l a t i o n o f cholinergic nerves. In five e x p e r i m e n t s prostaglandin E l p o t e n t i a t e d the responses in a dose d e p e n d e n t m a n n e r (fig. 6). Prostaglandin E, 2 X 10 -9 M, caused a 39.6 + 1.6% increase and prostaglandin E1 2 X 10 -s M, caused a 56.6 + 4.9% increase. In t w o e x p e r i m e n t s acetylsalicylic acid (2.5
h 2.5 x 1 0 - 4 M
E1
Fig. 6. E f f e c t s o f PGEI and acetylsalicylic acid (A) on the c o n s t r i c t o r responses o f the isolated guinea-pig trachea to t r a n s m u r a l electrical stimuli (0.2 msec, 20 Hz, 40 V for 15 sec). The p r e p a r a t i o n s were b a t h e d in Krebs s o l u t i o n with p r o p r a n o l o l h y d r o c h l o r i d e (1.7 x 10 -6 M). Bars r e p r e s e n t ~ S.E.M.
X 10 -4 M) caused a 38% decrease in the c o n s t r i c t o r responses. The e f f e c t was reversed by application o f prostaglandin E, (2 X 10 -s M).
3.8. Effects of PGE, and acetylsalicylic acid on the phrenic nerve--diaphragm preparation In six experiments prostaglandin E, (2 X I0 -9 to 10 -7 M) had no effect on the twitch height or tetanic response. Acetylsalicylic acid (2.5 X 10 -4 M) also had no effect on twitch height or tetanic tension.
4. Discussion One o f the main conclusions t h a t can be drawn f r o m our s t u d y on the ileum is t h a t prostaglandins facilitate the t w i t c h response to t r a n s m u r a l electrical stimuli t h r o u g h an action on the nerves in the gut wall. In o u r experiments, unlike those o f Harry ( 1 9 6 8 ) and Kadlec et al. ( 1 9 7 4 ) the responses to activation o f the muscle by a c e t y l c h o l i n e were n o t
PROSTAGLANDINS AND CHOLINERGIC TRANSMISSION potentiated. The difference between our findings and those of others can be explained as follows. We used doses of acetylcholine which matched in height the responses to transmural electrical stimuli. Harry (1968) and Kadlec et al. (1974) used large doses of acetylcholine. Acetylcholine is known to cause the release of prostaglandins from rabbit heart (Wennmalm and Junstad, 1973). Prostaglandins occur in the guinea-pig ileum (Ambache et al., 1966; Botting and Salzmann, 1974). It is possible that large doses of acetylcholine release sufficient prostaglandins from the ileum to cause, together with added prostaglandin, a contractile effect which would add to the acetylcholine response. The findings of Kadlec et al. (1974) that indomethacin blocked the 'potentiating' effect of added prostaglandin on acetylcholine responses would appear to support this suggestion. The observations on plexus-free strips confirm our findings with gut segments. As well as eliminating the cholinergic nerve plexus, this preparation also removes the problem of mechanical interaction between the muscle layers. The circular muscle is inhibited by prostaglandin E1 (Bennett et al., 1968; Fleshler and Bennett, 1969) and this could facilitate shortening of the longitudinal muscle. The absence of contraction in the plexus-free strips to prostaglandin E~ 10 -7 M, supports the suggestion that contraction partly depends on the release of acetylcholine from the nerves in the wall of the gut (Harry, 1968). Facilitated release of acetylcholine could explain the potentiation of the twitch response to nerve stimulation in fig. 1. A decrease in acetylcholine release in the absence of endogenous prostaglandins could explain the findings with acetylsalicylic acid. Measurement of acetylcholine o u t p u t from the transmurally stimulated ileum showed a significant decrease in the presence of acetylsalicylic acid. The dose of prostaglandin E~ which substantially reversed the effect of acetylsalicylic acid on the twitch response, also restored acetylcholine output. These findings seem to disagree with those of Botting and Salzmann
45
(1974) who found that indomethacin caused only a small transient reduction of the twitch response to electrical stimuli, with no significant alteration in the output of acetylcholine. The effects of indomethacin will depend on the dose used (Hall and O'Regan, 1974). In addition to blocking endogenous prostaglandin synthesis, indomethacin can increase cyclic AMP levels (Flores and Sharp, 1972). Increased cyclic AMP could facilitate neurotransmission in the gut (Collier and Roy, 1974). Unlike Botting and Salzmann (1974), Ehrenpreis et al. (1973) and Kadlec et al. (1974) found that indomethacin reduces the twitch response of the ileum to electrical stimulation. They employed different concentrations of indomethacin. At high dose levels, prostaglandin E1 caused an increase in tone followed by a period of transient inhibition of twitch height. Since a similar increase in tone caused by histamine was without effect on twitch height, we conclude that the inhibition of twitch height is n o t a direct consequence of the increase in tone. Two mechanisms can be suggested to explain it. It may be similar to that at the vagal nerve endings in rabbit heart (Wennmalm and Hedqvist, 1971) where prostaglandin El inhibits the release of acetylcholine. Alternatively it may be due to depletion of acetylcholine at the nerve endings since prostaglandin E~ contracts the ileum partly through acetylcholine release from the nerves in the wall (Harry, 1968). The similarity between the findings in the trachea and ileum suggest that prostaglandins play a similar role in cholinergic neurotransmission in the trachea. Prostaglandin E~ significantly reduced the negative chronotropic effect of vagal stimulation in the guinea-pig heart. This is in accord with the findings of Wennmalm and Hedqvist (1971) in rabbit heart. It is tempting to compare the effect of prostaglandin E~ on the heart with that of high doses in the ileum but in our experiments on the guinea-pig heart it is impossible to establish the concentration of prostaglandin E~ at its site of action. Approx-
46 i m a t e c a l c u l a t i o n s s u g g e s t t h a t it falls w i t h i n t h e r a n g e o f d o s e s w h i c h c a u s e p o t e n t i a t i o n in the ileum. Other differences between the cholinergic nerves of the gut and heart have been reported. For example, morphine blocks chol i n e r g i c t r a n s m i s s i o n in t h e i l e u m ( P a t o n , 1957) but has no effect on cholinergic transm i s s i o n in t h e h e a r t ( K o s t e r l i t z a n d T a y l o r , 1 9 5 9 ) . R e c e n t l y K a d l e c e t al. ( 1 9 7 4 ) h a v e suggested that prostaglandins may influence a c e t y l c h o ! i n e r e l e a s e in g u t t h r o u g h a n a c t i o n on the sympathetic nerves. We c o n c l u d e f r o m o u r s t u d y o f t h e p h r e n i c nerve--diaphragm preparation that prostagland i n Et p l a y s n o s i g n i f i c a n t r o l e in c h o l i n e r g i c n e u r o t r a n s m i s s i o n in t h i s t i s s u e . T h i s is in accord with the observations on the frog sciatic n e r v e - - s a r t o r i u s m u s c l e ( G i n s b o r g a n d Hirst, 1971). The range of prostaglandin levels e m p l o y e d b y us i n c l u d e d t h e a m o u n t s w h i c h can be released from the rat phrenic nerve-d i a p h r a g m p r e p a r a t i o n ( R a m w e l l e t al., 1 9 6 5 ; Laity, 1969). Horton and Main {1967) found t h a t p r o s t a g l a n d i n E1 d i m i n i s h e s t h e t w i t c h response of cat gastrocnemius muscle to stimu l a t i o n o f its m o t o r n e r v e . H o w e v e r , in t h i s s t u d y t h e d o s e s o f p r o s t a g l a n d i n E1 i n f u s e d into the artery supplying the muscle were v e r y large.
Acknowledgements The authors wish to acknowledge a gift of prostaglandin E1 (May and Baker Ltd.). P. O'N. was the holder of a student grant from the Medical Research Council of Ireland.
References Ambache, N., H.C. Brummer, J.G. Rose and J. Whiting, 1966, Thinlayer chromatography of spasmogenic unsaturated h y d r o x y acids from various tissues, J. Physiol. (London) 185, 77. Bennett, A., K.G. Eley and G.B. Scholes, 1968, Effects of prostaglandin El and E2 on human, guinea-pig and rat isolated small intestine, Brit. J. Pharmacol. 34, 630. Botting, J.H. and R. Salzmann, 1974, The effect of
w.J. HALL ET AL. indomethacin on the release of prostaglandin E2 and acetylcholine from guinea-pig isolated ileum at rest and during field stimulation, Brit. J. Pharmacol. 50, 119. Brody, M.J. and P.J. Kadowitz, 1974, Prostaglandins as modulators of the autonomic nervous system, Federation Proc. 33, 48. Collier, W.O.J. and A.C. Roy, 1974, Morphine-like drugs inhibit the stimulation by E prostaglandins of cyclic AMP formation by rat brain homogenate, Nature (London) 248, 24. Ehrenpreis, S., J. Greenberg and S. Belman, 1973, Prostaglandins reverse inhibition of electrically induced contractions of guinea-pig ileum by morphine, indomethacin and acetylsalicylic acid, Nature (New Biol.) 245, 280. Ferreira, S.H., S. Moncada and J.R. Vane, 1971, Indomethacin and aspirin abolish prostaglandin release from the spleen, Nature New Biol. 231, 237. Fleshier, B. and A. Bennett, 1969, Responses of human, guinea-pig and rat colonic circular muscle to prostaglandins, J. Lab. Clin. Med. 74, 872. Flores, A.G.A. and G.W.G. Sharp, 1972, Endogenous prostaglandins and osmotic water flow in the toad bladder, Amer. J. Physiol. 223, 1392. Flower, R.J., R. Greglewski, K. Herbaczynska-Cedro and J.R. Vane, 1972, Effects of anti-inflammatory drugs on prostaglandin biosynthesis, Nature New Biol. 238, 104. Ginsborg, B.L. and G.D.S. Hirst, 1971, Prostaglandin E1 and noradrenaline at the neuromuscular junction, Brit. J. Pharmacol. 42, 153. Hall, W.J. and M.G. O'Regan, 1974, The effects of indomethacin and theophylline on the response of frog skin to prostaglandin El, J. Physiol. (London) 236, 48. Harry, J., 1962, Effect of cooling, local anaesthetic compounds and botulinum toxin on the responses of and the acetylcholine output from the electrically transmurally stimulated isolated guinea-pig ileum, Brit. J. Pharmacol. 19, 42. Harry, J., 1968, The action of prostaglandin El on the guinea-pig isolated intestine, Brit. d. Pharmacol. 33, 213. Hedqvist, P., 1973, Prostaglandin mediated control of sympathetic neuroeffector transmission, Advan. Biosci. 9, 461. Horton, E.W., 1973, Prostaglandins at adrenergic nerve-endings, Brit. Med. Bull. 29, 148. Horton, E.W. and I.H.M. Main, 1967, Further observations on the central nervous actions of prostaglandins F2a and E l , Brit. J. Pharmacol. 30, 568. Jamieson, D., 1962, A method for the quantitative estimation of drugs on the isolated intact trachea, Brit. J. Pharmacol. 19, 286. Kadlec, O., K. Masek and I. Seferna, 1974, A modulating role of prostaglandins in contractions of the guinea-pig ileum, Brit. J. Pharmacol. 51, 565.
PROSTAGLANDINS AND CHOLINERGIC TRANSMISSION Kosterlitz, H.W. and D.W. Taylor, 1959, The effect of morphine on vagal inhibition of the heart, Brit. J. Pharmacol. 14, 209. Laity, J.L.H., 1969, The release of prostaglandin E l from the rat phrenic nerve--diaphragm preparation, Brit. J. Pharmacol. 37,698. Northover, B.J., 1971, Mechanism of the inhibitory action of indomethacin on smooth muscle, Brit, J. Pharmacol. 41, 540. Northover, B.J., 1972, The effects of indomethacin on calcium, potassium and magnesium fluxes in various tissues of the guinea pig, Brit. J. Pharmacol. 45, 651. Paton, W.D.M., 1955, The response of the guinea-pig ileum to electrical stimulation by coaxial electrodes, J. Physiol. (London) 127, 40. Paton, W.D.M., 1957, The action of morphine and related substances on contraction and on acetylcholine output of coaxially stimulated guinea-pig ileum, Brit. J. Pharmacol. 11, 119. Paton, W.D.M. and M.A. Zar, 1968, The origin of acetylcholine released from guinea-pig intestine and longitudinal muscle strips, J. Physiol. (London) 194, 13.
47
Paton, W.D.M. and E.S. Vizi, 1969, The inhibitory action of noradrenaline and adrenaline on acetylcholine output by guinea-pig ileum longitudinal muscle strip, Brit. J. Pharmacol. 35, 10. Ramwell, P.W., J.E. Shaw and J. Kucharski, 1965, Prostaglandin release from the rat phrenic nerve-diaphragm preparation, Science (N.Y.) 149, 1390. Rang, H.P., 1964, Stimulant actions of volatile anaesthetics on smooth muscle, Brit. J. Pharmacol. 22, 356. Smith, J.B. and A.L. Willis, 1971, Aspirin selectively inhibits prostaglandin production in human platelets, Nature New Biol. 231, 235. Vane, J.R., 1971, Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs, Nature New Biol. 231, 232. Wennmalm, A. and P. Hedqvist, 1971, Inhibition by prostaglandin E1 of parasympathetic neurotransmission in the rabbit heart, Life Sci. 10, 465. Wennmalm, A. and M. Junstad, 1973, Endogenous prostaglandin mediated inhibition of parasympathetic neurotransmission in the rabbit heart?, Acta Physiol. Scand. Suppl. 396, 22.
39
T H E R O L E O F P R O S T A G L A N D I N S IN C H O L I N E R G I C N E U R O T R A N S M I S S I O N IN T H E G U I N E A P I G WILLIAM J. HALL, PADRAIC O'NEILL and JOHN D. SHEEHAN Department of Physiology, University College, Cork, Ireland Received 25 March 1975, revised MS received 11 June 1975, accepted 24 June 1975
W.J. HALL, P. O'NEILL and J.D. SHEEHAN, The role of prostaglandins in cholinergic neurotransmission in the guinea pig, European J. Pharmacol. 34 (1975) 39--47. Prostaglandins have contrasting effects on neurotransmission at different cholinergic nerve endings. This is a report on the role of prostaglandins in a number of cholinergic preparations from the guinea pig. In the isolated ileum PGE1 (2 × 10-l° to 5 × 10-s M) potentiated the response to electrical stimulation of the cholinergic nerves. PGE1 (10 -~ M) caused an increase in tone followed by a period of transient inhibition of twitch height. Responses to stimulation of the ileum with drugs were not potentiated by PGE 1. Responses of atropinized or plexus-free muscle to electrical stimulation were also not potentiated by PGEI. Acetylsalicylic acid (2.5 × 10 -a M) diminished the twitch response and the output of acetylcholine from the ileum. Both effects were reversed by PGEI. Qualitatively similar observations were made on the trachea. It is concluded that prostaglandins facilitate acetylcholine release in the ilel~m and trachea. PGE1 diminished the effect of vagal stimulation on the heart rate. The response to stimulation of the phrenic nerve was not affected. Cholinergic neurotransmission
Acetylsalicylic acid
1. I n t r o d u c t i o n Prostaglandins regulate the response to s y m p a t h e t i c nerve s t i m u l a t i o n in a variety o f tissues (Hedqvist, 1 9 7 3 ; H o r t o n , 1 9 7 3 ; B r o d y and K a d o w i t z , 1 9 7 4 ) by altering t r a n s m i t t e r release at the s y m p a t h e t i c nerve terminals. This ability o f p r o s t a g l a n d i n s t o alter transm i t t e r release is n o t restricted t o adrenergic nerves. In the rabbit, p r o s t a g l a n d i n E1 inhibits the b r a d y c a r d i a caused b y vagal stimulation w i t h o u t altering t h a t p r o d u c e d b y an infusion of a c e t y l c h o l i n e ( W e n n m a l m and Hedqvist, 1971). The release o f prostaglandins f r o m the heart, following vagal stimulation, suggests a negative f e e d b a c k c o n t r o l b y t h e m on a c e t y l c h o l i n e release ( W e n n m a l m and Junstad, 1973). The few o t h e r studies on the e f f e c t o f prostaglandins on cholinergic n e u r o t r a n s m i s sion include the r e p o r t b y H a r r y ( 1 9 6 8 ) t h a t
Acetylcholine release
Prostaglandin Et
p r o s t a g l a n d i n E1 increases the t w i t c h response of the guinea-pig ileum to t r a n s m u r a l electrical stimuli. B o t t i n g and S a l z m a n n ( 1 9 7 4 ) investigated the role o f e n d o g e n o u s prostaglandins in the same tissue. T h e y r e p o r t t h a t i n d o m e t h a c i n caused only a small transient r e d u c t i o n in the t w i t c h response t o field stimuli and did n o t cause a significant reduction in the o u t p u t o f acetylcholine. Ehrenpreis et al. ( 1 9 7 3 ) f o u n d t h a t high doses o f i n d o m e t h a c i n did inhibit the c o n t r a c t i o n s o f the electrically s t i m u l a t e d guinea-pig ileum and c o n c l u d e t h a t e n d o g e n o u s prostaglandins facilitate cholinergic n e u r o t r a n s m i s s i o n in the ileum. In the p r e s e n t s t u d y evidence was s o u g h t for this h y p o t h e s i s , using acetylsalicylic acid as a n i n h i b i t o r o f p r o s t a g l a n d i n biosynthesis (Vane, 1 9 7 1 ; Ferreira et al., 1 9 7 1 ; S m i t h and Willis, 1 9 7 1 ; F l o w e r et al., 1972), rather than i n d o m e t h a c i n w h i c h has been s h o w n to have
40 other actions (Northover, 1971, 1972; Flores and Sharp, 1972). In addition, an attempt was made to evaluate the contribution of muscle and neural elements to the potentiation of the twitch response by added prostaglandin E l . Harry (1968) suggested both factors were involved but his investigations do not provide a definitive answer. Since the report of the inhibitory action of prostaglandin E~ on transmitter release from vagal fibres came from a study on rabbit heart (Wennmalm and Hedqvist, 1971) it was also decided to investigate the role of prostaglandins at other cholinergic sites in the guinea pig.
2. Materials and methods Guinea pigs of either sex, weighing 400-600 g, were used.
2.1. Gut segment preparation Guinea pigs were stunned by a blow on the head and bled out. A length of ileum was excised at least 12 cm above the ileo-caecal valve. This was m o u n t e d for electrical stimulation as described by Paton (1955) in a 10 ml organ bath containing Tyrode solution, gassed with 100% O2 and maintained at 37°C. The Tyrode solution had the following composition (mM): NaC1 137, KC1 2.7, CaC12 2, MgC12 0.5, NaH2PO4 0.4, NaHCO3 11.9 and glucose 5.6. Rectangular-wave stimuli were applied transmurally through platinum electrodes from a Grass $4 stimulator at a frequency of 0.1 Hz and a pulse duration of 0.2 msec. The twitch responses were recorded with an isometric transducer linked to an amplifier and pen-recorder. Activation of the muscle was also achieved by drugs or by adequate electrical stimulation of the atropinized preparation.
2. 2. Plexus-free gut strip preparation Plexus-free strips of longitudinal muscle from the ileum were prepared according to
W.J. HALL ET AL. the methods of Rang (1964) and Paton and Zar (1968). In addition to visual examination the absence of a nerve plexus was confirmed by pharmacological means at the end of each experiment. Field stimuli (4--12 V, 50 Hz for 10 sec) at 1 min intervals were used to produce submaximal twitch contractions which were recorded on the isometric system above.
2.3. Measurement of acetylcholine output from gut segment To determine acetylcholine release the methods of Paton and Zar (1968) and Paton and Vizi (1969) were followed in outline. Physostigmine sulphate (6 X 10 -6 M) was added to the Tyrode solution and the ileum was left for 2 hr to reach equilibrium, during which time it was washed continuously by the physostigmine-containing solution. The period allowed for the accumulation of acetylcholine was usually 10 min. The bath fluid was then withdrawn and assayed on guinea-pig ileum in Tyrode solution containing morphine sulphate (2.6 X 10 -s M) and physostigmine sulphate (1.5 X 10 -8 M). To increase specificity, methysergide maleate (2 X 10 -7 M) and mepyramine maleate (3 X 10 -s M) were also included in the bathing fluid. Each segment of gut served as its own control and the a m o u n t of activity released in each test period was expressed as a percentage of the control by a four-point assay. The results were analysed by the one-sample t-test.
2. 4. Heart preparation Guinea pigs were anaesthetised by i.p. administration of sodium pentobarbitone (35 mg/kg). The right carotid artery was cannulated and a fine polythene cannula was pushed towards the aortic valve. In two animals the tip of the cannula was found by post mortem examination at the level of the aortic valve and in two others it was found in the left ventricle. Prostaglandin E~, 5 pg/kg, was infused over a 30 sec period. Control infusions with saline were also made. The periph-
PROSTAGLANDINS AND CHOLINI~RGICTRANSMISSION eral end of the cut right vagus was stimulated electrically (0.1 msec, 20 Hz, 20--40 V) and the heart rate was measured from the simultaneously recorded electrocardiogram. 2. 5. Tracheal preparation The trachea was m o u n t e d according to the method of Jamieson (1962) in Krebs solution gassed with 5% CO 2 in O 2. The bath temperature was 37 °C. The composition of the Krebs solution was as follows (mM): NaC1 118, KC1 4.7, CaC12 2, NaHCO3 25, KH2PO4 1.2, MgSO4 1.2 and glucose 5.6. Propranolol hydrochloride (1.7 × 10 -6 M) was added to the reservoir. The fluid level in the graduated capillary was adjusted to a suitable level above the fluid in the organ bath. The upper end of the fluid column in the capillary consisted of Brodie manometric solution. This facilitated both the reading of the meniscus and the movements of the fluid. Changes of 0.5 pl in tracheal volume could be observed. Rectangular-wave stimuli (0.2 msec, 20 Hz, 40 V for 15 sec) from a Grass $4 stimulator were applied transmurally through platinum electrodes to excite the tissue. 2. 6. Phrenic nerve--diaphragm preparation The preparation was m o u n t e d in Krebs solution, maintained at 37°C and aerated with 5% CO2 in O2. Rectangular-wave stimuli (0.1 msec, 0.1 Hz and 20--40 V) were applied to the nerve and the twitch responses were recorded using an isometric transducer. The preparations were also stimulated at 50 Hz to produce tetanus. 2. 7. Drugs used Drugs were added to the fluid reservoir when lengthy application was required and to the bath fluid for briefer periods of application. The molar concentrations of drugs refer to the active component. The replacement of the bath fluid at least three times was standard procedure to wash out drugs. The fol-
41
lowing drugs were used: acetylcholine chloride, acetylsalicylic acid, atropine sulphate, physostigmine sulphate (Sigma); histamine phosphate (Koch--Light); morphine sulphate (Cahill & Co.); methysergide maleate (Sandoz); mepyramine maleate, prostaglandin E1 (May & Baker); propranolol hydrochloride (I.C.I.); procaine hydrochloride (Antigen). 3. Results 3.1. Effect of PGE1 on the response o f the ileum to nerve stimulation Transmural electrical stimulation of the ileum was found to give more satisfactory results than field stimulation. To ensure that nerves and not muscle were being stimulated a pulse width of 0.2 msec was employed (Harry, 1962). The responses could be blocked by atropine (1.2 > 10 -6 M) and thus it was concluded that cholinergic nerves were being stimulated. In doses ranging from 2 )< 10 -l 0 to 5 X 10 -s M, prostaglandin E1 potentiated the twitch response to transmural electrical stimuli in a dose dependent manner (fig. 1A). While potentiation was demonstrable in all six preparations used, the threshold dose and the degree of potentiation were variable. The maximum increase in twitch height ranged from 30 to 110% with a mean value of 71.1 + 9.3%. At high dose levels, 10 -7 M prostaglandin E i caused an increase in tone followed by a period of transient inhibition of twitch-height (fig. 1B). This inhibition was more evident if the calcium in the bathing fluid was reduced from 2 to 1 mM. A similar increase in tone, due to added histamine, was w i t h o u t effect on twitch height. 3.2. Effect o f PGE~ on the response of the ileum to drugs Responses to stimulation of the ileum by drugs were n o t potentiated by prostaglandin E l . In four gut segment preparations, responses to acetylcholine and histamine which
42
W.J. HALL ET AL.
t w o atropinized preparations, the responses to activation by electrical stimuli (12 V, 50 Hz, 10 sec) were n o t p o t e n t i a t e d by prostaglandin El. I n six p l e x u s - f r e e s t r i p s o f i l e a l l o n g i t u d i n a l muscle the twitch responses to electric field stimuli were not significantly affected by p r o s t a g l a n d i n E l (2 × 1 0 -9 t o 1 0 -7 M). T h e responses of the plexus-free strips to acetylc h o l i n e w e r e also u n a f f e c t e d b y a p p l i e d p r o s t a g l a n d i n E l o v e r a s i m i l a r d o s e r a n g e (fig. 2).
20
3g
.
50
.
.
.
.
.
.
.
A
l O0
~'~ii"~i~ii'~iii')~il~~!i!!~i~ii~~,~i!)! Fig. 1. Effects of PGE1 on the transmurally stimulated (0.2 msec, 0.1 Hz) gut segment. In panel A the twitch responses are potentiated. In panel B an increase m tone is followed by transient inhibition of twitch height. Time: 1 min. Doses of PGEI: x 10 -9 M.
B
20 l O0
Ig m a t c h e d in height the twitch responses to transmural electrical stimuli, were not affected b y p r o s t a g l a n d i n E1 (2 × 10 -9 t o 1 0 -7
M). 3.3. Effect o f PGEI on the response o f atropinized or plexus-free ileal muscle to electrical stimulation I n t w o g u t s e g m e n t s t h e r e s p o n s e s t o rectangular-wave electrical pulses of 0.2 msec duration were first abolished by atropine (1.2 X 10 -6 M). S u b s e q u e n t l y , in six t r i a l s o n t h e s e
x l O'9M ACh Fig. 2. Effect of PGE l on a plexus-free longitudinal muscle strip of guinea-pig ileum stimulated electrically (4 V, 50 Hz for 10 sec) at I min intervals (panel A) and by acetylcholine (panel B). The response to acetylcholine was unaffected and the change in twitch height was < 10%.
PROSTAGLANDINS AND CHOLINERGIC TRANSMISSION Unlike the gut segments, the strips were n o t c o n t r a c t e d b y p r o s t a g l a n d i n El 10 -7 M. In each o f the strips, a t r o p i n e (1.2 X 10 -6 M) or p r o c a i n e (7 × 10 -s M) did n o t alter the t w i t c h responses t o electric field stimuli. This was regarded as c o n f i r m i n g t o t a l r e m o v a l o f the cholinergic nerves.
3.4. Effect o f acetylsalicylic acid on the response of the ileum to nerve stimulation The e f f e c t o f the p r o s t a g l a n d i n s y n t h e t a s e inhibitor, acetylsalicylic acid (2.5 X 10 -4 M), was tested o n the t w i t c h response t o transmural electrical stimuli in six e x p e r i m e n t s . It caused an 84.9 + 6.5% r e d u c t i o n in the responses (p < 0.001). This e f f e c t o f acetylsalicylic acid on the t w i t c h response c o u l d be reversed by p r o s t a g l a n d i n E1 application (fig. 3). Prostaglandin Ej (10 -s M) restored the responses to 74.8 _+ 11.4% o f n o r m a l (p < 0.01). The response o f the ileum t o a c e t y l c h o line was u n a f f e c t e d by acetylsalicylic acid.
43
3.5. Effects o f acetylsalicylic acid and PGE~ acetylcholine o u t p u t from the ileum
on
The e f f e c t o f acetylsalicylic acid (2.5 X 10 .4 M) on the o u t p u t o f a c e t y l c h o l i n e f r o m the t r a n s m u r a l l y s t i m u l a t e d ileum (0.2 msec, 0.1 Hz, 40 V) was m e a s u r e d in six experiments. The o u t p u t o f a c e t y l c h o l i n e was significantly r e d u c e d in the presence o f acetylsalicylic acid t o 6 4 . 9 + 6.0% o f n o r m a l {p < 0.01). Prostaglandin El (10 -s M) restored acet y l c h o l i n e o u t p u t t o 99.0 -+ 5.1% o f n o r m a l (fig. 4). This dose of prostaglandin E1 also caused a significant reversal o f the e f f e c t o f acetylsalicylic acid on the t w i t c h response (fig. 3).
3. 6. Effect o f PGE~ on vagal bradycardia The over a rate in fusions
infusion o f p r o s t a g l a n d i n E l , 5 pg/kg, 30 sec p e r i o d h a d no e f f e c t on h e a r t f o u r preparations. H o w e v e r similar ino f p r o s t a g l a n d i n E] significantly re-
5-
100 - 100 P <0,01 'g
+
80
o
S" o
8O
6O
60 N
o
n= 5 P <0.01
II
4O
40 ::=
n=6 P
2o
/1 A
I
A A+E 1
Fig. 3. Effect of acetylsalicylic acid (A) on the twitch response of the gut segment to transmural electrical stimuli (0.2 msec, 0.1 Hz). The twitch response is expressed as a percentage of the control response, each gut serving as its own control. Acetylsalicylic acid (2.5 × 10 -4 M) caused a highly significant decrease in the response (p < 0.001). Prostaglandin El (10 -8 M) caused a significant reversal of the inhibition.
1
A+E 1
Fig. 4. Effect of acetylsalicylic acid (A) on acetylcholine output from the transmurally stimulated (0.2 msec, 0.1 Hz) gut segment. The output of acetylcholine is expressed as a percentage of the control output, each gut serving as its own control. Acetylsalicylic acid (2.5 × 10 -4 M) caused a significant decrease in acetylcholine output (p < 0.01). Prostaglandin E1 (10 -8 M) restored the output to 99.0 ± 5.1% of normal, p = probability level (t-test). Bars represent ± S.E.M.
44
W.J. H A L L ET AL. 90
n=5
-
I| tl
fl
70 -
<
50 -
'ic
30
n =6 P O,Ol
i-
2C
II
-
2
u
2O
x I0"9M PGE1 10
#= -2
-
~
n=2
-4C S
Fig. 5. E f f e c t o f PGE1 5 ug/kg, infused over a 30 see period on the negative e h r o n o t r o p i c e f f e c t o f stimulating the peripheral end of t h e cut right vagus nerve in the a n a e s t h e t i z e d guinea pig. The vagus was stimulated 30 see after the infusion o f saline or PGE1. S = c o n t r o l infusion of saline. Ej = PGE1 infusion. V.S. = vagal stimulation, p = p r o b a b i l i t y level (Ftest). Bars r e p r e s e n t + S.E.M.
d u c e d the negative c h r o n o t r o p i c effects o f vagal s t i m u l a t i o n in six trials f r o m an 82.9 +6.2% decrease in h e a r t rate in c o n t r o l conditions t o 40.6 :t 6.7% after the infusion o f prostaglandin E, (fig. 5). T h e tests were carried o u t 30 sec after the infusion o f saline or prostaglandin E,.
3. 7. Effects o f PGE, and acetylsalicylic acid on the response of the trachea to nerve stimulation P r o p r a n o l o l h y d r o c h l o r i d e was included in the Krebs solution so t h a t t r a n s m u r a l stimuli caused only c o n s t r i c t o r responses. T h e s e c o u l d be b l o c k e d b y a t r o p i n e (3.46 X 10 `7 M) and thus it was c o n c l u d e d t h a t the constriction was due to s t i m u l a t i o n o f cholinergic nerves. In five e x p e r i m e n t s prostaglandin E l p o t e n t i a t e d the responses in a dose d e p e n d e n t m a n n e r (fig. 6). Prostaglandin E, 2 X 10 -9 M, caused a 39.6 + 1.6% increase and prostaglandin E1 2 X 10 -s M, caused a 56.6 + 4.9% increase. In t w o e x p e r i m e n t s acetylsalicylic acid (2.5
h 2.5 x 1 0 - 4 M
E1
Fig. 6. E f f e c t s o f PGEI and acetylsalicylic acid (A) on the c o n s t r i c t o r responses o f the isolated guinea-pig trachea to t r a n s m u r a l electrical stimuli (0.2 msec, 20 Hz, 40 V for 15 sec). The p r e p a r a t i o n s were b a t h e d in Krebs s o l u t i o n with p r o p r a n o l o l h y d r o c h l o r i d e (1.7 x 10 -6 M). Bars r e p r e s e n t ~ S.E.M.
X 10 -4 M) caused a 38% decrease in the c o n s t r i c t o r responses. The e f f e c t was reversed by application o f prostaglandin E, (2 X 10 -s M).
3.8. Effects of PGE, and acetylsalicylic acid on the phrenic nerve--diaphragm preparation In six experiments prostaglandin E, (2 X I0 -9 to 10 -7 M) had no effect on the twitch height or tetanic response. Acetylsalicylic acid (2.5 X 10 -4 M) also had no effect on twitch height or tetanic tension.
4. Discussion One o f the main conclusions t h a t can be drawn f r o m our s t u d y on the ileum is t h a t prostaglandins facilitate the t w i t c h response to t r a n s m u r a l electrical stimuli t h r o u g h an action on the nerves in the gut wall. In o u r experiments, unlike those o f Harry ( 1 9 6 8 ) and Kadlec et al. ( 1 9 7 4 ) the responses to activation o f the muscle by a c e t y l c h o l i n e were n o t
PROSTAGLANDINS AND CHOLINERGIC TRANSMISSION potentiated. The difference between our findings and those of others can be explained as follows. We used doses of acetylcholine which matched in height the responses to transmural electrical stimuli. Harry (1968) and Kadlec et al. (1974) used large doses of acetylcholine. Acetylcholine is known to cause the release of prostaglandins from rabbit heart (Wennmalm and Junstad, 1973). Prostaglandins occur in the guinea-pig ileum (Ambache et al., 1966; Botting and Salzmann, 1974). It is possible that large doses of acetylcholine release sufficient prostaglandins from the ileum to cause, together with added prostaglandin, a contractile effect which would add to the acetylcholine response. The findings of Kadlec et al. (1974) that indomethacin blocked the 'potentiating' effect of added prostaglandin on acetylcholine responses would appear to support this suggestion. The observations on plexus-free strips confirm our findings with gut segments. As well as eliminating the cholinergic nerve plexus, this preparation also removes the problem of mechanical interaction between the muscle layers. The circular muscle is inhibited by prostaglandin E1 (Bennett et al., 1968; Fleshler and Bennett, 1969) and this could facilitate shortening of the longitudinal muscle. The absence of contraction in the plexus-free strips to prostaglandin E~ 10 -7 M, supports the suggestion that contraction partly depends on the release of acetylcholine from the nerves in the wall of the gut (Harry, 1968). Facilitated release of acetylcholine could explain the potentiation of the twitch response to nerve stimulation in fig. 1. A decrease in acetylcholine release in the absence of endogenous prostaglandins could explain the findings with acetylsalicylic acid. Measurement of acetylcholine o u t p u t from the transmurally stimulated ileum showed a significant decrease in the presence of acetylsalicylic acid. The dose of prostaglandin E~ which substantially reversed the effect of acetylsalicylic acid on the twitch response, also restored acetylcholine output. These findings seem to disagree with those of Botting and Salzmann
45
(1974) who found that indomethacin caused only a small transient reduction of the twitch response to electrical stimuli, with no significant alteration in the output of acetylcholine. The effects of indomethacin will depend on the dose used (Hall and O'Regan, 1974). In addition to blocking endogenous prostaglandin synthesis, indomethacin can increase cyclic AMP levels (Flores and Sharp, 1972). Increased cyclic AMP could facilitate neurotransmission in the gut (Collier and Roy, 1974). Unlike Botting and Salzmann (1974), Ehrenpreis et al. (1973) and Kadlec et al. (1974) found that indomethacin reduces the twitch response of the ileum to electrical stimulation. They employed different concentrations of indomethacin. At high dose levels, prostaglandin E1 caused an increase in tone followed by a period of transient inhibition of twitch height. Since a similar increase in tone caused by histamine was without effect on twitch height, we conclude that the inhibition of twitch height is n o t a direct consequence of the increase in tone. Two mechanisms can be suggested to explain it. It may be similar to that at the vagal nerve endings in rabbit heart (Wennmalm and Hedqvist, 1971) where prostaglandin El inhibits the release of acetylcholine. Alternatively it may be due to depletion of acetylcholine at the nerve endings since prostaglandin E~ contracts the ileum partly through acetylcholine release from the nerves in the wall (Harry, 1968). The similarity between the findings in the trachea and ileum suggest that prostaglandins play a similar role in cholinergic neurotransmission in the trachea. Prostaglandin E~ significantly reduced the negative chronotropic effect of vagal stimulation in the guinea-pig heart. This is in accord with the findings of Wennmalm and Hedqvist (1971) in rabbit heart. It is tempting to compare the effect of prostaglandin E~ on the heart with that of high doses in the ileum but in our experiments on the guinea-pig heart it is impossible to establish the concentration of prostaglandin E~ at its site of action. Approx-
46 i m a t e c a l c u l a t i o n s s u g g e s t t h a t it falls w i t h i n t h e r a n g e o f d o s e s w h i c h c a u s e p o t e n t i a t i o n in the ileum. Other differences between the cholinergic nerves of the gut and heart have been reported. For example, morphine blocks chol i n e r g i c t r a n s m i s s i o n in t h e i l e u m ( P a t o n , 1957) but has no effect on cholinergic transm i s s i o n in t h e h e a r t ( K o s t e r l i t z a n d T a y l o r , 1 9 5 9 ) . R e c e n t l y K a d l e c e t al. ( 1 9 7 4 ) h a v e suggested that prostaglandins may influence a c e t y l c h o ! i n e r e l e a s e in g u t t h r o u g h a n a c t i o n on the sympathetic nerves. We c o n c l u d e f r o m o u r s t u d y o f t h e p h r e n i c nerve--diaphragm preparation that prostagland i n Et p l a y s n o s i g n i f i c a n t r o l e in c h o l i n e r g i c n e u r o t r a n s m i s s i o n in t h i s t i s s u e . T h i s is in accord with the observations on the frog sciatic n e r v e - - s a r t o r i u s m u s c l e ( G i n s b o r g a n d Hirst, 1971). The range of prostaglandin levels e m p l o y e d b y us i n c l u d e d t h e a m o u n t s w h i c h can be released from the rat phrenic nerve-d i a p h r a g m p r e p a r a t i o n ( R a m w e l l e t al., 1 9 6 5 ; Laity, 1969). Horton and Main {1967) found t h a t p r o s t a g l a n d i n E1 d i m i n i s h e s t h e t w i t c h response of cat gastrocnemius muscle to stimu l a t i o n o f its m o t o r n e r v e . H o w e v e r , in t h i s s t u d y t h e d o s e s o f p r o s t a g l a n d i n E1 i n f u s e d into the artery supplying the muscle were v e r y large.
Acknowledgements The authors wish to acknowledge a gift of prostaglandin E1 (May and Baker Ltd.). P. O'N. was the holder of a student grant from the Medical Research Council of Ireland.
References Ambache, N., H.C. Brummer, J.G. Rose and J. Whiting, 1966, Thinlayer chromatography of spasmogenic unsaturated h y d r o x y acids from various tissues, J. Physiol. (London) 185, 77. Bennett, A., K.G. Eley and G.B. Scholes, 1968, Effects of prostaglandin El and E2 on human, guinea-pig and rat isolated small intestine, Brit. J. Pharmacol. 34, 630. Botting, J.H. and R. Salzmann, 1974, The effect of
w.J. HALL ET AL. indomethacin on the release of prostaglandin E2 and acetylcholine from guinea-pig isolated ileum at rest and during field stimulation, Brit. J. Pharmacol. 50, 119. Brody, M.J. and P.J. Kadowitz, 1974, Prostaglandins as modulators of the autonomic nervous system, Federation Proc. 33, 48. Collier, W.O.J. and A.C. Roy, 1974, Morphine-like drugs inhibit the stimulation by E prostaglandins of cyclic AMP formation by rat brain homogenate, Nature (London) 248, 24. Ehrenpreis, S., J. Greenberg and S. Belman, 1973, Prostaglandins reverse inhibition of electrically induced contractions of guinea-pig ileum by morphine, indomethacin and acetylsalicylic acid, Nature (New Biol.) 245, 280. Ferreira, S.H., S. Moncada and J.R. Vane, 1971, Indomethacin and aspirin abolish prostaglandin release from the spleen, Nature New Biol. 231, 237. Fleshier, B. and A. Bennett, 1969, Responses of human, guinea-pig and rat colonic circular muscle to prostaglandins, J. Lab. Clin. Med. 74, 872. Flores, A.G.A. and G.W.G. Sharp, 1972, Endogenous prostaglandins and osmotic water flow in the toad bladder, Amer. J. Physiol. 223, 1392. Flower, R.J., R. Greglewski, K. Herbaczynska-Cedro and J.R. Vane, 1972, Effects of anti-inflammatory drugs on prostaglandin biosynthesis, Nature New Biol. 238, 104. Ginsborg, B.L. and G.D.S. Hirst, 1971, Prostaglandin E1 and noradrenaline at the neuromuscular junction, Brit. J. Pharmacol. 42, 153. Hall, W.J. and M.G. O'Regan, 1974, The effects of indomethacin and theophylline on the response of frog skin to prostaglandin El, J. Physiol. (London) 236, 48. Harry, J., 1962, Effect of cooling, local anaesthetic compounds and botulinum toxin on the responses of and the acetylcholine output from the electrically transmurally stimulated isolated guinea-pig ileum, Brit. J. Pharmacol. 19, 42. Harry, J., 1968, The action of prostaglandin El on the guinea-pig isolated intestine, Brit. d. Pharmacol. 33, 213. Hedqvist, P., 1973, Prostaglandin mediated control of sympathetic neuroeffector transmission, Advan. Biosci. 9, 461. Horton, E.W., 1973, Prostaglandins at adrenergic nerve-endings, Brit. Med. Bull. 29, 148. Horton, E.W. and I.H.M. Main, 1967, Further observations on the central nervous actions of prostaglandins F2a and E l , Brit. J. Pharmacol. 30, 568. Jamieson, D., 1962, A method for the quantitative estimation of drugs on the isolated intact trachea, Brit. J. Pharmacol. 19, 286. Kadlec, O., K. Masek and I. Seferna, 1974, A modulating role of prostaglandins in contractions of the guinea-pig ileum, Brit. J. Pharmacol. 51, 565.
PROSTAGLANDINS AND CHOLINERGIC TRANSMISSION Kosterlitz, H.W. and D.W. Taylor, 1959, The effect of morphine on vagal inhibition of the heart, Brit. J. Pharmacol. 14, 209. Laity, J.L.H., 1969, The release of prostaglandin E l from the rat phrenic nerve--diaphragm preparation, Brit. J. Pharmacol. 37,698. Northover, B.J., 1971, Mechanism of the inhibitory action of indomethacin on smooth muscle, Brit, J. Pharmacol. 41, 540. Northover, B.J., 1972, The effects of indomethacin on calcium, potassium and magnesium fluxes in various tissues of the guinea pig, Brit. J. Pharmacol. 45, 651. Paton, W.D.M., 1955, The response of the guinea-pig ileum to electrical stimulation by coaxial electrodes, J. Physiol. (London) 127, 40. Paton, W.D.M., 1957, The action of morphine and related substances on contraction and on acetylcholine output of coaxially stimulated guinea-pig ileum, Brit. J. Pharmacol. 11, 119. Paton, W.D.M. and M.A. Zar, 1968, The origin of acetylcholine released from guinea-pig intestine and longitudinal muscle strips, J. Physiol. (London) 194, 13.
47
Paton, W.D.M. and E.S. Vizi, 1969, The inhibitory action of noradrenaline and adrenaline on acetylcholine output by guinea-pig ileum longitudinal muscle strip, Brit. J. Pharmacol. 35, 10. Ramwell, P.W., J.E. Shaw and J. Kucharski, 1965, Prostaglandin release from the rat phrenic nerve-diaphragm preparation, Science (N.Y.) 149, 1390. Rang, H.P., 1964, Stimulant actions of volatile anaesthetics on smooth muscle, Brit. J. Pharmacol. 22, 356. Smith, J.B. and A.L. Willis, 1971, Aspirin selectively inhibits prostaglandin production in human platelets, Nature New Biol. 231, 235. Vane, J.R., 1971, Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs, Nature New Biol. 231, 232. Wennmalm, A. and P. Hedqvist, 1971, Inhibition by prostaglandin E1 of parasympathetic neurotransmission in the rabbit heart, Life Sci. 10, 465. Wennmalm, A. and M. Junstad, 1973, Endogenous prostaglandin mediated inhibition of parasympathetic neurotransmission in the rabbit heart?, Acta Physiol. Scand. Suppl. 396, 22.