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Effects of neuronal polypeptides on intestinal smooth muscle; a comparison with non-adrenergic, non-cholinergic nerve stimulation and ATP
European Journal of Pharmacology, 54 (1979) 251--259 © Elsevier/North-Holland Biomedical Press
251
E F F E C T S OF N E U R O N A L POLYPEPTIDES ON I N T E S T I N A L SMOOTH MUSCLE; A COMPARISON WITH NON-ADRENERGIC, NON-CHOLINERGIC NERVE STIMULATION AND ATP THOMAS COCKS and GEOFFREY BURNSTOCK Department of Anatomy and Embryology, University College London, Gower Street, London, WCIE 6BT, U.K.
Received 15 August 1978, revised MS received 22 November 1978, accepted 1 December 1978
T. COCKS and G. BURNSTOCK, Effects of neuronal polypeptides on intestinal smooth muscle; a comparison with non-adrenergic, non-cholinergic nerve stimulation and A TP, European J. Pharmacol. 54 (1979) 251--259. Substance P, somatostatin, enkephalin and vasoactive intestinal polypeptide (VIP) did not mimic the inhibitory responses to non-adrenergic, non-cholinergic nerve stimulation. Substance P (0.1--10 pg/ml) always caused contraction, enkephalin (0.1--10 pg/ml) and somatostatin (0.1--10 gg/ml) were inactive, while VIP (0.01--1 pg/ml) produced very slow relaxation, taking about 4 min to reach a maximum after a latency of about 60 sec. Low concentrations of neurotensin (1--10 ng/mg) caused contraction, but at higher concentrations (50--1000 ng/ml) it produced a biphasic response which consisted of an initial contraction followed by a slow relaxation. In high tone preparations, the slow relaxation did not mimic the nerve-mediated response, taking approximately 43 sec. to reach maximum, after a long latency of about 15 sec. In contrast, ATP (0.1--50 gg/ml) mimicked closely the rapid responses to non-adrenergic, non-cholinergic nerve stimulation in all preparations, whether the tone was low, medium or high. The time for the inhibitory response to reach maximum was about 15 sec after a latency of approximately 1 sec. Indomethacin (3.4--34 pg/ml) did not unmask any inhibitory responses to any of the peptides. It is concluded that ATP remains the most likely substance to be the inhibitory transmitter released from non-adrenergic, non-cholinergic nerves supplying the smooth muscle of the taenia coli. Taenia coli
Purinergic
ATP
Gastrointestinal peptides
1. I n t r o d u c t i o n A p a r t f r o m classical cholinergic and adrenergic n e u r o n e s , t h e a u t o n o m i c i n n e r v a t i o n o f the intestine includes intrinsic n e u r o n e s w h i c h release o t h e r t r a n s m i t t e r s u b s t a n c e s (see B u r n s t o c k , 1 9 6 9 , 1 9 7 9 ; Campbell, 1 9 7 0 ; Furness and Costa, 1973). A d e n o s i n e 5'-trip h o s p h a t e ( A T P ) or a related purine n u c l e o t i d e has been p r o p o s e d as an i n h i b i t o r y t r a n s m i t t e r s u b s t a n c e in s o m e o f these nerves, w h i c h have been called " p u r i n e r g i c " (see B u r n s t o c k , 1972, 1975, 1979). R e c e n t i m m u n o h i s t o c h e m i c a l studies have d e m o n s t r a t e d t h a t s o m e biologically active p o l y p e p t i d e s are localized w i t h i n enteric n e u r o n e s o f the gastrointestinal t r a c t : these
include S u b s t a n c e P (Nilsson et al., 1 9 7 5 ; Pearse and Polak, 1 9 7 5 ; HSkfelt et al., 1 9 7 7 b ; Mroz and L e e m a n , 1 9 7 7 ; S u n d l e r et al., 1977), s o m a t o s t a t i n ( H S k f e l t et al., 1 9 7 5 a , b ; Costa et al., 1 9 7 7 ) , e n k e p h a l i n (Elde et al., 1 9 7 6 ; Polak et al., 1 9 7 7 ; J o h a n s s o n et al., 1978), and vasoactive intestinal p o l y p e p t i d e (VIP) (Larsson et al., 1 9 7 6 ; B r y a n t et al., 1 9 7 6 ; F u x e et al., 1 9 7 7 ; Larsson, 1 9 7 7 ) . O t h e r vasoactive p o l y p e p t i d e s such as n e u r o t e n s i n have also been f o u n d in the gut {Carraway a n d L e e m a n , 1 9 7 6 ; L e e m a n et al., 1 9 7 7 ) , a l t h o u g h n o t localized h i s t o c h e m i c a l l y within enteric n e u r o n e s (Orci et al., 1 9 7 6 ; Polak et al., 1977}. O n the basis o f these studies it has been claimed t h a t t h e n o n - a d r e n e r g i c , n o n - c h o l i n -
252 ergic nerves s u p p l y i n g intestinal s m o o t h m u s c l e are " p e p t i d e r g i c " r a t h e r t h a n " p u r i n e r g i c " ( B l o o m and Polak, 1 9 7 8 ; H u m p h r e y and Fischer, 1978). An i m p o r t a n t c r i t e r i o n for the i d e n t i f i c a t i o n o f a n e u r o t r a n s m i t t e r is that exogenous application of the substance m i m i c s t h e r e s p o n s e to n e r v e s t i m u l a t i o n . Thus, t h e a i m o f o u r e x p e r i m e n t s was to c o m p a r e t h e e f f e c t s on t h e s m o o t h m u s c l e o f t h e guinea-pig t a e n i a coli, o f t h o s e polyp e p t i d e s s h o w n to be localised in intestinal nerves, with t h e responses to s t i m u l a t i o n o f non-adrenergic, non-cholinergic inhibitory nerves and ATP.
2. Materials a n d m e t h o d s G u i n e a pigs ( 3 0 0 - - 4 5 0 g) o f either sex were s t u n n e d b y a b l o w to t h e b a c k o f t h e h e a d a n d e x s a n g u i n a t e d . 2--3 c m strips o f taenia coli w i t h u n d e r l y i n g A u e r b a c h ' s p l e x u s and circular m u s c l e a t t a c h e d w e r e r a p i d l y dissected and s u s p e n d e d in a 10 ml organ b a t h , c o n t a i n i n g a m o d i f i e d K r e b s s o l u t i o n (Biilbring, 1 9 5 3 ) k e p t at 3 7 ° C and c o n t i n u a l l y gassed w i t h a m i x t u r e o f 95% 02, 5% CO2. O n e end o f t h e p r e p a r a t i o n was a t t a c h e d t o a rigid s u p p o r t and t h e o t h e r to a Grass F T 1 0 f o r c e t r a n s d u c e r via a 1 g / c m spring. Muscle activity was r e c o r d e d a u x o t o n i c a l l y o n a Grass m o d e l 7D p o l y g r a p h . An initial l o a d o f lg was placed o n t h e p r e p a r a t i o n a n d 60 m i n a l l o w e d for e q u i l i b r a t i o n b e f o r e beginning t h e experim e n t . Electrical field s t i m u l a t i o n was a c h i e v e d b y m e a n s o f a pair o f p l a t i n u m ring e l e c t r o d e s ( a b o u t 3 m m apart) p l a c e d a r o u n d t h e tissue a n d r e c t a n g u l a r pulses w e r e delivered b y a Grass $9 e l e c t r o n i c s t i m u l a t o r . Drugs used were: a d e n o s i n e 5 ' - t r i p h o s p h a t e ( A T P : Sigma), l e u - e n k e p h a l i n ( B e c k m a n ) , i n d o m e t h a c i n (Sigma), n e u r o t e n s i n , s o m a t o statin ( C a l b i o c h e m ) , s u b s t a n c e P ( B e c k m a n ) and vasoactive intestinal p o l y p e p t i d e . I n d o m e t h a c i n was dissolved in 70% a l c o h o l in c o n c e n t r a t i o n s w h i c h a l l o w e d 10 pl a d d i t i o n s to the organ b a t h . All o t h e r drugs w e r e dissolved in distilled w a t e r in s t o c k c o n c e n t r a -
T. COCKS, G. BURNSTOCK tions w h i c h allowed 1 0 - - 1 0 0 pl a d d i t i o n s to the organ b a t h . Drugs were w a s h e d f r o m the b a t h b y replacing t h e s o l u t i o n twice.
3. Results
3.1. Responses to non-adrenergic, non cholinergic inhibitory nerve stimulation and A T P In t h e p r e s e n c e o f a t r o p i n e (1 p g / m l ) and g u a n e t h i d i n e (1 p g / m l ) responses o f t h e t a e n i a coli t o l o w f r e q u e n c y electrical field stimulat i o n (0.2 m s e c d u r a t i o n pulses delivered at 0 . 5 - - 5 Hz a n d s u p r a m a x i m a l voltage f o r 1 0 - 3 0 s e c ) and t o A T P were f o u n d t o v a r y d e p e n d i n g on t h e " t o n e " o f t h e p r e p a r a t i o n (figs. l a , b ; and see also C a m p b e l l , 1 9 6 6 ; and B u r n s t o c k et al., 1970). P r e p a r a t i o n s w e r e r e g a r d e d as having high t o n e if t h e r e s p o n s e s to b o t h stimuli c o n s i s t e d o f large well m a i n t a i n e d r e l a x a t i o n s f o l l o w e d b y little or no " r e b o u n d TABLE 1 Comparison of the time course of inhibitory responses to intramural nerve stimulation (NS: 0.2 msec duration pulses and supramaximal voltage (40--60 V) delivered at the frequency shown), ATP, VIP and neurotensin (NT). Atropine (1 ug/ml) and guanethidine (1 t~g/ml) were present throughout, n = number of preparations and figures represent the mean ± S.E.M. The initial rate of relaxation was calculated over the first 5 sec of the stimulation period. Stimulus
Latency of onset of relaxation (sec)
Initial rate of relaxation (mm/sec)
NS (2 Hz) n=8 ATP ( 1 ug/ml) n=8 VIP (0.5 ~g/ml) n=3 NT (1 ~g/ml) n=3
<1
5.6 ± 0.7
12±
5.7 -+ 0.7
17-+ 0.7
1.3-+ 0.1 60
+- 30
0.07-+ 0.02
15
-+ 4
0.3 -+ 0.2
Time to maximum relaxation (sec) 5.5
225 ± 25 43±
5
PURINERGIC AND PEPTIDERGIC EFFECTS ON GUT (a)NERVE STIM ( b ) A T P
(c)SUB P
253
(d)SST
(e)leu-ENK
(f)N T
HIGH TONE
MEDIUM TONE
LOW TONE
lmin
Fig. 1. Comparison of the responses of the guinea-pig taenia coli to (a) intramural nerve stimulation (nerve stim: 0.2 msec duration pulses delivered at 5 Hz and supramaximal voltage (40 V), (b) ATP (5 pg/ml), (c) Substance P (Sub-P: 10 ng/ml), (d) somatostatin (SST: 10 ftg/ml), (e) leu-enkephalin (leu-ENK: 1 pg/ml)and (f)neurotensin (NT: 10 ng/ml), in high, medium and low tone preparations. The same preparation was used for successive stimuli. Atropine (1 pg/ml) and guanethidine (1 pg/ml) were present throughout.
c o n t r a c t i o n s " (fig. l a ) . P r e p a r a t i o n s having m e d i u m t o n e w e r e t h o s e w h i c h gave significant r e l a x a t i o n s t o b o t h stimuli, f o l l o w e d b y "rebound contractions" which were often similar in m a g n i t u d e t o t h e i n h i b i t o r y responses. In t h e s e preparations during extended periods of stimulation "breakthrough" contractions were often observed f o l l o w i n g t h e initial i n h i b i t o r y response. L o w t o n e p r e p a r a t i o n s were t h o s e in w h i c h little or n o r e l a x a t i o n o c c u r r e d during s t i m u l a t i o n , b u t t h e r e was a large " r e b o u n d c o n t r a c t i o n " u p o n cessation o f t h e stimulus. In all p r e p a r a t i o n s (n = 8) t h e r e s p o n s e to 0 . 1 - - 5 0 p g / m l
A T P m i m i c k e d closely t h e r e s p o n s e to electrical field s t i m u l a t i o n o f n o n - a d r e n e r g i c , i n h i b i t o r y nerves (fig. l b ) . F o r high a n d average t o n e p r e p a r a t i o n s , t h e l a t e n c y in o n s e t o f r e l a x a t i o n , t h e initial r a t e o f relaxat i o n a n d t h e t i m e to r e a c h m a x i m u m are given in t a b l e 1.
3.2. Effects of polypep tides 3.2.1. Substance P In all p r e p a r a t i o n s (n = 5), S u b s t a n c e P ( 0 . 0 1 - - 1 p g / m l ) caused a c o n t r a c t i o n w h i c h was r a p i d in o n s e t a n d m a i n t a i n e d (fig. l c ) .
254
T. COCKS, G. BURNSTOCK b
T h e degree o f c o n t r a c t i o n for a n y p a r t i c u l a r c o n c e n t r a t i o n o f S u b s t a n c e P increased as t h e t o n e decreased.
3.2.2. Sornatostatin S o m a t o s t a t i n ( 0 . 1 - - 1 0 p g / m l ) rarely prod u c e d a response, regardless o f t h e t o n e o f t h e p r e p a r a t i o n (fig. l d ) . H o w e v e r , in 2 p r e p a r a tions ( o u t o f 10) w i t h m e d i u m t o n e , s o m a t o statin (10 p g / m l ) caused a biphasic r e s p o n s e which consisted o f an initial small r e l a x a t i o n , f o l l o w e d b y a t o n i c c o n t r a c t i o n , a n d in o n e low t o n e p r e p a r a t i o n t h e s a m e c o n c e n t r a t i o n of s o m a t o s t a t i n gave a " r e b o u n d c o n t r a c t i o n " (fig. l d ) .
d
f
g
3.2.3. Enkephalin In all p r e p a r a t i o n s {n = 5) l e u - e n k e p h a l i n ( 0 . 1 - - 1 0 p g / m l ) failed t o give a n y d i r e c t r e s p o n s e (see fig. l e ) . As e n k e p h a l i n s h a v e b e e n s h o w n to be p o t e n t i n h i b i t o r s o f electrically s t i m u l a t e d c o n t r a c t i o n o f s m o o t h m u s c l e o f t h e guinea-pig i l e u m ( H u g h e s et al., 1 9 7 5 a ) , p r e s u m a b l y acting b y h y p e r polarization of excitatory neurones within t h e m y e n t e r i c p l e x u s ( N o r t h and Williams, 1976), t h e e f f e c t o f l e u - e n k e p h a l i n on the i n h i b i t o r y r e s p o n s e t o b o t h electrical field s t i m u l a t i o n a n d A T P in t h e guinea-pig t a e n i a coli was e x a m i n e d . In t h e a b o v e c o n c e n t r a t i o n range, l e u - e n k e p h a l i n did n o t a f f e c t t h e i n h i b i t o r y r e s p o n s e t o either low f r e q u e n c y field s t i m u l a t i o n (0.2 m s e c d u r a t i o n pulses delivered at 0 . 5 - - 5 H z a n d s u p r a m a x i m a l voltage) or A T P ( 1 - - 1 0 g g / m l ) . N a l o x o n e (1 p g / m l ) , an a n t a g o n i s t o f e n k e p h a l i n s , also had n o e f f e c t on t h e i n h i b i t o r y r e s p o n s e s t o field s t i m u l a t i o n . 3.2.4. Neurotensin T h e r e s p o n s e t o l o w c o n c e n t r a t i o n s of n e u r o t e n s i n ( 1 - - 1 0 n g / m l ) was always a cont r a c t i o n regardless o f t o n e (fig. l f ) . T h e r e s p o n s e t o higher c o n c e n t r a t i o n s o f neurotensin ( 5 0 - 1 0 0 0 n g / m l ) was a c o n t r a c t i o n in l o w a n d m e d i u m t o n e p r e p a r a t i o n s , b u t in high t o n e p r e p a r a t i o n s (n = 3) t h e c o n t r a c t i o n s h o w e d fade w h i c h d e v e l o p e d i n t o a slow
I
1rain
i
Fig. 2. Responses of a high tone preparation of the guinea-pig taenia coli to (a) 0.001, (b) 0.01, (c) 0.05, (d) 0.1, (e) 0.5 and (f) 1 /lg/ml neurotensin (horizontal bars). Note: the gradual change from a sustained contraction to a delayed, slow relaxation. In (g) the tone has fallen to a lower level and under these conditions neurotensin (1 pg/ml) causes an initial rapid contraction followed by a slow relaxation. A second application of the same dose of neurotensin (1 pg/ml) while the tone is high, now causes a delayed slow relaxation similar to that in (f). Atropine (1 pg/ ml) and guanethidine (1 pg/ml) were present throughout.
i n h i b i t o r y r e s p o n s e (fig. 2c,d), or a slow r e l a x a t i o n with a l a t e n c y in o n s e t o f a b o u t 15 sec and a t i m e to m a x i m u m r e s p o n s e of a b o u t 4 0 - - 5 0 sec (fig. 2e,f a n d t a b l e 1).
3.2.5. Vasoactive intestinal polypeptide (VIP) In high and average t o n e p r e p a r a t i o n s (n = 3), V I P ( 0 . 0 1 - - 1 p g / m l ) caused slow relaxat i o n s ( t i m e t o m a x i m u m 2 0 0 - - 2 5 0 sec) w i t h a l a t e n c y in o n s e t o f a b o u t 60 sec (fig. 3 a n d t a b l e 1). T h e r e s p o n s e s t o electrical field
PURINERGIC AND PEPTIDERGIC EFFECTS ON GUT
VIP HIGH~
TONE V
ATP
NS
255 b o t h intramural nerve stimulation and ATP, and also in the one experi m ent in which somatostatin caused a " r e b o u n d c o n t r a c t i o n " .
I
4. Discussion
MEDIUM~TONE
TONE I ! 5min
Fig. 3. Comparison of the inhibitory responses of the guinea-pig taenia coli to vasoactive intestinal polypeptide (VIP: 0.5//g/ml), ATP (0.5 pg/ml) and intramural nerve stimulation (NS: 0.2 msec duration pulses delivered at 5Hz and supramaximal voltage (50 V) for 30 sec) in high, medium and low tone preparations. Atropine (1 pg/ml) and guanethidine (1 pg/ml) were present throughout.
stimulation and ATP compared with that to VIP are shown in fig. 3. Also, in the above c o n c e n tr atio n range, VIP did n o t affect the responses to either electrical field stimulation or ATP.
3.3. Effects o f indemethacin Incubation with indomethacin (3.4--34 pg/ ml) for 60 min did n o t affect the direct contractile responses to the polypeptides tested whereas it blocked t he " r e b o u n d contract i o n s " following the inhibitory responses to
The response of the guinea-pig taenia coli to ATP has been f o u n d to mimic closely the inhibitory response to non-adrenergic, noncholinergic "puri nergi c" nerve stimulation, confirming the earlier report of Burnstock et al. (1970). Both responses had a latency of about i sec and a relaxation that reached maxim u m in 12 to 17 sec; in preparations which had low t o medium tone, both inhibitory responses were followed by a " r e b o u n d cont r a c t i o n " {see Burnstock et al., 1963; Campbell, 1966; Bennett, 1966; this paper). Further, the ionic mechanisms underlying muscle membrane hyperpolarization following application of ATP have been shown to be similar to those associated with the inhibitory junction potentials elicited in response to stimulation of non-adrenergic, non-cholinergic nerves (Tomita and Watanabe, 1973; Jager, 1974). The low frequencies of stimulation used (0.5--5 Hz) selectively activate purinergic nerves and n o t adrenergic nerves. Furthermore, guanethidine, in the concentrations used, completely abolishes the response to adrenergic nerve stimulation {Burnstock et al., 1966). None of the polypeptides which have been shown to be localised in enteric nerves with immunofluorescence m et hods (see Introduction for references) produced responses which resembled those to non-adrenergic, noncholinergic nerve stimulation; Substance P was excitatory, enkephalin and somatostatin were inactive, while VIP produced a very slow relaxation reaching a m a x i m u m in 200--250 sec after a latency of about 60 sec. Low concentrations of neurotensin produced contraction of the taenia coli; higher concentrations in high t one preparations p r o d u c e d a relaxation, but this relaxation was slow (time to
256 maximum 40--50 sec) and did not mimic that produced by stimulation of intramural inhibitory nerves. Thus, on the basis of the similarity of postjunctional action, ATP is the most likely of the substances tested to be the transmitter released from non-adrenergic, non-cholinergic inhibitory nerves supplying the smooth muscle of the taenia coli. This conclusion is supported by evidence that ATP satisfies four other criteria required to establish a neurotransmitter, namely: synthesis and storage in nerve terminals; release during nerve stimulation; an inactivation mechanism involving both enzymes and high affinity re-uptake; and drugs that produce parallel effects on the responses to both nerve stimulation and exogenous transmitter (see Burnstock, 1972, 1975, 1979). Since Substance P, enkephalin, somatostatin and VIP are clearly not the transmitters released from the non-adrenergic, non-cholinergic efferent nerves supplying the intestinal musculature, other possible roles for these intraneuronal polypeptides should be considered. It is unlikely that the contractile responses to the peptides tested were produced by excitatory prostaglandins which masked any inhibitory effects. Such a role for prostaglandins has been proposed for the contractile response of the anococcygeus to exogenous ATP (Burnstock et al., 1978). However, in the present study no inhibitory responses to peptides were unmasked following inhibition of prostaglandin synthesis (see also Bury and Mashford, 1977). There is strong evidence that Substance P is a neurotransmitter in primary afferent fibres in the spinal cord and skin (see Lembeck, 1953; HSkfelt et al., 1977b; Otsuka and Konishi, 1977) so that is is possible that it is also localized in sensory fibres in the gut (see also HSkfelt et al., 1977b). However, Substance P is known to have a variety of peripheral actions including contraction of intestinal longitudinal muscle (Euler and Gaddum, 1931; Pernow, 1960; Franco and Costa, 1978) and dilatation of blood vessels
T. COCKS, G. BURNSTOCK (Euler and Gaddum, 1931). The endogenous opiate ligands, enkephalins, have potent inhibitory actions on electricallystimulated contractions of both the mouse vas deferens and the guinea-pig ileum (Hughes et al., 1975a,b). Also, both enkephalins and morphine have been shown to inhibit neuronal firing in Auerbach's plexus (Dingledine and Goldstein, 1976; North and Williams, 1976) by causing membrane hyperpolarization (North and Tonini, 1976). Therefore, it seems likely that enkephalins are released from interneurones involved in modulation of nervemediated excitation of the intestine. Neither enkephalin nor naloxone, a potent opiate antagonist (see Klee, 1977) had any effect on the inhibitory response to non-adrenergic intramural nerve stimulation in the guinea-pig taenia coli. It has also been suggested that neurones which display somatostatin immunoreactivity are interneurones within the enteric nervous system (Costa et al., 1977). This is supported by the finding that somatostatin inhibits the release of acetylcholine from nerves within Auerbach's plexus of the small iptestine (Guillemin, 1976) and no somatostatin immunoreactive fibres were found in either the longitudinal or circular muscle of the ileum (Costa et al., 1977). Intrinsic VIP-containing neurones found throughout the gut may also be interneurones, since they have been shown to innervate nerve cells in both Meissner's and Auerbach's plexus (Fuxe et al., 1977). VIP-positive nerve fibres have been observed within the circular muscle layer and muscularis mucosae of the rat stomach, duodenum and colon, but these appear to be en route to the mucosal epithelium (Fuxe et al. 1977). The role of the very slow inhibitory response of the taenia coli to VIP is not clear, since it does not mimic the nerve-mediated response. Similar slow relaxations to VIP have been reported in the gall bladder, trachea and stomach (Piper et al., 1970) and canine small intestine (Kachelhoffer et al., 1976). Finally, the possibility should be considered that polypeptides are stored together with
PURINERGIC AND PEPTIDERGIC EFFECTS ON GUT neurotransmitters in e n t e r i c n e r v e s . B u r n stock (1976) has recently discussed the concept that some nerves contain more than one transmitter. H S k f e l t e t al. ( 1 9 7 7 a ) h a v e s h o w n t h a t s o m e a d r e n e r g i c n e u r o n e s in peripheral sympathetic ganglia contain somatostatin together with noradrenaline and earlier Euler (1963) showed that splenic n e r v e s c o n t a i n e d v e s i c u l a r S u b s t a n c e P. F u r ther, up to nine different types of enteric nerves have been identified on an ultrastruct u r a l b a s i s in t h e g a s t r o i n t e s t i n a l t r a c t , s e v e r a l of them containing combinations of different vesicle types (Gabella, 1972; Cook and Burnstock, 1976).
Acknowledgements We are grateful to Dr. S. Leeman, Harvard Medical School, Boston, and Professor V. Mutt, Karolinska Institute, Stockholm, for the gifts of neurotensin and purified porcine vasoactive intestinal polypeptide respectively.
References Bennett, M.R., 1966, Rebound excitation of the smooth muscle cells of the guinea-pig taenia coli after stimulation of intramural inhibitory nerves, J. Physiol. (London) 185, 124. Bloom, S.R. and J.M. Polak, 1978, Peptidergic versus purinergic, The Lancet 1, 94. Bryant, M.D., J.M. Polak, I. Modlin, S.R. Bloom, R.H. Albuquerque and A.G.E. Pearse, 1976, Possible dual role for vasoactive intestinal peptide as gastrointestinal hormone and neurotransmitter substance, Lancet I, 991. Bulbring, E., 1953, Measurements of oxygen consumption in smooth muscle, J. Physiol. (London) 122,111. Burnstock, G., 1969, Evolution of the autonomic innervation of visceral and cardio-vascular systems in vertebrates, Pharmacol. Rev. 21,247. Burnstock, G. 1972, Purinergic nerves, Pharmacol. Rev. 24,509. Burnstock, G., 1975, Purinergic transmission, in: Handbook of Psychopharmacology, Vol. 5, Synaptic Modulators, eds. L.L. Iversen, S.D. Iversen and S.H. Snyder (Plenum Press, New York) p. 131. Burnstock, G., 1976, Do some nerve cells release more than one transmitter?, Neuroscience 1, 239.
257 Burnstock, G., 1979, Past and current evidence for the purinergic nerve hypothesis, Symposium on: Physiological and Regulatory Function of Adenosine and Adenine Nucleotides, Banff, Canada. (Raven Press, New York) (in press). Burnstock, G., G. Campbell, M. Bennett and M.E. Holman, 1963, Inhibition of the smooth muscle of the taenia eoli, Nature 200, 581. Burnstock, G., G. Campbell and M.J. Rand, 1966, The inhibitory innervation of the taenia of the guinea pig eaccum, J. Physiol. (London) 182,504. Burnstock, G., G. Campbell, D.G. Satchell and A. Smythe, 1970, Evidence that adenosine triphosphate or a related nucleotide is the transmitter substance released by non-adrenergic inhibitory nerves in the gut, Brit. J. Pharmacol. Chemotherap. 40, 668. Burnstock, G., T. Cocks and R. Crowe, 1978, Evidence for purinergic innervation of the anococygeus muscle, Brit. J. Pharmacol. 64, 13. Bury, R.W. and M.L. Mashford, 1977, A pharmacological investigation of synthetic substance P on the isolated guinea-pig ileum, Clin. Exptl. Pharmacot. Physiol. 4 , 4 5 3 . Campbell, G., 1966, Nerve-mediated excitation of the taenia of the guinea-pig caecum, J. Physiol. (London) 185, 148. Campbell, G., 1970, Autonomic nervous supply to effector tissues, in: Smooth Muscle, eds. E. Billbring, A.F. Brading, A.W. Jones and T. Tomita (Edward Arnold, London) p. 451. Carraway, R. and S.E. Leeman, 1976, Characterization of radioimmunoassayable neurotensin in the rat -- its differential distribution in the central nervous system, small intestine and stomach, J. Biol. Chem. 251, 7045. Cook, R.D. and G. Burnstock, 1976, The ultrastructure of Auerbach's plexus in the guinea-pig. 1. Neural elements, J. Neurocytol. 5,171. Costa, M., Y. Patel, J.B. Furness and A. Arimura, 1977, Evidence that some intrinsic neurones of the intestine contain somatostatin, Neuroscienee. Lett. 6, 215. Dingledine, R. and A. Goldstein, 1976, Effects of synaptic transmission blockade on morphine action in the guinea-pig myenteric plexus, J. Pharmacol. Exptl. Therap. 196, 205. Elde, R. T. HSkfelt, O. Johansson and L. Terenius, 1976, Immunohistochemical studies using antibodies to leucine-enkephalin. Initial observations on the nervous system of the rat, Neuroscience 1, 349. Franco, R. and M. Costa, 1978, The Effect of substance P on intestinal nerves and muscle, Proc. Aust. Physiol. Pharmacol. Soc. (May, 1978), 10P. Furness, J.B. and M. Costa, 1973, The nervous release and the action of substances which affect intestinal
258 smooth muscle through neither adrenoceptor nor cholinoreceptors, Phil. Trans, Roy. Soc. London, Series B 265, 123. Fuxe, K., T. HSkfelt, S.I. Said and V. Mutt, 1977, Vasoactive intestinal polypeptide and the nervous system. Immunohistochemical evidence for localization in central and peripheral neurones, particularly intracortical neurons of the cerebral cortex, Neuroscience, Lett. 5,241. Gabella, G., 1972, Fine structure of the myenteric plexus in the guinea-pig ileum, J. Anat. London 111, 69. Guillemin, R., 1976, Somatostatin inhibits the release of acetylcholine induced electrically in the myenteric plexus, Endocrinology 99, 1653. HSkfelt, T., S. Efendic, C. Hellerstrom, O. Johansson, R. Luft and A. Arimura, 1975a, Cellular localization of somatostatin in endocrine-like cells and neurons of the rat with special reference to the AI cells of the pancreatic islets and to the hypothalamus, Acta. Endocr. (Kbh), Suppl. 200, 1. H6kfelt, T. L.G. Elfvin, R. Elde, M. Schultzberg, M. Goldstein and R. Luft, 1977a, Occurrence of somatostatin-like immuno-reactivity in some peripheral sympathetic noradrenergic neurons, Proc. Nat. Acad. Sci. U.S.A. 74, 3587. HSkfelt, T., O. Johansson, S. Efendic, R. Luft and A. Arimura, 1975b, Are there somatostatincontaining nerves in the rat gut? Immunohistochemical evidence for a new type peripheral nerve, Experentia 31,852. HSkfelt, T., O. Johansson, J.O. Kellerth, ~,. Ljungdahl, G. Nilsson, A. Nyg~rds and B. Pernow, 1977b, Immunohistochemical distribution of substance P, in: Substance P. Nobel Symposium 37, eds. U.S. Von Euler and B. Pernow (Raven Press, New York) p. 117. Hughes, J., T.W. Smith, H.W. Kosterlitz, L.A. Fothergill, B.A. Morgan and H.R. Morris, 1975a, Identification of two related pentapeptides from the brain with potent opiate agonist activity, Nature 258,577. Hughes, J., T. Smith, B. Morgan and L. Fothergill, 1975b, Purification and properties of enkephalin -- the possible endogenous ligand for the morphine receptor, Life Sci. 16, 1753. Humphrey, C.S. and J.E. Fischer, 1978, Peptidergic versus purinergic nerves, The Lancet 1,390. Jager, L.P., 1974, The effect of catecholamines and ATP on the smooth muscle cell membrane of the guinea-pig taenia coli, European J. Pharmacoh 25, 372. Johansson, O. T, H6kfelt, R.P. Elde, M. Schultzberg and L. Terenius, 1978, Immunohistochemical distribution of enkephalin neurons, in: Advances in Biochemical Psychopharmacology, Vol. 18, eds. E. Costa and M. Trabucchi (Raven Press, New York) p. 51.
T. COCKS, G. BURNSTOCK Kachelhoffer, J., C. Mendel, J. Dauchel, D. Hohmatter, and J.F. Grenier, 1976, The effects of VIP on intestinal motility. Study on ex vivo perfused isolated canine jejunal loops, Amer. J. Digest. Dis. 21,957. Klee, W.A., 1977, Endogenous opiate peptides, in: Peptides in Neurobiology, ed. H. Gainer (Plenum Press, New York) p. 357. Larsson, L.-I., 1977, Ultrastructural localization of a new neuronal peptide (VIP), Histochem. 54,173. Larsson, L.-I., J. Fahrenkrug, O. Schaffalitzky de Muckadell, F. Sundler, R. H~/kanson and J.F. Rehfeld, 1976, Localization of vasoactive intestinal polypeptide (VII') to central and peripheral neurons, Proc. Nat. Acad. Sci. U.S.A. 73, 3197. Leeman, S.E., E.A. Mroz and R.E. Carraway, 1977, Substance P and neurotensin, in: Peptides in Neurobiology, ed. H. Gainer (Plenum Press, New York) p. 99. Lembeck, F., 1953, Zur Frage der zentralen 0bertragung afferenter Impulse. III. Mitteilung, Das Vorkommen und die Bedeutung der Substanz P in den dorsalen Wurzeln des Riickenmarks, Naunyn-Schmiedeb. Arch. Pharmacol. 219, 197. Mroz, E.A. and S.E. Leeman, 1977, Substance P, in: Vitamins and Hormones, Vol. 35, eds. P.L. Munson, J. Glover, E. Diczfalusy and R.E. Olson, (Academic Press, New York, San Francisco, London) p. 209. Nilsson, G., L.-I. Larsson, R. Hgkanson, E. Brodin, F. Sundler and B. Pernow, 1975, Localization of substance P-like immunoreactivity in mouse gut, Histochem. 43, 97. North, R.A. and M. Tonini, 1976, Hyperpolarization by morphine of myenterie neurones, in: Opiates and Endogenous Opioid Peptides ed. H.W. Kosterlitz (North Holland, Amsterdam) p. 205. North, R.A. and J.T. Williams, 1976, Enkephalin inhibits firing of myenteric neurones, Nature 264, 460. Orci, L., D. Baetens, C. Rufener, M. Brown, W. Vale and R. Guillemin, 1976, Evidence for immunoreactive neurotensin in dog intestinal mucosa, Life Sci. 19, 559. Otsuka, M. and S. Konishi, 1977, Electrophysiological and neurochemical evidence for substance P as a transmitter of primary sensory neurons, in: Substance P: Nobel Symposium 37, eds. U.S. Von Euler and B. Pernow (Raven Press, New York) p. 207. Paddle, B.M. and G. Burnstoek, 1974, Release of ATP from perfused heart during coronary vasodilatation, Blood Vessels 2, 110. Pearse, A.G.E. and J.M. Polak, 1975, Immunohistochemical localization of substance P in mammalian intestine, Histochem. 4 1 , 3 7 3 . Pernow, B., 1960, Effect of substance P on smooth muscle, in: Polypeptides which Affect Smooth
PURINERGIC AND PEPTIDERGIC EFFECTS ON GUT Muscle and Blood Vessels, ed. M. Schechter (Pergamon Press, Elsmford, New York) p. 163. Piper, P.J., S.I. Said and J.R. Vane, 1970, Effects on smooth muscle preparations of unidentified vasoactive peptides from intestine and lung, Nature 225, 1144. Polak, J.M., S.N. Sullivan, S.R. Bloom, A.M.J. Buchan, P. Facer, M.R. Brown and A.G.E. Pearse, 1977, Specific localization of neurotensin to the N cell in human intestine by radioimmunoassay and immunocytochemistry, Nature 270, 183. Polak, J.M., S.N. Sullivan, S.R. Bloom, P. Facer and A.G.E. Pearse, 1977, Enkephalin-like immunoreactivity in the human gastrointestinal tract, Lancet I, 972.
259 Sundler, F., R. H~/kanson, L.-I. Larsson, E. Brodin and G. Nilsson, 1977, in: Substance P: Nobel Symposium 37, eds. U.S. Von Euler and B. Pernow (Raven Press, New York) p. 59. Tomita, T. and H. Watanabe, 1973, A comparison of the effects of adenosine triphosphate with noradrenaline and with the inhibitory potential of the guinea-pig taenia coli, J. Physiol. (London) 231,167. Von Euler, U.S., 1963, Substance P in subcellular particles in peripheral nerves, Ann. N.Y. Acad. Sci. 104,449. Von Euler, U.S. and J.H. Gaddum, 1931, An unidentified depressor substance in certain tissue extracts, J. Physiol. (London) 72, 74.
251
E F F E C T S OF N E U R O N A L POLYPEPTIDES ON I N T E S T I N A L SMOOTH MUSCLE; A COMPARISON WITH NON-ADRENERGIC, NON-CHOLINERGIC NERVE STIMULATION AND ATP THOMAS COCKS and GEOFFREY BURNSTOCK Department of Anatomy and Embryology, University College London, Gower Street, London, WCIE 6BT, U.K.
Received 15 August 1978, revised MS received 22 November 1978, accepted 1 December 1978
T. COCKS and G. BURNSTOCK, Effects of neuronal polypeptides on intestinal smooth muscle; a comparison with non-adrenergic, non-cholinergic nerve stimulation and A TP, European J. Pharmacol. 54 (1979) 251--259. Substance P, somatostatin, enkephalin and vasoactive intestinal polypeptide (VIP) did not mimic the inhibitory responses to non-adrenergic, non-cholinergic nerve stimulation. Substance P (0.1--10 pg/ml) always caused contraction, enkephalin (0.1--10 pg/ml) and somatostatin (0.1--10 gg/ml) were inactive, while VIP (0.01--1 pg/ml) produced very slow relaxation, taking about 4 min to reach a maximum after a latency of about 60 sec. Low concentrations of neurotensin (1--10 ng/mg) caused contraction, but at higher concentrations (50--1000 ng/ml) it produced a biphasic response which consisted of an initial contraction followed by a slow relaxation. In high tone preparations, the slow relaxation did not mimic the nerve-mediated response, taking approximately 43 sec. to reach maximum, after a long latency of about 15 sec. In contrast, ATP (0.1--50 gg/ml) mimicked closely the rapid responses to non-adrenergic, non-cholinergic nerve stimulation in all preparations, whether the tone was low, medium or high. The time for the inhibitory response to reach maximum was about 15 sec after a latency of approximately 1 sec. Indomethacin (3.4--34 pg/ml) did not unmask any inhibitory responses to any of the peptides. It is concluded that ATP remains the most likely substance to be the inhibitory transmitter released from non-adrenergic, non-cholinergic nerves supplying the smooth muscle of the taenia coli. Taenia coli
Purinergic
ATP
Gastrointestinal peptides
1. I n t r o d u c t i o n A p a r t f r o m classical cholinergic and adrenergic n e u r o n e s , t h e a u t o n o m i c i n n e r v a t i o n o f the intestine includes intrinsic n e u r o n e s w h i c h release o t h e r t r a n s m i t t e r s u b s t a n c e s (see B u r n s t o c k , 1 9 6 9 , 1 9 7 9 ; Campbell, 1 9 7 0 ; Furness and Costa, 1973). A d e n o s i n e 5'-trip h o s p h a t e ( A T P ) or a related purine n u c l e o t i d e has been p r o p o s e d as an i n h i b i t o r y t r a n s m i t t e r s u b s t a n c e in s o m e o f these nerves, w h i c h have been called " p u r i n e r g i c " (see B u r n s t o c k , 1972, 1975, 1979). R e c e n t i m m u n o h i s t o c h e m i c a l studies have d e m o n s t r a t e d t h a t s o m e biologically active p o l y p e p t i d e s are localized w i t h i n enteric n e u r o n e s o f the gastrointestinal t r a c t : these
include S u b s t a n c e P (Nilsson et al., 1 9 7 5 ; Pearse and Polak, 1 9 7 5 ; HSkfelt et al., 1 9 7 7 b ; Mroz and L e e m a n , 1 9 7 7 ; S u n d l e r et al., 1977), s o m a t o s t a t i n ( H S k f e l t et al., 1 9 7 5 a , b ; Costa et al., 1 9 7 7 ) , e n k e p h a l i n (Elde et al., 1 9 7 6 ; Polak et al., 1 9 7 7 ; J o h a n s s o n et al., 1978), and vasoactive intestinal p o l y p e p t i d e (VIP) (Larsson et al., 1 9 7 6 ; B r y a n t et al., 1 9 7 6 ; F u x e et al., 1 9 7 7 ; Larsson, 1 9 7 7 ) . O t h e r vasoactive p o l y p e p t i d e s such as n e u r o t e n s i n have also been f o u n d in the gut {Carraway a n d L e e m a n , 1 9 7 6 ; L e e m a n et al., 1 9 7 7 ) , a l t h o u g h n o t localized h i s t o c h e m i c a l l y within enteric n e u r o n e s (Orci et al., 1 9 7 6 ; Polak et al., 1977}. O n the basis o f these studies it has been claimed t h a t t h e n o n - a d r e n e r g i c , n o n - c h o l i n -
252 ergic nerves s u p p l y i n g intestinal s m o o t h m u s c l e are " p e p t i d e r g i c " r a t h e r t h a n " p u r i n e r g i c " ( B l o o m and Polak, 1 9 7 8 ; H u m p h r e y and Fischer, 1978). An i m p o r t a n t c r i t e r i o n for the i d e n t i f i c a t i o n o f a n e u r o t r a n s m i t t e r is that exogenous application of the substance m i m i c s t h e r e s p o n s e to n e r v e s t i m u l a t i o n . Thus, t h e a i m o f o u r e x p e r i m e n t s was to c o m p a r e t h e e f f e c t s on t h e s m o o t h m u s c l e o f t h e guinea-pig t a e n i a coli, o f t h o s e polyp e p t i d e s s h o w n to be localised in intestinal nerves, with t h e responses to s t i m u l a t i o n o f non-adrenergic, non-cholinergic inhibitory nerves and ATP.
2. Materials a n d m e t h o d s G u i n e a pigs ( 3 0 0 - - 4 5 0 g) o f either sex were s t u n n e d b y a b l o w to t h e b a c k o f t h e h e a d a n d e x s a n g u i n a t e d . 2--3 c m strips o f taenia coli w i t h u n d e r l y i n g A u e r b a c h ' s p l e x u s and circular m u s c l e a t t a c h e d w e r e r a p i d l y dissected and s u s p e n d e d in a 10 ml organ b a t h , c o n t a i n i n g a m o d i f i e d K r e b s s o l u t i o n (Biilbring, 1 9 5 3 ) k e p t at 3 7 ° C and c o n t i n u a l l y gassed w i t h a m i x t u r e o f 95% 02, 5% CO2. O n e end o f t h e p r e p a r a t i o n was a t t a c h e d t o a rigid s u p p o r t and t h e o t h e r to a Grass F T 1 0 f o r c e t r a n s d u c e r via a 1 g / c m spring. Muscle activity was r e c o r d e d a u x o t o n i c a l l y o n a Grass m o d e l 7D p o l y g r a p h . An initial l o a d o f lg was placed o n t h e p r e p a r a t i o n a n d 60 m i n a l l o w e d for e q u i l i b r a t i o n b e f o r e beginning t h e experim e n t . Electrical field s t i m u l a t i o n was a c h i e v e d b y m e a n s o f a pair o f p l a t i n u m ring e l e c t r o d e s ( a b o u t 3 m m apart) p l a c e d a r o u n d t h e tissue a n d r e c t a n g u l a r pulses w e r e delivered b y a Grass $9 e l e c t r o n i c s t i m u l a t o r . Drugs used were: a d e n o s i n e 5 ' - t r i p h o s p h a t e ( A T P : Sigma), l e u - e n k e p h a l i n ( B e c k m a n ) , i n d o m e t h a c i n (Sigma), n e u r o t e n s i n , s o m a t o statin ( C a l b i o c h e m ) , s u b s t a n c e P ( B e c k m a n ) and vasoactive intestinal p o l y p e p t i d e . I n d o m e t h a c i n was dissolved in 70% a l c o h o l in c o n c e n t r a t i o n s w h i c h a l l o w e d 10 pl a d d i t i o n s to the organ b a t h . All o t h e r drugs w e r e dissolved in distilled w a t e r in s t o c k c o n c e n t r a -
T. COCKS, G. BURNSTOCK tions w h i c h allowed 1 0 - - 1 0 0 pl a d d i t i o n s to the organ b a t h . Drugs were w a s h e d f r o m the b a t h b y replacing t h e s o l u t i o n twice.
3. Results
3.1. Responses to non-adrenergic, non cholinergic inhibitory nerve stimulation and A T P In t h e p r e s e n c e o f a t r o p i n e (1 p g / m l ) and g u a n e t h i d i n e (1 p g / m l ) responses o f t h e t a e n i a coli t o l o w f r e q u e n c y electrical field stimulat i o n (0.2 m s e c d u r a t i o n pulses delivered at 0 . 5 - - 5 Hz a n d s u p r a m a x i m a l voltage f o r 1 0 - 3 0 s e c ) and t o A T P were f o u n d t o v a r y d e p e n d i n g on t h e " t o n e " o f t h e p r e p a r a t i o n (figs. l a , b ; and see also C a m p b e l l , 1 9 6 6 ; and B u r n s t o c k et al., 1970). P r e p a r a t i o n s w e r e r e g a r d e d as having high t o n e if t h e r e s p o n s e s to b o t h stimuli c o n s i s t e d o f large well m a i n t a i n e d r e l a x a t i o n s f o l l o w e d b y little or no " r e b o u n d TABLE 1 Comparison of the time course of inhibitory responses to intramural nerve stimulation (NS: 0.2 msec duration pulses and supramaximal voltage (40--60 V) delivered at the frequency shown), ATP, VIP and neurotensin (NT). Atropine (1 ug/ml) and guanethidine (1 t~g/ml) were present throughout, n = number of preparations and figures represent the mean ± S.E.M. The initial rate of relaxation was calculated over the first 5 sec of the stimulation period. Stimulus
Latency of onset of relaxation (sec)
Initial rate of relaxation (mm/sec)
NS (2 Hz) n=8 ATP ( 1 ug/ml) n=8 VIP (0.5 ~g/ml) n=3 NT (1 ~g/ml) n=3
<1
5.6 ± 0.7
12±
5.7 -+ 0.7
17-+ 0.7
1.3-+ 0.1 60
+- 30
0.07-+ 0.02
15
-+ 4
0.3 -+ 0.2
Time to maximum relaxation (sec) 5.5
225 ± 25 43±
5
PURINERGIC AND PEPTIDERGIC EFFECTS ON GUT (a)NERVE STIM ( b ) A T P
(c)SUB P
253
(d)SST
(e)leu-ENK
(f)N T
HIGH TONE
MEDIUM TONE
LOW TONE
lmin
Fig. 1. Comparison of the responses of the guinea-pig taenia coli to (a) intramural nerve stimulation (nerve stim: 0.2 msec duration pulses delivered at 5 Hz and supramaximal voltage (40 V), (b) ATP (5 pg/ml), (c) Substance P (Sub-P: 10 ng/ml), (d) somatostatin (SST: 10 ftg/ml), (e) leu-enkephalin (leu-ENK: 1 pg/ml)and (f)neurotensin (NT: 10 ng/ml), in high, medium and low tone preparations. The same preparation was used for successive stimuli. Atropine (1 pg/ml) and guanethidine (1 pg/ml) were present throughout.
c o n t r a c t i o n s " (fig. l a ) . P r e p a r a t i o n s having m e d i u m t o n e w e r e t h o s e w h i c h gave significant r e l a x a t i o n s t o b o t h stimuli, f o l l o w e d b y "rebound contractions" which were often similar in m a g n i t u d e t o t h e i n h i b i t o r y responses. In t h e s e preparations during extended periods of stimulation "breakthrough" contractions were often observed f o l l o w i n g t h e initial i n h i b i t o r y response. L o w t o n e p r e p a r a t i o n s were t h o s e in w h i c h little or n o r e l a x a t i o n o c c u r r e d during s t i m u l a t i o n , b u t t h e r e was a large " r e b o u n d c o n t r a c t i o n " u p o n cessation o f t h e stimulus. In all p r e p a r a t i o n s (n = 8) t h e r e s p o n s e to 0 . 1 - - 5 0 p g / m l
A T P m i m i c k e d closely t h e r e s p o n s e to electrical field s t i m u l a t i o n o f n o n - a d r e n e r g i c , i n h i b i t o r y nerves (fig. l b ) . F o r high a n d average t o n e p r e p a r a t i o n s , t h e l a t e n c y in o n s e t o f r e l a x a t i o n , t h e initial r a t e o f relaxat i o n a n d t h e t i m e to r e a c h m a x i m u m are given in t a b l e 1.
3.2. Effects of polypep tides 3.2.1. Substance P In all p r e p a r a t i o n s (n = 5), S u b s t a n c e P ( 0 . 0 1 - - 1 p g / m l ) caused a c o n t r a c t i o n w h i c h was r a p i d in o n s e t a n d m a i n t a i n e d (fig. l c ) .
254
T. COCKS, G. BURNSTOCK b
T h e degree o f c o n t r a c t i o n for a n y p a r t i c u l a r c o n c e n t r a t i o n o f S u b s t a n c e P increased as t h e t o n e decreased.
3.2.2. Sornatostatin S o m a t o s t a t i n ( 0 . 1 - - 1 0 p g / m l ) rarely prod u c e d a response, regardless o f t h e t o n e o f t h e p r e p a r a t i o n (fig. l d ) . H o w e v e r , in 2 p r e p a r a tions ( o u t o f 10) w i t h m e d i u m t o n e , s o m a t o statin (10 p g / m l ) caused a biphasic r e s p o n s e which consisted o f an initial small r e l a x a t i o n , f o l l o w e d b y a t o n i c c o n t r a c t i o n , a n d in o n e low t o n e p r e p a r a t i o n t h e s a m e c o n c e n t r a t i o n of s o m a t o s t a t i n gave a " r e b o u n d c o n t r a c t i o n " (fig. l d ) .
d
f
g
3.2.3. Enkephalin In all p r e p a r a t i o n s {n = 5) l e u - e n k e p h a l i n ( 0 . 1 - - 1 0 p g / m l ) failed t o give a n y d i r e c t r e s p o n s e (see fig. l e ) . As e n k e p h a l i n s h a v e b e e n s h o w n to be p o t e n t i n h i b i t o r s o f electrically s t i m u l a t e d c o n t r a c t i o n o f s m o o t h m u s c l e o f t h e guinea-pig i l e u m ( H u g h e s et al., 1 9 7 5 a ) , p r e s u m a b l y acting b y h y p e r polarization of excitatory neurones within t h e m y e n t e r i c p l e x u s ( N o r t h and Williams, 1976), t h e e f f e c t o f l e u - e n k e p h a l i n on the i n h i b i t o r y r e s p o n s e t o b o t h electrical field s t i m u l a t i o n a n d A T P in t h e guinea-pig t a e n i a coli was e x a m i n e d . In t h e a b o v e c o n c e n t r a t i o n range, l e u - e n k e p h a l i n did n o t a f f e c t t h e i n h i b i t o r y r e s p o n s e t o either low f r e q u e n c y field s t i m u l a t i o n (0.2 m s e c d u r a t i o n pulses delivered at 0 . 5 - - 5 H z a n d s u p r a m a x i m a l voltage) or A T P ( 1 - - 1 0 g g / m l ) . N a l o x o n e (1 p g / m l ) , an a n t a g o n i s t o f e n k e p h a l i n s , also had n o e f f e c t on t h e i n h i b i t o r y r e s p o n s e s t o field s t i m u l a t i o n . 3.2.4. Neurotensin T h e r e s p o n s e t o l o w c o n c e n t r a t i o n s of n e u r o t e n s i n ( 1 - - 1 0 n g / m l ) was always a cont r a c t i o n regardless o f t o n e (fig. l f ) . T h e r e s p o n s e t o higher c o n c e n t r a t i o n s o f neurotensin ( 5 0 - 1 0 0 0 n g / m l ) was a c o n t r a c t i o n in l o w a n d m e d i u m t o n e p r e p a r a t i o n s , b u t in high t o n e p r e p a r a t i o n s (n = 3) t h e c o n t r a c t i o n s h o w e d fade w h i c h d e v e l o p e d i n t o a slow
I
1rain
i
Fig. 2. Responses of a high tone preparation of the guinea-pig taenia coli to (a) 0.001, (b) 0.01, (c) 0.05, (d) 0.1, (e) 0.5 and (f) 1 /lg/ml neurotensin (horizontal bars). Note: the gradual change from a sustained contraction to a delayed, slow relaxation. In (g) the tone has fallen to a lower level and under these conditions neurotensin (1 pg/ml) causes an initial rapid contraction followed by a slow relaxation. A second application of the same dose of neurotensin (1 pg/ml) while the tone is high, now causes a delayed slow relaxation similar to that in (f). Atropine (1 pg/ ml) and guanethidine (1 pg/ml) were present throughout.
i n h i b i t o r y r e s p o n s e (fig. 2c,d), or a slow r e l a x a t i o n with a l a t e n c y in o n s e t o f a b o u t 15 sec and a t i m e to m a x i m u m r e s p o n s e of a b o u t 4 0 - - 5 0 sec (fig. 2e,f a n d t a b l e 1).
3.2.5. Vasoactive intestinal polypeptide (VIP) In high and average t o n e p r e p a r a t i o n s (n = 3), V I P ( 0 . 0 1 - - 1 p g / m l ) caused slow relaxat i o n s ( t i m e t o m a x i m u m 2 0 0 - - 2 5 0 sec) w i t h a l a t e n c y in o n s e t o f a b o u t 60 sec (fig. 3 a n d t a b l e 1). T h e r e s p o n s e s t o electrical field
PURINERGIC AND PEPTIDERGIC EFFECTS ON GUT
VIP HIGH~
TONE V
ATP
NS
255 b o t h intramural nerve stimulation and ATP, and also in the one experi m ent in which somatostatin caused a " r e b o u n d c o n t r a c t i o n " .
I
4. Discussion
MEDIUM~TONE
TONE I ! 5min
Fig. 3. Comparison of the inhibitory responses of the guinea-pig taenia coli to vasoactive intestinal polypeptide (VIP: 0.5//g/ml), ATP (0.5 pg/ml) and intramural nerve stimulation (NS: 0.2 msec duration pulses delivered at 5Hz and supramaximal voltage (50 V) for 30 sec) in high, medium and low tone preparations. Atropine (1 pg/ml) and guanethidine (1 pg/ml) were present throughout.
stimulation and ATP compared with that to VIP are shown in fig. 3. Also, in the above c o n c e n tr atio n range, VIP did n o t affect the responses to either electrical field stimulation or ATP.
3.3. Effects o f indemethacin Incubation with indomethacin (3.4--34 pg/ ml) for 60 min did n o t affect the direct contractile responses to the polypeptides tested whereas it blocked t he " r e b o u n d contract i o n s " following the inhibitory responses to
The response of the guinea-pig taenia coli to ATP has been f o u n d to mimic closely the inhibitory response to non-adrenergic, noncholinergic "puri nergi c" nerve stimulation, confirming the earlier report of Burnstock et al. (1970). Both responses had a latency of about i sec and a relaxation that reached maxim u m in 12 to 17 sec; in preparations which had low t o medium tone, both inhibitory responses were followed by a " r e b o u n d cont r a c t i o n " {see Burnstock et al., 1963; Campbell, 1966; Bennett, 1966; this paper). Further, the ionic mechanisms underlying muscle membrane hyperpolarization following application of ATP have been shown to be similar to those associated with the inhibitory junction potentials elicited in response to stimulation of non-adrenergic, non-cholinergic nerves (Tomita and Watanabe, 1973; Jager, 1974). The low frequencies of stimulation used (0.5--5 Hz) selectively activate purinergic nerves and n o t adrenergic nerves. Furthermore, guanethidine, in the concentrations used, completely abolishes the response to adrenergic nerve stimulation {Burnstock et al., 1966). None of the polypeptides which have been shown to be localised in enteric nerves with immunofluorescence m et hods (see Introduction for references) produced responses which resembled those to non-adrenergic, noncholinergic nerve stimulation; Substance P was excitatory, enkephalin and somatostatin were inactive, while VIP produced a very slow relaxation reaching a m a x i m u m in 200--250 sec after a latency of about 60 sec. Low concentrations of neurotensin produced contraction of the taenia coli; higher concentrations in high t one preparations p r o d u c e d a relaxation, but this relaxation was slow (time to
256 maximum 40--50 sec) and did not mimic that produced by stimulation of intramural inhibitory nerves. Thus, on the basis of the similarity of postjunctional action, ATP is the most likely of the substances tested to be the transmitter released from non-adrenergic, non-cholinergic inhibitory nerves supplying the smooth muscle of the taenia coli. This conclusion is supported by evidence that ATP satisfies four other criteria required to establish a neurotransmitter, namely: synthesis and storage in nerve terminals; release during nerve stimulation; an inactivation mechanism involving both enzymes and high affinity re-uptake; and drugs that produce parallel effects on the responses to both nerve stimulation and exogenous transmitter (see Burnstock, 1972, 1975, 1979). Since Substance P, enkephalin, somatostatin and VIP are clearly not the transmitters released from the non-adrenergic, non-cholinergic efferent nerves supplying the intestinal musculature, other possible roles for these intraneuronal polypeptides should be considered. It is unlikely that the contractile responses to the peptides tested were produced by excitatory prostaglandins which masked any inhibitory effects. Such a role for prostaglandins has been proposed for the contractile response of the anococcygeus to exogenous ATP (Burnstock et al., 1978). However, in the present study no inhibitory responses to peptides were unmasked following inhibition of prostaglandin synthesis (see also Bury and Mashford, 1977). There is strong evidence that Substance P is a neurotransmitter in primary afferent fibres in the spinal cord and skin (see Lembeck, 1953; HSkfelt et al., 1977b; Otsuka and Konishi, 1977) so that is is possible that it is also localized in sensory fibres in the gut (see also HSkfelt et al., 1977b). However, Substance P is known to have a variety of peripheral actions including contraction of intestinal longitudinal muscle (Euler and Gaddum, 1931; Pernow, 1960; Franco and Costa, 1978) and dilatation of blood vessels
T. COCKS, G. BURNSTOCK (Euler and Gaddum, 1931). The endogenous opiate ligands, enkephalins, have potent inhibitory actions on electricallystimulated contractions of both the mouse vas deferens and the guinea-pig ileum (Hughes et al., 1975a,b). Also, both enkephalins and morphine have been shown to inhibit neuronal firing in Auerbach's plexus (Dingledine and Goldstein, 1976; North and Williams, 1976) by causing membrane hyperpolarization (North and Tonini, 1976). Therefore, it seems likely that enkephalins are released from interneurones involved in modulation of nervemediated excitation of the intestine. Neither enkephalin nor naloxone, a potent opiate antagonist (see Klee, 1977) had any effect on the inhibitory response to non-adrenergic intramural nerve stimulation in the guinea-pig taenia coli. It has also been suggested that neurones which display somatostatin immunoreactivity are interneurones within the enteric nervous system (Costa et al., 1977). This is supported by the finding that somatostatin inhibits the release of acetylcholine from nerves within Auerbach's plexus of the small iptestine (Guillemin, 1976) and no somatostatin immunoreactive fibres were found in either the longitudinal or circular muscle of the ileum (Costa et al., 1977). Intrinsic VIP-containing neurones found throughout the gut may also be interneurones, since they have been shown to innervate nerve cells in both Meissner's and Auerbach's plexus (Fuxe et al., 1977). VIP-positive nerve fibres have been observed within the circular muscle layer and muscularis mucosae of the rat stomach, duodenum and colon, but these appear to be en route to the mucosal epithelium (Fuxe et al. 1977). The role of the very slow inhibitory response of the taenia coli to VIP is not clear, since it does not mimic the nerve-mediated response. Similar slow relaxations to VIP have been reported in the gall bladder, trachea and stomach (Piper et al., 1970) and canine small intestine (Kachelhoffer et al., 1976). Finally, the possibility should be considered that polypeptides are stored together with
PURINERGIC AND PEPTIDERGIC EFFECTS ON GUT neurotransmitters in e n t e r i c n e r v e s . B u r n stock (1976) has recently discussed the concept that some nerves contain more than one transmitter. H S k f e l t e t al. ( 1 9 7 7 a ) h a v e s h o w n t h a t s o m e a d r e n e r g i c n e u r o n e s in peripheral sympathetic ganglia contain somatostatin together with noradrenaline and earlier Euler (1963) showed that splenic n e r v e s c o n t a i n e d v e s i c u l a r S u b s t a n c e P. F u r ther, up to nine different types of enteric nerves have been identified on an ultrastruct u r a l b a s i s in t h e g a s t r o i n t e s t i n a l t r a c t , s e v e r a l of them containing combinations of different vesicle types (Gabella, 1972; Cook and Burnstock, 1976).
Acknowledgements We are grateful to Dr. S. Leeman, Harvard Medical School, Boston, and Professor V. Mutt, Karolinska Institute, Stockholm, for the gifts of neurotensin and purified porcine vasoactive intestinal polypeptide respectively.
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