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Comparative effects of opiate agonists on proximal and distal colonic motility in dogs
European Journal of Pharmacology, 110 (1985) 329-334 Elsevier
329
C O M P A R A T I V E EFFECTS OF O P I A T E A G O N I S T S ON P R O X I M A L AND D I S T A L C O L O N I C M O T I L I T Y IN D O G S THIERRY BARDON and YVES RUCKEBUSCH * Department of Physiology, National Veterinary School 31076 Toulouse, France
Received 5 October 1984, revised MS received 18 December 1984, accepted 15 January 1985
Th. BARDON and Y. RUCKEBUSCH, Comparatiue effects of opiate agonists on proximal and distal colonic motility in dogs, European J. Pharmacol. 110 (1985) 329-334. The colonic motility index was measured in dogs, by using strain-gauge transducers, before and after administration of opiate agonists. Fentanyl, a /~-compound, ethylketazocine (EKC), a r-compound, and [D-Ala2,LeuS]enkephalin (DADLE), a 8-agonist, were administered by intravenous (i.v.) or intrathecal (i.t.) routes. Fentanyl (2-10 nmol. kg-1 i.v.) induced a dose-related period of hyperactivity characterized by an increase in both tone and frequency of contractions in the distal portion of the colon, whereas a period of hypomotility following a short-lived period of increased activity was elicited on the proximal colon. Fentanyl (0.1, 0.2 nmol. kg- 1 i.t.) had inhibitory effects on both proximal and distal colon. Small doses of EKC (2-4 nmol- kg -~) administered i.t. or larger doses (20-40 nmol - kg-a i.v.) inhibited colonic motility for a dose-related duration, the effects of EKC being more marked on the distal colon than on the proximal colon. The administration of DADLE, 1-2 nmol. kg- 1 i.t., and 40-20 nmol- kg 1 i.v., inhibited and stimulated the colonic contractions, respectively, in both proximal and distal colon. The results demonstrated that the colonic opiate-mediated responses may vary according to the route of administration, the portion of the colon studied and the opioid agonist used. Opiate agonists when administered centrally show a tendency to modulate colonic activity in a similar way whatever the type of agonist. Opiate agonists
Colonic motility
Sites of action
1. I n t r o d u c t i o n
The existence of widespread, multiple types of opioid receptors has suggested differences in the role of local, peripheral and central components of opiate modulation of gastrointestinal motility. The increased transit time induced in rats by morphine given intraperitoneally (i.p.) or intravenously (i.v.) seems to be primarily gut-mediated (Tavani et al., 1980; Bianchi et al., 1983), although small doses of morphine administered intracerebroventricularly (i.c.v.) were also effective in altering small intestinal motility (Galligan and Burks, 1983). The opioid peptides which modify small bowel motor function * To whom all correspondence should be addressed: Service Physiologie, Ecole Nationale V6t6rinaire, 23 chemin des Capelles, 31076 Toulouse C6dex, France 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
Dog
may act either by a central mechanism (Galligan and Burks, 1982) or via peripheral binding sites as evidenced by in vitro (Burks et al., 1982) and in vivo studies (Burks et al., 1984). Moreover, the spinal cord, which is a site of opioid effects on intestinal transit in the mouse (Porreca and Burks, 1983) is not involved in the rat (Vaught et al., 1983). For the latter species, the s.c. but not the i.c.v, administration of the x-type opioid agonist, ethylketazocine (EKC), delayed intestinal transit (Porreca et al., 1983). A plurality of sites of action and species variations for opioid agonists such as demonstrated for small intestine might also exist for motor function of the large bowel. Colonic motility has been stimulated in the dog by morphine (Adler and Ivy, 1940), N-allyl-normorphine (Bu6no et al., 1981) and an enkephalin analogue (Bickel, 1983), but
330 little is known about the characteristics of the opioid-induced colonic motor responses. The indirect demonstration of the involvement of an endogenous opioid system in human constipation and its relief by naloxone treatment (Kreek et al., 1983) suggests similar sites of action between endogenous and exogenous opioid agonists on large bowel motility. Recent studies suggest differences in the response of the proximal and distal portions of the rabbit colon to opioid agonists (Pairet and Ruckebusch, 1984). In addition, 6- and /~-type opioid agonists, which have similar effects on the small bowel when administered i.c.v, in fed rats, have opposite motor effects on the large bowel following an intrathecal (i.t.) injection (Ruckebusch et al., 1984). The aim of these experiments was to compare the motor effects of different opioid agonists, administered either centrally or peripherally, on two distinct parts of the colon of the dog.
2. Materials and methods 2.1. Animal preparation Six mongrel dogs (15-18 kg) were surgically equipped with 2 curved strain-gauge force transducers (1.5 x 1.5 cm) under halothane anesthesia. The gauges, calibrated before implantation, were fixed transversally on the proximal colon, 5 cm distal to the ileocecal junction (ICJ), and on the distal colon, 10 cm proximal to the rectal ampulla. The free ends of the strain gauge wires were directed s.c. along the flank to emerge between the scapulas. A silastic catheter (0.1 mm inner diam.) fitted with distilled water was inserted between the fourth and fifth lumbar vertebrae into the subarachnoid space using a needle as a trocar and anchored so that the tip remained at the level of the twelfth thoracic vertebra.
2.2. Recordings After a 10 day recovery period, each dog was placed in a metabolic cage to which it was adapted and given free access to water. All dogs were fed canned food (600 g / d a y , 4% N D F - non digestible fiber) once daily at 17:00 h. Mechanical activity was recorded from the transducers for 20 con-
secutive hours by connecting each gauge to a Wheatstone bridge amplifier (VT 2100. Vishay, France) and a potentiometric recorder. Mechanical changes were evaluated by measuring the area between the baseline and the contractile curves, The product of the amplitude expressed in grams and the time in minutes was continuously stored and analyzed using an Apple lie microcomputer as a digital integrator and data logging system after A / D conversion (Latour and Ferr6, 1985). The developed printing program indicated consecutive values every 5, 30 and 60 rain as motility indexes.
2.3. Drugs The following opioid agonists were administered in random fashion at 8:00 h with at least a 3-day interval between evaluations in each dog: fentanyl, as a /~-agonist (courtesy of Janssen Pharmaceutica, Beerse, Belgium) at the dosage of 2, 4, 10 n m o l . k g 1 i.v. and 0.1, 0.2 n m o l . k g i i.t.. diluted in sterilized saline (5 ml) or distilled water (0.5 ml), respectively; ethylketazocine (EKC) as a ~-agonist (courtesy of Sterling-Winthrop Research Institute, Rensselaer, NY, U.S.A.) (20-40 n m o l . k g i i.v. and 2-4 n m o l . k g -t i.t.): [DAla2,D-LeuS]enkephalin (DADLE) as a 6-agonist (purchased from Sigma-Interchim, Montluqon, France) (20-40 nmol • kg 1 i.v. and 1-2 nmol - kg 1 i.t.). Four measurements of colonic motility were made on the proximal colon and in each dog for each agent administered 10-15 rain after the occurrence of a phase of contraction. Saline and water used as placebo had no effect on colonic motility after i.v. or i.t. administration, respectively. The agonist at the dosage used i.t. was also administered i.v. Values of motility indexes were expressed as mean percentage of control activity and were compared by using the non-parametric Wilcoxon test.
3. Results 3.1. Normal colonic motor profile Motor activity of the colon obtained from the most proximal strain gauge was characterized by
331 TABLE 1 Variations of the motility index (MI) a following the administration of opioid agonists in 6 dogs. Expressed as a mean percentage of control activity (4 measurements for each dosage) Opioid agonists
Route
Fentanyl (/~)
i.v.
Fentanyl (~)
i.t.
EKC (K)
i.v.
EKC (K)
i.t.
DADLE (8)
i.v.
DADLE (8)
i.t.
Doses
MI variations (%)
(nmol • kg- 1)
Proximal colon
Distal colon
2
- 63
+ 22
4
-87
+44
10 0.1 0.2
-93 - 55 - 65
+61 - 15 - 20
20 40 2
- 33 -54 - 29
- 71 -86 - 65
4
- 49
- 87
+12 h + 25 -20 h -33
+19 b + 38 -25 -38
10 20 1 2
a The motility index corresponds to measurements of the area between the baseline and the contractile curves, over a period of 60 min before and after drug injection, b All other values significantly different from control values at P < 0.05.
contractions in series lasting 16-20 min and recurring at a frequency of 2.8 + 0.4/h (mean + S.D., n = 20) in dogs fasted for 10-15 h. On the distal portion of the colon, the contraction frequency was 2.2 + 0 . 5 / h (mean + S.D., n = 20) during the same period, i.e. 15-20% of the contractions detected by the proximal gauge were not recorded on the distal gauge. An increase in baseline tension was associated with contractions on both the proximal and distal colon.
3.2. Effects of fentanyl Disruption of the fasted pattern following administration of fentanyl was characterized by a marked and dose-related change in the motility index (MI) (table 1). An i.v. bolus of fentanyl (2 nmol. kg -~) triggered 1-2 contractions on the proximal and distal colon which lasted approxi-
Proximal
Proximal
_6g
colon
Distal Distal
colon
colon
colon
L IFENTANYL
2 nmoles
F'ENTANYL
0.2 nmoles.kg" IT.
kg" I V i,,
I hour I
I hour
i
I
Fig. 1. Effects of intravenous (i.v.) administration of fentanyl on colonic motor activity in the conscious dog. A period of inhibition occurred on the proximal portion of the colon during the hyperactivity recorded on the distal colon.
Fig. 2. Effects of intrathecal (i.t.) administration of fentanyl on colonic motor activity. Both the proximal and distal portions of the colon exhibited a period of lower activity. The increase in the baseline on the distal colon presumably corresponded to a change in resting muscle tone.
332 mately 5 rain in the 15 min period following the injection. The proximal colon was then inhibited whereas prolonged contractile hyperactivity, associated with a sustained increase in the baseline, occurred on the distal colon (fig. 1). A higher dose of i.v. fentanyl (10 n m o l - k g ~) induced rapid defecation during approximately 50% of the observations and in all cases the dogs became restless during the hour following injection. After i.t. administration of fentanyl, the proximal colon contractions were inhibited with no change in baseline whereas those of the distal colon were associated with a prolonged increase in baseline tension lasting 30 and 60 min for 0.1 and 0.2 nmol (fig. 2).
3.4. Effects of enkephalm An inconstant (50% of observations) and transient (30 min) phase of hyperactivity of both the proximal and distal colon was recorded without behavioral changes following D A D L E at 10 nmol • kg i i.v. (table 1). A 45 rain period of inhibition was recorded on distal colon following D A D L E at 1 n m o l , kg 1 i.t. These opposite effects depending on the route of administration were significant on both proximal and distal colon and lasted approximately 60 min for higher doses: 20 n m o l . kg t i.v. a n d 2 n m o l . k g i i.t.
4. Discussion
3.3. Effects of ethylketazocine The ~-agonist E K C did not enhance colonic m o t o r activity by any route or at any dosage evaluated. E K C caused relaxation of the distal colon following i.v. administration• The proximal colon was less sensitive and its activity was unchanged by 4 n m o l . kg 1 (fig. 3). The minimal dosage of E K C that caused consistent relaxation of both the proximal and distal colon was 20 n m o l . k g 1 i.v. a n d 2 n m o l . k g 1 i.t. T h e M I w a s reduced by 60-70% for up to 60 min in these cases without evidence of behavioral changes (table 1). PROXJMAL
DISTAL
Three prototype opioid agonists were used to study changes in the motor profile of two distinct parts of the dog colon. The results as shown in fig. 4 indicate for the first time to our knowledge important differences in the responses induced in the proximal and in the distal portions of the large bowel• The data lend support to the hypothesis that there exists a heterogenous distribution of opiate receptor subtypes related to colonic motility, and they suggest a spinal site of action for agonists involved in opiate-induced colomc motor events.
COLON
COLON
.
i t
E K C 4 n moles kg -' I V
I
I
Fig. 3. Effects of intravenous (i.v.) administration of ethylketazocine (EKC) on colonic motor activity. The distal portion of the colon, presumably more sensitive to EKC than the proximal colon, exhibited a period of inhibition characterized by contractions of lower magnitude and a decrease in resting muscle tone.
333 Mot ili t 5 imi
index .~
4
3~
PROXIMAL COLON Cont rock1ons per hour
[,.-
2
I
g 0
0
.-
-~
....
12
COLON tENTANYL 2nmoles kg-' I V
3Ore,Jn
Fig. 4. Proximal versus distal colonic responses (mean_+S.D. for n = 4) followingi.v. administration of fentanyl in same dog. The motility index represented as columns at 30 min intervals for 6 h and the frequency of contractions per h (dotted lines) varied in an opposite way on the proximal versus distal portion of the colon.
The i.v. fentanyl-induced stimulation of the distal colon and associated inhibition of the proximal colon may be explained either by a different distribution of the /.t-type receptors or by a neural interaction between the proximal and distal colon (Fukai and Fukuda, 1984). The long-lasting inhibition of contractions in the proximal colon and increased baseline tension on the distal segment after i.t. fentanyl injection presumably reflected a more complex response involving a spinal spite of action again with differences between the proximal and distal colon. Both D A D L E and EKC seemed to act at more specific sites. Whatever the route of administration, D A D L E induced similar effects on the proximal and distal colon. The trivial motor effects of i.v. D A D L E contrast with the pronounced stimulatory effect of the synthetic enkephalin analog, H O E 825, on the colon of conscious dogs
(Bickel, 1983). The #-agonist properties of H O E 825, more prominent than those of DADLE, could explain this apparent paradoxical result. In the rat, where most (90%) of the binding is attributed to ~and 8-sites in both brain and spinal cord (Mack et al., 1984), enkephalins administered centrally were shown to trigger a biphasic colonic response characterized by an immediate and short period of excitation followed by a long-lasting inhibition (Pascaud et al., 1980). Inhibition associated with enkephalin was also seen in the guinea-pig ileum (Clark and Smith, 1983). Although the distribution of enkephalin binding sites within the spinal cord remains unknown in dogs, the presence of 8-receptors is likely, based on results in other species studied including humans (La Motte and De Lanerolle, 1981), rats (Finley et al., 1981) and cats (Glazer and Basbaum, 1981). Since D A D L E inhibited colonic motor activity at dosages ineffective by the i.v. route at both the proximal and distal level when administered i.t. a spinal site of action may be postulated. Although r-binding sites in the colon or spinal cord of dogs have not been defined, our data indicate a specific action for EKC, i.e. inhibition of colonic contractions regardless of the route of administration and at doses similar to those involved in the medullary-mediated hyperalgesia linked to K-receptors (Wu et al., 1983). Since xagonists whether i.t. or i.c.v, were ineffective to alter gastrointestinal transit in mice (Porreca et al., 1983), our data in dogs are consistent with an interspecies variation of opiate-induced disturbances in digestive motor activity. Morphine is well known to produce analgesia when injected into the spinal subarachnoid space via a local mechanism (Glick et al., 1977) and colonic motility, which is usually disturbed following traumatic spinal cord injury (Connell et al., 1963) is probably related to an increase in endogenous opioid levels within the spinal cord (Faden et ai., 1983, Glick et al., 1984). The relative specificity of the opioid agonists evaluated in this study and the lack of precision regarding binding sites for opioid subtypes prevented any firm conclusion. However, the data are consistent with the existence of an opioid sensitive structure in the spinal cord involved in the modu-
334
lation of colonic motor events and, at least for fentanyl, of well defined different responses in the proximal and distal portions of the canine colon. That the #-agonist, fentanyk and the 3-agonist, D A D L E produced equivalent effects when given i.t. on both proximal and distal colonic activity is in line with the observation of Porreca et al. (1984) that the spinal cord contains both p~- and 6-receptors related to gastrointestinal function. The specificity of the effects observed in the present study is evident from the observations that i.v. administration of fentanyl produced excitation in the distal colon and i.v. administration of D A D L E produced excitation in both portions of the colon. The lack of precision offered by the motility index as an indicator of motor events does not argue against the observations.
References Adler, H.F. and A.C. Ivy, 1940, Morphine atropine antagonism on colon motility in the dog, J. Pharmacol. Exp. Ther. 70, 454. Bianchi, G., P. Ferretti, M. Recchia, M. Rocchetti, A. Tavani and L. Manara, 1983, Morphine tissue levels and reduction of gastrointestinal transit in rats, Gastroenterology 85, 852. Bickel, M., 1983, Stimulation of colonic motility in dogs and rats by an enkephalin analogue pentapeptide, Life Sci. 33, Suppl. I, 469. BuGno, L., J. Fioramonti and M. Ruckebusch, 1981, Comparative effects of morphine and nalorphine on colonic motility in the conscious dog, European J. Pharmacol. 79, 239. Burks, T.F., J.J. Galligan, L.D. Hirning and F. Porreca, 1984, Spinal, supraspinal and peripheral sites of opioid intestinal actions, in: Gastrointestinal Motility, ed. C. Roman (MTP Press, Lancaster) p. 569. Burks, T.F., L.D. Hirning, J.J. Galligan and T.P. Davis, 1982, Motility effects of opioid peptides in dog intestine, Life Sci. 31, 2237. Clark, S.J. and T.W. Smith, 1983, The release of met-enkephalin from the guinea pig ileum at rest and during peristaltic activity, Life Sci. 33, Suppl. I, 465. Connell, A.M., H. Frankel and L. Guttmen, 1963, The motility of the pelvic colon following complete lesions of the spinal cord, Analgesia 1, 98. Faden, A.I., T.P. Jacobs, G.P. Smith, B. Green and J.A. Zivin, 1983, Neuropeptides in spinal cord injury: comparative experimental models, Peptides 4, 631. Finley, J.C.W., J.M. Maderdrut and P. Petrusz, 1981, The immunocytochemical localization of enkephalin in the central nervous system of the rat, J. Comp. Neurol. 198, 541. Fukai, K. and H. Fukuda, 1984, The intramural pelvic nerves in the colon of dogs. J. Physiol. (London) 354, 89.
Galligan, J.J. and T.F. Burks, 1982, Opioid peptides inhibit intestinal transit in the rat by a central mechanism, European J. Pharmacol. 85, 61. Galligan, J.J. and T.F. Burks, 1983, Centrally mediated inhibition of small intestinal transit and motility by morphine in the rat, J. Pharmacol. Exp. Ther. 2, 356. Glazer, E.J. and A.I. Basbaum, 1981, lmmunohistochemical localization of leucine enkephalin in the spinal cord of the cat: enkephalin containing marginal neurons and pain modulation, J. Comp. Neurol. 196, 377. Glick, M.E., H. Meshkinpour, S. Haldeman, F. Hoehler, N. Downey and W.E. Bradley, 1984~ Colonic dysfunction in patients with thoracic spinal cord injury, Gastroenterology 86, 287. Kreek, M.J., E.F. Hahn, R.A. Schaefer and J. Fishman, 1983, Naloxone, a specific opioid antagonist, reverses chronic idiopathic constipation, Lancet 8319, 261. La Motte, C.C. and N.C. De Lanerolle, 1981, Human spinal neurons: innervation by both substance P and enkephalin, Neuroscience 6, 713. Latour, A. and J.P. FerrG, 1985, Computer-aided analysis of gastrointestinal myoelectric activity, J. Biomed. Eng. (in press). Mack, K.J., A. Killian and J.A. Weyhenmeyer, 1984, Comparison of mu~ delta, kappa opiate binding sites in rat brain and spinal cord, Life Sci. 34, 281. Pairet, M. and Y. Ruckebusch, 1984, Opioid receptor agonists in the rabbit colon: comparison of in vivo and in vitro studies, Life Sci. 35, 1653. Pascaud, X., M. Genton, G. Remond and M. Vincent, 1980, Antral to colonic motility responses to ICV administration of D-Ala2-enkephalinamide, fl endorphine, met-enkephaline and fentanyl in anesthetized rats, in: Gastrointestinal Motility, ed. J. Christensen (Raven Press, New York) p. 459. Porreca, F. and T.F. Burks, 1983, The spinal cord as a site of drug action on gastrointestinal transit, Gastroenterology 89, 1274.
Porreca, F., A. Filla and T.F. Burks, 1983, Spinal cord mediated opiate effects on gastrointestinal transit in mice, European J. Pharmacol. 86, 135. Porreca, F., H.I. Mosberg, T. Hurst, V.J. Hruby and T.S. Burks, 1984, Role of mu, delta, kappa opioid receptors in spinal and supraspinal mediation on gastrointestinal transit effects and hot-plate analgesia in the mouse, J. Pharmacol. Exp. Ther. 230, 341. Ruckebusch, Y., J.P. Ferr6 and Ch. Du, 1984, In vivo modulation of intestinal motility and sites of opioid effects in the rat, Regul. Peptides 9, 109. Tavani, A., G. Bianchi, P. Ferneti and L. Manara, 1980, Morphine is most effective on GI propulsion in rats by IP route: evidence for local action, Life Sci. 27, 2211. Vaught, J.L., A. Cowan and D.E. Gmerek, 1983, A species difference in the slowing effect of intrathecal morphine on gastrointestinal tract, European J. Pharmacol. 94, 181. Wu, K.M., W.R. Martin, S.G. Kamerling and S.G. Wenstein, 1983, Possible medullary K hyperalgesic mechanism. I. A new potential role for endogenous opioid peptide in pain perception. II. The effects of EKC administered into the fourth cerebral ventricle of the conscious dog, Life Sci. 33, 1831, 1839.
329
C O M P A R A T I V E EFFECTS OF O P I A T E A G O N I S T S ON P R O X I M A L AND D I S T A L C O L O N I C M O T I L I T Y IN D O G S THIERRY BARDON and YVES RUCKEBUSCH * Department of Physiology, National Veterinary School 31076 Toulouse, France
Received 5 October 1984, revised MS received 18 December 1984, accepted 15 January 1985
Th. BARDON and Y. RUCKEBUSCH, Comparatiue effects of opiate agonists on proximal and distal colonic motility in dogs, European J. Pharmacol. 110 (1985) 329-334. The colonic motility index was measured in dogs, by using strain-gauge transducers, before and after administration of opiate agonists. Fentanyl, a /~-compound, ethylketazocine (EKC), a r-compound, and [D-Ala2,LeuS]enkephalin (DADLE), a 8-agonist, were administered by intravenous (i.v.) or intrathecal (i.t.) routes. Fentanyl (2-10 nmol. kg-1 i.v.) induced a dose-related period of hyperactivity characterized by an increase in both tone and frequency of contractions in the distal portion of the colon, whereas a period of hypomotility following a short-lived period of increased activity was elicited on the proximal colon. Fentanyl (0.1, 0.2 nmol. kg- 1 i.t.) had inhibitory effects on both proximal and distal colon. Small doses of EKC (2-4 nmol- kg -~) administered i.t. or larger doses (20-40 nmol - kg-a i.v.) inhibited colonic motility for a dose-related duration, the effects of EKC being more marked on the distal colon than on the proximal colon. The administration of DADLE, 1-2 nmol. kg- 1 i.t., and 40-20 nmol- kg 1 i.v., inhibited and stimulated the colonic contractions, respectively, in both proximal and distal colon. The results demonstrated that the colonic opiate-mediated responses may vary according to the route of administration, the portion of the colon studied and the opioid agonist used. Opiate agonists when administered centrally show a tendency to modulate colonic activity in a similar way whatever the type of agonist. Opiate agonists
Colonic motility
Sites of action
1. I n t r o d u c t i o n
The existence of widespread, multiple types of opioid receptors has suggested differences in the role of local, peripheral and central components of opiate modulation of gastrointestinal motility. The increased transit time induced in rats by morphine given intraperitoneally (i.p.) or intravenously (i.v.) seems to be primarily gut-mediated (Tavani et al., 1980; Bianchi et al., 1983), although small doses of morphine administered intracerebroventricularly (i.c.v.) were also effective in altering small intestinal motility (Galligan and Burks, 1983). The opioid peptides which modify small bowel motor function * To whom all correspondence should be addressed: Service Physiologie, Ecole Nationale V6t6rinaire, 23 chemin des Capelles, 31076 Toulouse C6dex, France 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.
Dog
may act either by a central mechanism (Galligan and Burks, 1982) or via peripheral binding sites as evidenced by in vitro (Burks et al., 1982) and in vivo studies (Burks et al., 1984). Moreover, the spinal cord, which is a site of opioid effects on intestinal transit in the mouse (Porreca and Burks, 1983) is not involved in the rat (Vaught et al., 1983). For the latter species, the s.c. but not the i.c.v, administration of the x-type opioid agonist, ethylketazocine (EKC), delayed intestinal transit (Porreca et al., 1983). A plurality of sites of action and species variations for opioid agonists such as demonstrated for small intestine might also exist for motor function of the large bowel. Colonic motility has been stimulated in the dog by morphine (Adler and Ivy, 1940), N-allyl-normorphine (Bu6no et al., 1981) and an enkephalin analogue (Bickel, 1983), but
330 little is known about the characteristics of the opioid-induced colonic motor responses. The indirect demonstration of the involvement of an endogenous opioid system in human constipation and its relief by naloxone treatment (Kreek et al., 1983) suggests similar sites of action between endogenous and exogenous opioid agonists on large bowel motility. Recent studies suggest differences in the response of the proximal and distal portions of the rabbit colon to opioid agonists (Pairet and Ruckebusch, 1984). In addition, 6- and /~-type opioid agonists, which have similar effects on the small bowel when administered i.c.v, in fed rats, have opposite motor effects on the large bowel following an intrathecal (i.t.) injection (Ruckebusch et al., 1984). The aim of these experiments was to compare the motor effects of different opioid agonists, administered either centrally or peripherally, on two distinct parts of the colon of the dog.
2. Materials and methods 2.1. Animal preparation Six mongrel dogs (15-18 kg) were surgically equipped with 2 curved strain-gauge force transducers (1.5 x 1.5 cm) under halothane anesthesia. The gauges, calibrated before implantation, were fixed transversally on the proximal colon, 5 cm distal to the ileocecal junction (ICJ), and on the distal colon, 10 cm proximal to the rectal ampulla. The free ends of the strain gauge wires were directed s.c. along the flank to emerge between the scapulas. A silastic catheter (0.1 mm inner diam.) fitted with distilled water was inserted between the fourth and fifth lumbar vertebrae into the subarachnoid space using a needle as a trocar and anchored so that the tip remained at the level of the twelfth thoracic vertebra.
2.2. Recordings After a 10 day recovery period, each dog was placed in a metabolic cage to which it was adapted and given free access to water. All dogs were fed canned food (600 g / d a y , 4% N D F - non digestible fiber) once daily at 17:00 h. Mechanical activity was recorded from the transducers for 20 con-
secutive hours by connecting each gauge to a Wheatstone bridge amplifier (VT 2100. Vishay, France) and a potentiometric recorder. Mechanical changes were evaluated by measuring the area between the baseline and the contractile curves, The product of the amplitude expressed in grams and the time in minutes was continuously stored and analyzed using an Apple lie microcomputer as a digital integrator and data logging system after A / D conversion (Latour and Ferr6, 1985). The developed printing program indicated consecutive values every 5, 30 and 60 rain as motility indexes.
2.3. Drugs The following opioid agonists were administered in random fashion at 8:00 h with at least a 3-day interval between evaluations in each dog: fentanyl, as a /~-agonist (courtesy of Janssen Pharmaceutica, Beerse, Belgium) at the dosage of 2, 4, 10 n m o l . k g 1 i.v. and 0.1, 0.2 n m o l . k g i i.t.. diluted in sterilized saline (5 ml) or distilled water (0.5 ml), respectively; ethylketazocine (EKC) as a ~-agonist (courtesy of Sterling-Winthrop Research Institute, Rensselaer, NY, U.S.A.) (20-40 n m o l . k g i i.v. and 2-4 n m o l . k g -t i.t.): [DAla2,D-LeuS]enkephalin (DADLE) as a 6-agonist (purchased from Sigma-Interchim, Montluqon, France) (20-40 nmol • kg 1 i.v. and 1-2 nmol - kg 1 i.t.). Four measurements of colonic motility were made on the proximal colon and in each dog for each agent administered 10-15 rain after the occurrence of a phase of contraction. Saline and water used as placebo had no effect on colonic motility after i.v. or i.t. administration, respectively. The agonist at the dosage used i.t. was also administered i.v. Values of motility indexes were expressed as mean percentage of control activity and were compared by using the non-parametric Wilcoxon test.
3. Results 3.1. Normal colonic motor profile Motor activity of the colon obtained from the most proximal strain gauge was characterized by
331 TABLE 1 Variations of the motility index (MI) a following the administration of opioid agonists in 6 dogs. Expressed as a mean percentage of control activity (4 measurements for each dosage) Opioid agonists
Route
Fentanyl (/~)
i.v.
Fentanyl (~)
i.t.
EKC (K)
i.v.
EKC (K)
i.t.
DADLE (8)
i.v.
DADLE (8)
i.t.
Doses
MI variations (%)
(nmol • kg- 1)
Proximal colon
Distal colon
2
- 63
+ 22
4
-87
+44
10 0.1 0.2
-93 - 55 - 65
+61 - 15 - 20
20 40 2
- 33 -54 - 29
- 71 -86 - 65
4
- 49
- 87
+12 h + 25 -20 h -33
+19 b + 38 -25 -38
10 20 1 2
a The motility index corresponds to measurements of the area between the baseline and the contractile curves, over a period of 60 min before and after drug injection, b All other values significantly different from control values at P < 0.05.
contractions in series lasting 16-20 min and recurring at a frequency of 2.8 + 0.4/h (mean + S.D., n = 20) in dogs fasted for 10-15 h. On the distal portion of the colon, the contraction frequency was 2.2 + 0 . 5 / h (mean + S.D., n = 20) during the same period, i.e. 15-20% of the contractions detected by the proximal gauge were not recorded on the distal gauge. An increase in baseline tension was associated with contractions on both the proximal and distal colon.
3.2. Effects of fentanyl Disruption of the fasted pattern following administration of fentanyl was characterized by a marked and dose-related change in the motility index (MI) (table 1). An i.v. bolus of fentanyl (2 nmol. kg -~) triggered 1-2 contractions on the proximal and distal colon which lasted approxi-
Proximal
Proximal
_6g
colon
Distal Distal
colon
colon
colon
L IFENTANYL
2 nmoles
F'ENTANYL
0.2 nmoles.kg" IT.
kg" I V i,,
I hour I
I hour
i
I
Fig. 1. Effects of intravenous (i.v.) administration of fentanyl on colonic motor activity in the conscious dog. A period of inhibition occurred on the proximal portion of the colon during the hyperactivity recorded on the distal colon.
Fig. 2. Effects of intrathecal (i.t.) administration of fentanyl on colonic motor activity. Both the proximal and distal portions of the colon exhibited a period of lower activity. The increase in the baseline on the distal colon presumably corresponded to a change in resting muscle tone.
332 mately 5 rain in the 15 min period following the injection. The proximal colon was then inhibited whereas prolonged contractile hyperactivity, associated with a sustained increase in the baseline, occurred on the distal colon (fig. 1). A higher dose of i.v. fentanyl (10 n m o l - k g ~) induced rapid defecation during approximately 50% of the observations and in all cases the dogs became restless during the hour following injection. After i.t. administration of fentanyl, the proximal colon contractions were inhibited with no change in baseline whereas those of the distal colon were associated with a prolonged increase in baseline tension lasting 30 and 60 min for 0.1 and 0.2 nmol (fig. 2).
3.4. Effects of enkephalm An inconstant (50% of observations) and transient (30 min) phase of hyperactivity of both the proximal and distal colon was recorded without behavioral changes following D A D L E at 10 nmol • kg i i.v. (table 1). A 45 rain period of inhibition was recorded on distal colon following D A D L E at 1 n m o l , kg 1 i.t. These opposite effects depending on the route of administration were significant on both proximal and distal colon and lasted approximately 60 min for higher doses: 20 n m o l . kg t i.v. a n d 2 n m o l . k g i i.t.
4. Discussion
3.3. Effects of ethylketazocine The ~-agonist E K C did not enhance colonic m o t o r activity by any route or at any dosage evaluated. E K C caused relaxation of the distal colon following i.v. administration• The proximal colon was less sensitive and its activity was unchanged by 4 n m o l . kg 1 (fig. 3). The minimal dosage of E K C that caused consistent relaxation of both the proximal and distal colon was 20 n m o l . k g 1 i.v. a n d 2 n m o l . k g 1 i.t. T h e M I w a s reduced by 60-70% for up to 60 min in these cases without evidence of behavioral changes (table 1). PROXJMAL
DISTAL
Three prototype opioid agonists were used to study changes in the motor profile of two distinct parts of the dog colon. The results as shown in fig. 4 indicate for the first time to our knowledge important differences in the responses induced in the proximal and in the distal portions of the large bowel• The data lend support to the hypothesis that there exists a heterogenous distribution of opiate receptor subtypes related to colonic motility, and they suggest a spinal site of action for agonists involved in opiate-induced colomc motor events.
COLON
COLON
.
i t
E K C 4 n moles kg -' I V
I
I
Fig. 3. Effects of intravenous (i.v.) administration of ethylketazocine (EKC) on colonic motor activity. The distal portion of the colon, presumably more sensitive to EKC than the proximal colon, exhibited a period of inhibition characterized by contractions of lower magnitude and a decrease in resting muscle tone.
333 Mot ili t 5 imi
index .~
4
3~
PROXIMAL COLON Cont rock1ons per hour
[,.-
2
I
g 0
0
.-
-~
....
12
COLON tENTANYL 2nmoles kg-' I V
3Ore,Jn
Fig. 4. Proximal versus distal colonic responses (mean_+S.D. for n = 4) followingi.v. administration of fentanyl in same dog. The motility index represented as columns at 30 min intervals for 6 h and the frequency of contractions per h (dotted lines) varied in an opposite way on the proximal versus distal portion of the colon.
The i.v. fentanyl-induced stimulation of the distal colon and associated inhibition of the proximal colon may be explained either by a different distribution of the /.t-type receptors or by a neural interaction between the proximal and distal colon (Fukai and Fukuda, 1984). The long-lasting inhibition of contractions in the proximal colon and increased baseline tension on the distal segment after i.t. fentanyl injection presumably reflected a more complex response involving a spinal spite of action again with differences between the proximal and distal colon. Both D A D L E and EKC seemed to act at more specific sites. Whatever the route of administration, D A D L E induced similar effects on the proximal and distal colon. The trivial motor effects of i.v. D A D L E contrast with the pronounced stimulatory effect of the synthetic enkephalin analog, H O E 825, on the colon of conscious dogs
(Bickel, 1983). The #-agonist properties of H O E 825, more prominent than those of DADLE, could explain this apparent paradoxical result. In the rat, where most (90%) of the binding is attributed to ~and 8-sites in both brain and spinal cord (Mack et al., 1984), enkephalins administered centrally were shown to trigger a biphasic colonic response characterized by an immediate and short period of excitation followed by a long-lasting inhibition (Pascaud et al., 1980). Inhibition associated with enkephalin was also seen in the guinea-pig ileum (Clark and Smith, 1983). Although the distribution of enkephalin binding sites within the spinal cord remains unknown in dogs, the presence of 8-receptors is likely, based on results in other species studied including humans (La Motte and De Lanerolle, 1981), rats (Finley et al., 1981) and cats (Glazer and Basbaum, 1981). Since D A D L E inhibited colonic motor activity at dosages ineffective by the i.v. route at both the proximal and distal level when administered i.t. a spinal site of action may be postulated. Although r-binding sites in the colon or spinal cord of dogs have not been defined, our data indicate a specific action for EKC, i.e. inhibition of colonic contractions regardless of the route of administration and at doses similar to those involved in the medullary-mediated hyperalgesia linked to K-receptors (Wu et al., 1983). Since xagonists whether i.t. or i.c.v, were ineffective to alter gastrointestinal transit in mice (Porreca et al., 1983), our data in dogs are consistent with an interspecies variation of opiate-induced disturbances in digestive motor activity. Morphine is well known to produce analgesia when injected into the spinal subarachnoid space via a local mechanism (Glick et al., 1977) and colonic motility, which is usually disturbed following traumatic spinal cord injury (Connell et al., 1963) is probably related to an increase in endogenous opioid levels within the spinal cord (Faden et ai., 1983, Glick et al., 1984). The relative specificity of the opioid agonists evaluated in this study and the lack of precision regarding binding sites for opioid subtypes prevented any firm conclusion. However, the data are consistent with the existence of an opioid sensitive structure in the spinal cord involved in the modu-
334
lation of colonic motor events and, at least for fentanyl, of well defined different responses in the proximal and distal portions of the canine colon. That the #-agonist, fentanyk and the 3-agonist, D A D L E produced equivalent effects when given i.t. on both proximal and distal colonic activity is in line with the observation of Porreca et al. (1984) that the spinal cord contains both p~- and 6-receptors related to gastrointestinal function. The specificity of the effects observed in the present study is evident from the observations that i.v. administration of fentanyl produced excitation in the distal colon and i.v. administration of D A D L E produced excitation in both portions of the colon. The lack of precision offered by the motility index as an indicator of motor events does not argue against the observations.
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Porreca, F., A. Filla and T.F. Burks, 1983, Spinal cord mediated opiate effects on gastrointestinal transit in mice, European J. Pharmacol. 86, 135. Porreca, F., H.I. Mosberg, T. Hurst, V.J. Hruby and T.S. Burks, 1984, Role of mu, delta, kappa opioid receptors in spinal and supraspinal mediation on gastrointestinal transit effects and hot-plate analgesia in the mouse, J. Pharmacol. Exp. Ther. 230, 341. Ruckebusch, Y., J.P. Ferr6 and Ch. Du, 1984, In vivo modulation of intestinal motility and sites of opioid effects in the rat, Regul. Peptides 9, 109. Tavani, A., G. Bianchi, P. Ferneti and L. Manara, 1980, Morphine is most effective on GI propulsion in rats by IP route: evidence for local action, Life Sci. 27, 2211. Vaught, J.L., A. Cowan and D.E. Gmerek, 1983, A species difference in the slowing effect of intrathecal morphine on gastrointestinal tract, European J. Pharmacol. 94, 181. Wu, K.M., W.R. Martin, S.G. Kamerling and S.G. Wenstein, 1983, Possible medullary K hyperalgesic mechanism. I. A new potential role for endogenous opioid peptide in pain perception. II. The effects of EKC administered into the fourth cerebral ventricle of the conscious dog, Life Sci. 33, 1831, 1839.