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The release of met-enkephalin from the guinea-pig ileum at rest and during peristaltic activity
Life Sciences, Vol. 33, Sup. I, 1983, pp. 465-468 Printed in the U.S.A.
Pergamon Press
THE RELEASE OF MET-ENKEPHALIN FROM THE GUINEA-PIG ILEUM AT REST AND DURING PERISTALTIC ACTIVITY S.J. Clark and T.W. Smith
D e p a r t m e n t of Pharmacology, Wellcome Research Laboratories, Langley Court, Beckenham, Kent, England. (Received in final form June 26, 1983)
Summary Segments of guinea-pig ileum maintained at an intraluminal pressure of Ocm H?0 released methionine-enkephalin-like material (ME) into the bathing fluid. Elevation of intraluminal pressure to 5 cm H20 induced peristalsis and reduced ME release both in the absence and presence of naloxone (0.27 llM). Following prolonged distension, tissues developed intermittent peristaltic activity. ME release during intermittency was consistently and significantly (p < 0.05) greater than that observed from tissues exhibiting continuous peristalsis. These results suggest that ME release is associated with nonpropulsive activity in the guinea-pig ileum.
The well-established e f f e c t s of enkephalin on e l e c t r i c a l l y - and distension-induced contractions of guinea-pig isolated ileum are thought to be mediated via presynaptic inhibition of cholinergic a c t i v i t y (1). Endogenous opioid peptides located in the intrinsic plexi (2) may t h e r e f o r e function as inhibitory n e u r o t r a n s m i t t e r s or neuromodulators of gastrointestinal motility. In support of this notion, naloxone has been shown to increase the output of acetylcholine from e l e c t r i c a l l y stimulated m y e n t e r i c plexus-longitudinal muscle (MPLM) preparations (3). F u r t h e r m o r e , opiate antagonists f a c i l i t a t e p e r i s t a l t i c a c t i v i t y in vitro in a variety of species (4). In general, however, e f f e c t s of naloxone on the reflex are only observed following prolonged distension and the development of i n t e r m i t t e n t a c t i v i t y i.e. bursts of contractions alternating with p e r i s t a l s i s - f r e e intervals (5). These observations t h e r e f o r e suggest that activation of endogenous opioid mechanisms is related to an i n t e r m i t t e n t , but not continuous p a t t e r n of peristalsis. It has recently been demonstrated that the release of methionine-enkephalin-like m a t e r i a l (ME) from guinea-pig ileum is significantly reduced during periods of peristalsis (6). These studies have now been extended to investigate the release of ME in the presence of naloxone. In addition, ME release during continuous peristalsis and i n t e r m i t t e n t a c t i v i t y have been compared. Materials and Methods Whole segments of guinea-pig ileum (Scm) were suspended under a 2g load in a 3m1 organ bath. Each segment was continuously washed (3.0 - 4.5 ml/min) with warm (33°C) Krebs solution containing bovine serum albumin (0.025% w/v). The release of ME from resting and distended tissues was studied using techniques described elsewhere (6). Briefly, tissues were equilibrated (50 min) at a resting intraluminal pressure of Ocm H2O prior to the induction of peristalsis by radial distension (intraluminal pressure = 5cm H?0). A f t e r 30 rain distension, intraluminal pressure was returned to Ocm H20. In control- experiments, resting conditions were maintained throughout. Samples ~30-40 ml) of effluent were collected on ice into hydrochloric acid (final concentration 8.1N) according to the schedule shown in Fig. 1. In other experiments, ME release was determined in the presence of naloxone hydrochloride (0.27 llM). 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
466
Enkephalin Release From Guinea Pig Ileum
Vol. 33, Sup. I, 1983
The effects of prolonged distension on ME release were also investigated by collecting effluent samples (30ml) during consecutive 10 min periods for 80-90 rain. ME in each sample was isolated by adsorption onto purified Amberlite XAD-2 (250rag), eluted with methanol (2ml) and evaporated under reduced pressure. The dried eluates were redissolved in 0.2 ml 50 mM Tris-HC1 buffer (pH 7.4 at 25°C) and ME content determined by radioimmunoassay.
5 min .................... ::
5cm[
...........................
.................... :::u,,,r
........................
J
. . . .
A
B
C
D
FIG. i
Schedule for collection of effluent. Samples (50-/40 ml) of effluent were collected during the periods A-D (hatched areas). Peristalsis (indicated by horizontal bar) was induced by elevating intraluminal pressure to 5cm H20. Control tissues were maintained at Ocm H20. The upper traces are continuous records of longitudinal muscle tension and intraluminal pressure. Results
Control segments (group 1), maintained at an intraluminal pressure of Ocm H?0, released ME into the bathing fluid (Table i). The ME content in successive effluent samples progressively decreased, but only the final sample ([D; 0.51 -+ 0.25 pmol/g tissue) contained significantly less ME than the initial sample (A; 2.48 + 1.15 pmol/g; p < 0.05). During periods (B and C) of distension (5cm H20; group 2), ME release was consistently and significantly (p < 0.05) reduced in compamson to the initial resting period (A; 3.5 + 1.4 pmol/g). Qn resetting intraluminal pressure to 0cm H?0, peristalsis was terminated and detectable levels of ME reappeared (D; 0.37 ± 0.12 pmbl/g), albeit at a significantly reduced level (p < 0.05). A similar pattern of ME release was obtained from segments incubated with naloxone (0.27 I~M). Although initial resting levels (A; 1.59 + 0.52 pmol/g) tended to be lower in this group of experiments, elevation of intraluminal pressure decreased ME release from each segment. When resting conditions were resumed, ME release (D; 1.i3 + 0./47 pmol/g) was considerably greater than that of the preceding period of distension (C; 0.16 + 0.15 pmol/g). The overall decrease in resting release, however, was less marked than that which occurred in the absence of naloxone. Segments (n=3) of ileum subjected to prolonged distension in Krebs solution developed intermittent activity after 37 + 1/4 rain. In these experiments, effluent samples (3) collected in the initial 10 min period of distension (corresponding to period A) contained 0.61 +_ 0.34 pmol of IVlE/g (Fig. 2). This level was similar to the mean ME content (0.53 + 0.21 pmol/g) of all samples (13) collected during continuous peristaltic activity. The onset of intermittency, however, was accompanied by a significant (p < 0.05) increase in lvlE release (1.48 + 0.41 proof/g; 13 samples) from these tissues into the bathing fluid. Naloxone (0.82 ~tM), administered at the end of each experiment, increased the duration, but not magnitude, of reflex activity.
Vol. 33, Sup. I, 1983
Enkephalin Release From Guinea Pig Ileum
467
TABLE I ME Release from Resting and Distended Segments of Guinea-Pig Ileum
Group
Treatment
n
COLLECTION PERIOD A
B
C
D
1.48+0.76
1.31+0.71
0.5[+_0.23"
i
Krebs Alone
4
2.48+-1.15
2
Krebs Alone
4
3.3 +_i.4
0.41+0.24" (54+7)
0* (30+9)
0.37+-0.12"
3
Krebs + Naloxone (0.271~M)
3
1.59+0.52
1.11+0.73 (47+_3)
0.i6+0.15 (42+4)
1.13+0.47
Group i
A-D shows the release of ME, in pmol/g tissue, when resting conditions (intraluminal pressure 0cm H20) were maintained throughout.
Groups 2 and }
Release of ME in the absence and presence of naloxone (0.2711M) respectively. A and D show the release of ME from tissues under resting conditions. B and C show ME release when intraluminal pressure was raised to 5cm H20. All results are expressed as the mean + standard error. * p < 0.05 (Significant change from period A, using the matched-pair Wi]coxon test). Figures in parentheses are the number of peristaltic contractions during the periods of distension.
MEpmolg-l(mean±s.e) Resting mContinuousactivity ~lntermittent activity
4"0t
3.O[
IIn=3
~=8
II
n=13 n=13 FIG. 2
LEFT HISTOGRAM: The closed column represents ME in the initial sample of effluent (corresponding to A) collected from tissues (n=3) exhibiting continuous peristalsis. ME released by resting tissues (0cm H20; n=8) during this period is indicated by the open column. RIGHT HISTOGRAM: ME release during prolonged (80-100 min) elevation of intraluminal pressure (Scm H20). The closed column shows the ME content of effluent samples (n=i3) collected from 3 tissues exhibiting continuous reflex activity. The hatched column shows ME in samples (n=13) of effluent following the development of intermittency. * p < 0.05 (Mann-Whitney test for grouped data).
468
Enkephalin Release From Guinea Pig Ileum
Vol. 33, Sup. I, 1983
Discussion An essential criterion for establishing a substance as a n e u r o t r a n s m i t t e r agent is the demonstration of its release under physiological conditions. In this respect, both direct (7) and indirect evidence (8) for the release of endogenous opioids from e l e c t r i c a l l y stimulated intestinal preparations have been reported. Although these studies suggest modulation of c o n t r a c t i l e a ct i v i t y , the present experiments indicate that ME release is a t t e n u a t e d during continuous peristalsis in the presence and absence of naloxone (0.27 I~M). Similarly, radial distension of guinea-pig ileum has been shown to reduce the release of immunoreactive dynorphin (9). Elevation of intraluminal pressure t h e r e f o r e appears to remove the inhibitory influence of endogenous opioids, and may account for the fact that naloxone does not i n t er f er e with normal peristaltic contractions (5). It is of interest that initial resting levels of ME in the presence of naloxone tended to be less than those observed in Krebs alone. Hence, naloxoneinduced decreases in the intraluminal pressure required to elicit peristalsis in vitro and in situ (10) could be due to a local reduction in resting enkephalin release. ME release in the final resting period, however, appeared g r e a t e r in those tissues incubated with naloxone. Whether this r e f l e c t s prolonged ME availability under these conditions requires further investigation. A f t e r prolonged distension, tissues develop i n t e r m i t t e n c y . Exposure to naloxone during a peristalsis-free interval restores normal reflex activity (5). In the present experiments, ME release following the development of i n t e r m i t t e n c y was significantly g reat er than that observed from tissues exhibiting continuous peristalsis. Thus ME release was associated with non-propulsive activity both in distended and non-distended tissues. In summary, the present demonstrations of ME release have provided further evidence for an inhibitory role of endogenous opioids in the guinea-pig ileum. Rel ease data from e l e c t r i c a l l y stimulated MPLM preparations may not be r e p r e s e n t a t i v e of the complex, integrated reflex activity of the intestine. The physiological phenomenon of peristalsis is t h e r e f o r e to be recommended for future studies involving the release of gastrointestinal peptides, and of their actions and interactions.
Acknowledgement The gift of methionine-enkephalin antiserum from Dr. T. Ashwood (University of Bristol) is gratefully acknowledged. References 1. 2. 3. 4. 5. 6. 7. 8. 9.
A.A. WATERFIELD, R.W.J. SMOKCUM, 3. HUGHES, H.W. KOSTERLITZ and G. HENDERSON, Eur. g. Pharmaeol. 43, 107-116 (1977). J. HUGHES, H.W. KOSTERLITZ and T.W. SMITH, Brit..3. Pharmacol. 61, 639-647 (1977). A.A. WATERFIELD and H.W. KOSTERLITZ, Life Sci. 16, 1787-1792 (1975). W. KROMER, W. PRETZLAFF and E. SCHEIBLHUBER, Endoqenous and Exogenous Opiate Agonists and Antagonists, pp 337-340, Pergamon Press, New York (1980). J.M. VAN NUETEN, P.A.J. JANSSEN and J. FONTAINE, Life Sci. 18, 803-810 (1976). S.J. CLARK and T.W. SMITH, Eur. 3. Pharmacol. 70, 421-424 (1981). R. SCHULZ, M. WUSTER, R. S[MANTOV, S. SNYDER and A. HERZ, Eur. J. Pharmacol. 41, 347-548 (1977). M.M. PUIG 9 P. GASCON, G.L. CRAVISO and 3.M. MUSACCHIO, Science, 195, 419-420 (1976). W. KROMER, V. HOLLT,~H. SCHMIDT and A. HERZ, Neurosci. Lett. 25, 53-56 (1981).
10.
S.J. CLARK and T.W. SMITH, Brit. 3. Pharmacol. 74, 953P-954P (1981).
Pergamon Press
THE RELEASE OF MET-ENKEPHALIN FROM THE GUINEA-PIG ILEUM AT REST AND DURING PERISTALTIC ACTIVITY S.J. Clark and T.W. Smith
D e p a r t m e n t of Pharmacology, Wellcome Research Laboratories, Langley Court, Beckenham, Kent, England. (Received in final form June 26, 1983)
Summary Segments of guinea-pig ileum maintained at an intraluminal pressure of Ocm H?0 released methionine-enkephalin-like material (ME) into the bathing fluid. Elevation of intraluminal pressure to 5 cm H20 induced peristalsis and reduced ME release both in the absence and presence of naloxone (0.27 llM). Following prolonged distension, tissues developed intermittent peristaltic activity. ME release during intermittency was consistently and significantly (p < 0.05) greater than that observed from tissues exhibiting continuous peristalsis. These results suggest that ME release is associated with nonpropulsive activity in the guinea-pig ileum.
The well-established e f f e c t s of enkephalin on e l e c t r i c a l l y - and distension-induced contractions of guinea-pig isolated ileum are thought to be mediated via presynaptic inhibition of cholinergic a c t i v i t y (1). Endogenous opioid peptides located in the intrinsic plexi (2) may t h e r e f o r e function as inhibitory n e u r o t r a n s m i t t e r s or neuromodulators of gastrointestinal motility. In support of this notion, naloxone has been shown to increase the output of acetylcholine from e l e c t r i c a l l y stimulated m y e n t e r i c plexus-longitudinal muscle (MPLM) preparations (3). F u r t h e r m o r e , opiate antagonists f a c i l i t a t e p e r i s t a l t i c a c t i v i t y in vitro in a variety of species (4). In general, however, e f f e c t s of naloxone on the reflex are only observed following prolonged distension and the development of i n t e r m i t t e n t a c t i v i t y i.e. bursts of contractions alternating with p e r i s t a l s i s - f r e e intervals (5). These observations t h e r e f o r e suggest that activation of endogenous opioid mechanisms is related to an i n t e r m i t t e n t , but not continuous p a t t e r n of peristalsis. It has recently been demonstrated that the release of methionine-enkephalin-like m a t e r i a l (ME) from guinea-pig ileum is significantly reduced during periods of peristalsis (6). These studies have now been extended to investigate the release of ME in the presence of naloxone. In addition, ME release during continuous peristalsis and i n t e r m i t t e n t a c t i v i t y have been compared. Materials and Methods Whole segments of guinea-pig ileum (Scm) were suspended under a 2g load in a 3m1 organ bath. Each segment was continuously washed (3.0 - 4.5 ml/min) with warm (33°C) Krebs solution containing bovine serum albumin (0.025% w/v). The release of ME from resting and distended tissues was studied using techniques described elsewhere (6). Briefly, tissues were equilibrated (50 min) at a resting intraluminal pressure of Ocm H2O prior to the induction of peristalsis by radial distension (intraluminal pressure = 5cm H?0). A f t e r 30 rain distension, intraluminal pressure was returned to Ocm H20. In control- experiments, resting conditions were maintained throughout. Samples ~30-40 ml) of effluent were collected on ice into hydrochloric acid (final concentration 8.1N) according to the schedule shown in Fig. 1. In other experiments, ME release was determined in the presence of naloxone hydrochloride (0.27 llM). 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
466
Enkephalin Release From Guinea Pig Ileum
Vol. 33, Sup. I, 1983
The effects of prolonged distension on ME release were also investigated by collecting effluent samples (30ml) during consecutive 10 min periods for 80-90 rain. ME in each sample was isolated by adsorption onto purified Amberlite XAD-2 (250rag), eluted with methanol (2ml) and evaporated under reduced pressure. The dried eluates were redissolved in 0.2 ml 50 mM Tris-HC1 buffer (pH 7.4 at 25°C) and ME content determined by radioimmunoassay.
5 min .................... ::
5cm[
...........................
.................... :::u,,,r
........................
J
. . . .
A
B
C
D
FIG. i
Schedule for collection of effluent. Samples (50-/40 ml) of effluent were collected during the periods A-D (hatched areas). Peristalsis (indicated by horizontal bar) was induced by elevating intraluminal pressure to 5cm H20. Control tissues were maintained at Ocm H20. The upper traces are continuous records of longitudinal muscle tension and intraluminal pressure. Results
Control segments (group 1), maintained at an intraluminal pressure of Ocm H?0, released ME into the bathing fluid (Table i). The ME content in successive effluent samples progressively decreased, but only the final sample ([D; 0.51 -+ 0.25 pmol/g tissue) contained significantly less ME than the initial sample (A; 2.48 + 1.15 pmol/g; p < 0.05). During periods (B and C) of distension (5cm H20; group 2), ME release was consistently and significantly (p < 0.05) reduced in compamson to the initial resting period (A; 3.5 + 1.4 pmol/g). Qn resetting intraluminal pressure to 0cm H?0, peristalsis was terminated and detectable levels of ME reappeared (D; 0.37 ± 0.12 pmbl/g), albeit at a significantly reduced level (p < 0.05). A similar pattern of ME release was obtained from segments incubated with naloxone (0.27 I~M). Although initial resting levels (A; 1.59 + 0.52 pmol/g) tended to be lower in this group of experiments, elevation of intraluminal pressure decreased ME release from each segment. When resting conditions were resumed, ME release (D; 1.i3 + 0./47 pmol/g) was considerably greater than that of the preceding period of distension (C; 0.16 + 0.15 pmol/g). The overall decrease in resting release, however, was less marked than that which occurred in the absence of naloxone. Segments (n=3) of ileum subjected to prolonged distension in Krebs solution developed intermittent activity after 37 + 1/4 rain. In these experiments, effluent samples (3) collected in the initial 10 min period of distension (corresponding to period A) contained 0.61 +_ 0.34 pmol of IVlE/g (Fig. 2). This level was similar to the mean ME content (0.53 + 0.21 pmol/g) of all samples (13) collected during continuous peristaltic activity. The onset of intermittency, however, was accompanied by a significant (p < 0.05) increase in lvlE release (1.48 + 0.41 proof/g; 13 samples) from these tissues into the bathing fluid. Naloxone (0.82 ~tM), administered at the end of each experiment, increased the duration, but not magnitude, of reflex activity.
Vol. 33, Sup. I, 1983
Enkephalin Release From Guinea Pig Ileum
467
TABLE I ME Release from Resting and Distended Segments of Guinea-Pig Ileum
Group
Treatment
n
COLLECTION PERIOD A
B
C
D
1.48+0.76
1.31+0.71
0.5[+_0.23"
i
Krebs Alone
4
2.48+-1.15
2
Krebs Alone
4
3.3 +_i.4
0.41+0.24" (54+7)
0* (30+9)
0.37+-0.12"
3
Krebs + Naloxone (0.271~M)
3
1.59+0.52
1.11+0.73 (47+_3)
0.i6+0.15 (42+4)
1.13+0.47
Group i
A-D shows the release of ME, in pmol/g tissue, when resting conditions (intraluminal pressure 0cm H20) were maintained throughout.
Groups 2 and }
Release of ME in the absence and presence of naloxone (0.2711M) respectively. A and D show the release of ME from tissues under resting conditions. B and C show ME release when intraluminal pressure was raised to 5cm H20. All results are expressed as the mean + standard error. * p < 0.05 (Significant change from period A, using the matched-pair Wi]coxon test). Figures in parentheses are the number of peristaltic contractions during the periods of distension.
MEpmolg-l(mean±s.e) Resting mContinuousactivity ~lntermittent activity
4"0t
3.O[
IIn=3
~=8
II
n=13 n=13 FIG. 2
LEFT HISTOGRAM: The closed column represents ME in the initial sample of effluent (corresponding to A) collected from tissues (n=3) exhibiting continuous peristalsis. ME released by resting tissues (0cm H20; n=8) during this period is indicated by the open column. RIGHT HISTOGRAM: ME release during prolonged (80-100 min) elevation of intraluminal pressure (Scm H20). The closed column shows the ME content of effluent samples (n=i3) collected from 3 tissues exhibiting continuous reflex activity. The hatched column shows ME in samples (n=13) of effluent following the development of intermittency. * p < 0.05 (Mann-Whitney test for grouped data).
468
Enkephalin Release From Guinea Pig Ileum
Vol. 33, Sup. I, 1983
Discussion An essential criterion for establishing a substance as a n e u r o t r a n s m i t t e r agent is the demonstration of its release under physiological conditions. In this respect, both direct (7) and indirect evidence (8) for the release of endogenous opioids from e l e c t r i c a l l y stimulated intestinal preparations have been reported. Although these studies suggest modulation of c o n t r a c t i l e a ct i v i t y , the present experiments indicate that ME release is a t t e n u a t e d during continuous peristalsis in the presence and absence of naloxone (0.27 I~M). Similarly, radial distension of guinea-pig ileum has been shown to reduce the release of immunoreactive dynorphin (9). Elevation of intraluminal pressure t h e r e f o r e appears to remove the inhibitory influence of endogenous opioids, and may account for the fact that naloxone does not i n t er f er e with normal peristaltic contractions (5). It is of interest that initial resting levels of ME in the presence of naloxone tended to be less than those observed in Krebs alone. Hence, naloxoneinduced decreases in the intraluminal pressure required to elicit peristalsis in vitro and in situ (10) could be due to a local reduction in resting enkephalin release. ME release in the final resting period, however, appeared g r e a t e r in those tissues incubated with naloxone. Whether this r e f l e c t s prolonged ME availability under these conditions requires further investigation. A f t e r prolonged distension, tissues develop i n t e r m i t t e n c y . Exposure to naloxone during a peristalsis-free interval restores normal reflex activity (5). In the present experiments, ME release following the development of i n t e r m i t t e n c y was significantly g reat er than that observed from tissues exhibiting continuous peristalsis. Thus ME release was associated with non-propulsive activity both in distended and non-distended tissues. In summary, the present demonstrations of ME release have provided further evidence for an inhibitory role of endogenous opioids in the guinea-pig ileum. Rel ease data from e l e c t r i c a l l y stimulated MPLM preparations may not be r e p r e s e n t a t i v e of the complex, integrated reflex activity of the intestine. The physiological phenomenon of peristalsis is t h e r e f o r e to be recommended for future studies involving the release of gastrointestinal peptides, and of their actions and interactions.
Acknowledgement The gift of methionine-enkephalin antiserum from Dr. T. Ashwood (University of Bristol) is gratefully acknowledged. References 1. 2. 3. 4. 5. 6. 7. 8. 9.
A.A. WATERFIELD, R.W.J. SMOKCUM, 3. HUGHES, H.W. KOSTERLITZ and G. HENDERSON, Eur. g. Pharmaeol. 43, 107-116 (1977). J. HUGHES, H.W. KOSTERLITZ and T.W. SMITH, Brit..3. Pharmacol. 61, 639-647 (1977). A.A. WATERFIELD and H.W. KOSTERLITZ, Life Sci. 16, 1787-1792 (1975). W. KROMER, W. PRETZLAFF and E. SCHEIBLHUBER, Endoqenous and Exogenous Opiate Agonists and Antagonists, pp 337-340, Pergamon Press, New York (1980). J.M. VAN NUETEN, P.A.J. JANSSEN and J. FONTAINE, Life Sci. 18, 803-810 (1976). S.J. CLARK and T.W. SMITH, Eur. 3. Pharmacol. 70, 421-424 (1981). R. SCHULZ, M. WUSTER, R. S[MANTOV, S. SNYDER and A. HERZ, Eur. J. Pharmacol. 41, 347-548 (1977). M.M. PUIG 9 P. GASCON, G.L. CRAVISO and 3.M. MUSACCHIO, Science, 195, 419-420 (1976). W. KROMER, V. HOLLT,~H. SCHMIDT and A. HERZ, Neurosci. Lett. 25, 53-56 (1981).
10.
S.J. CLARK and T.W. SMITH, Brit. 3. Pharmacol. 74, 953P-954P (1981).