5-HT4 receptor agonists and delta -opioid receptor antagonists act synergistically to stimulate colonic propulsion

A. E. Foxx-Orenstein, J.-G. Jin, and J. R. Grider

Departments of Medicine and Physiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0551

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Opioid neurons exert a tonic restraint on inhibitory VIP/PACAP/NOS motoneurons of the enteric nervous system. A decrease in opioid peptide release during the descending phase of the peristaltic reflex, which underlies propulsive activity, leads to an increase in vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), and nitric oxide (NO) release and circular muscle relaxation. These effects are accentuated by opioid receptor antagonists. Endogenous opioid peptides and selective opioid delta -, kappa - and µ-receptor agonists decreased the velocity of pellet propulsion in isolated segments of guinea pig colon, whereas selective antagonists increased velocity in a concentration-dependent fashion with an order of potency indicating preferential involvement of delta -receptors. 5-HT4 agonists (HTF-919 and R-093877), which also increase the velocity of propulsion, acted synergistically with the delta -receptor antagonist naltrindole; a threshold concentration of naltrindole (10 nM) shifted the concentration-response curve to HTF-919 to the left by 70-fold. A combination of 10 nM naltrindole with threshold concentrations of the 5-HT4 agonists caused significant increases in the velocity of propulsion (50 ± 7 to 77 ± 8%). We conclude that 5-HT4 agonists and opioid delta -receptor antagonists act synergistically to facilitate propulsive activity in isolated colonic segments.

intestinal smooth muscle; enteric nervous system; gut motility

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

OPIOID PEPTIDES OF THE GUT, predominant derivatives of proenkephalin ([Met]enkephalin, and COOH-terminal-extended derivatives of [Met]enkephalin) (24) and to a lesser extent (10%) of prodynorphin (dynorphin-13 and [Leu]enkephalin-related peptides) (18), are present in and released from neurons of the enteric nervous system (5, 6, 11, 13, 22). Distinct neural and muscular actions of opioid agonists have been identified in the gut. These include direct contraction of smooth muscle cells of the circular layer (15, 21) and inhibition of neuronal activity reflected by decreases in the release of both excitatory (acetylcholine and tachykinins) (3, 9, 19) and inhibitory [vasoactive intestinal peptide (VIP) and nitric oxide (NO)] neurotransmitters (11, 14). Suppression of VIP and NO release by opioid agonists eliminates inhibitory junction potentials and restores the intrinsic rhythmic electrical and contractile ac-tivity of sphincteric and intestinal smooth muscles; the effects are predominantly mediated by delta -opioid receptors (1, 2, 14, 16). Neurally mediated effects underlie the ability of opioid agonists to induce segmenting, nonpropulsive contractions and delay transit throughout the gut.

During reflex neuromuscular activity, opioid peptides are released in a coordinated fashion at precise locations to modulate sphincteric and peristaltic activity. The continuous restraint exerted by opioid neurons on VIP/NO synthase (NOS) neurons innervating circular muscle decreases during the descending phase of the peristaltic reflex leading to decrease in [Met]enkephalin and dynorphin release (4, 6, 11, 13) and increase in VIP and NO release (11, 13); the reverse occurs during the ascending phase of the peristaltic reflex. Consistent with this notion, opioid peptides decrease VIP and NO release and inhibit descending relaxation with an order of potency [Met]enkephalin (delta -receptor agonist) > dynorphin-13 (kappa -receptor agonist) >=  morphiceptin (µ-receptor agonist), whereas opioid antagonists have the reverse effect (10-13).

Inhibition of opioid peptide release facilitates the descending phase of the peristaltic reflex and should enhance intestinal propulsion. Studies of peristaltic activity using the Trendelenburg preparation show that the opioid antagonists naloxone and nor-binaltorphimine increased peristaltic contractions (20, 30) and that naltrindole increased gastrointestinal transit in mice treated with croton oil (26). In contrast, oral or parenteral administration of opioid agonists leads to rhythmic, uncoordinated, nonpropulsive muscle contractions (29), presumably mediated by inhibition of the release of inhibitory neurotransmitters (1, 2, 13).

Our recent studies (8, 12) in compartmented, flat-sheet preparations of rat and guinea pig colon and human small intestine have shown that selective 5-HT4 agonists (HTF-919 and R-093877) can trigger peristaltic activity by activating intrinsic sensory calcitonin gene-related peptide (CGRP) neurons. These neurons are normally activated by 5-HT released from mucosal enterochromaffin cells. Studies of pellet propulsion in isolated segments of guinea pig colon showed that 5-HT and selective 5-HT4 agonists increased the velocity of propulsion in a concentration-dependent fashion (17). We have postulated that opioid receptor antagonists could also enhance the velocity of propulsion. In the present study, we have examined the effect of selective opioid receptor agonists and antagonists on the velocity of propulsion and characterized the synergism between the effects of opioid antagonists and 5-HT4 agonists.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

The distal colon of male guinea pigs (weight 150-200 g) was incubated at 37°C for 30 min in Krebs-bicarbonate medium to allow spontaneous evacuation of fecal pellets. The composition of the medium was (in mM) 118 NaCl, 4.8 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2.5 CaCl2, 25 NaHCO3, and 11 glucose. The colon was then cut into two equal segments; each segment was secured with pins placed at intervals through the attached mesentery as described previously (7).

Artificial clay pellets, which mimicked natural colonic pellets in size and shape (10 mm long × 4 mm wide), were used to measure the velocity of propulsion. Control (i.e., basal) velocity was measured by inserting a pellet into the orad end of a colonic segment and allowing it to exit spontaneously through the caudad opening of the segment. The velocity was calculated from the time taken by a pellet to traverse a marked 3-cm segment. At 5-min intervals, a second and then a third pellet were inserted into the orad end, and the measurement of velocity was repeated. Control velocity was taken as the mean velocity of propulsion of three successive pellets. The segments were then allowed to equilibrate again for 30 min in fresh Krebs-bicarbonate solution. After the equilibration period, various opioid receptor agonists or antagonists were added for 15 min to a Krebs-bicarbonate medium containing 0.1% bovine serum albumin bathing the serosa, and the velocity of propulsion of three successive pellets was measured. The effect of the following selective opioid receptor agonists and antagonists was determined: CTOP (µ-receptor antagonist) (25), naltrindole (delta -receptor antagonist) (28), and nor-binaltorphimine (kappa -receptor antagonist) (27); DAMGO (µ-receptor agonist), DPDPE and [Met]enkephalin (delta -receptor agonists), and U-69593 and dynorphin-13 (kappa -receptor agonists). The effect of [Leu]enkephalin, which can act as delta - or µ-receptor agonist in different tissues (neurons, smooth muscle cells), was also determined (10, 15, 21).

In separate studies the effect of 5-HT4 agonists alone and in combination with naltrindole on pellet propulsion was determined. In some experiments both the 5-HT4 agonist and naltrindole were perfused intraluminally, whereas in other experiments the 5-HT4 agonists were perfused intraluminally and naltrindole was added to the serosal bathing medium. Previous studies had shown that 5-HT and 5-HT4 agonists were not effective when added to the serosal bathing medium (17). The colonic segments were perfused for a 30-min equilibration period with Krebs-bicarbonate medium at the optimal rate of 0.25 ml/min using a PE-10 catheter inserted through the caudad end. As previously shown, this perfusion rate did not by itself affect the velocity of pellet propulsion (17). After the equilibration period, 5-HT4 agonists (HTF-919 and R-093877) alone and in combination with naltrindole were added to the luminal perfusate. The perfusion of 5-HT4 agonists was begun 2 min before and that of naltrindole 15 min before insertion of pellets into the orad end of the colonic segment. In all studies the response to a given concentration of agonist or antagonist was measured in a single segment.

Data analysis. Results were expressed as percent of control (i.e., basal) velocity in millimeters per second. The concentration causing 50% of maximal response (EC50) was calculated from the fit of concentration-response curves using the p.fit 6.0 program (Elsevier, Cambridge). Separate colonic segments were used for each agonist or antagonist at each concentration. Values are means ± SE of n experiments, where n represents the number of colonic segments. Statistical significance was evaluated using Student's t-test for paired or unpaired data.

Materials. Naltrindole and nor-binaltorphimine were purchased from Research Biochemicals International (Natick, MA); [Met]enkephalin, [Leu]enkephalin, dynorphin-13, [D-Pen2,5]enkephalin (DPDPE), [D-Ala2, N-Me-Phe4, Gly5-ol] enkephalin (DAMGO) were from Bachem (Torrance, CA); U-69593 and all other chemicals were from Sigma Chemical (St. Louis, MO). HTF-919 was a gift from Drs. D. Romer and H.-J. Pfannkuche, Novartis (Basel, Switzerland). R-093877 was a gift from Drs. J. Schuurkes and M. Janssen, Janssen Research Foundation (Beerse, Belgium), and CTOP was a gift from Dr. V. Hruby, University of Arizona (Tucson, AZ).

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Control velocity of propulsion. As shown in earlier studies (7), the control (basal) velocity of pellet propulsion was constant for segments obtained from the same colon but differed from one colon to another (range 0.6 to 3.6 mm/s with a mean of 1.7 ± 0.1 mm/s, n = 462 pellets in 154 experiments). Intraluminal perfusion with Krebs-bicarbonate buffer had no significant effect on the control velocity (range 0.5 to 3.2 mm/s with a mean of 1.4 ± 0.3 mm/s, n = 87 pellets in 29 experiments).

Decrease in velocity of propulsion induced by endogenous and synthetic opioid peptides. Addition of endogenous opioid peptides to the serosal bathing medium caused a concentration-dependent decrease in the velocity of pellet propulsion with an order of potency of [Met]enkephalin (EC50 11 ± 12 nM) >=  dynorphin-13 (EC50 17 ± 8 nM) > [Leu]enkephalin (EC50 130 ± 24 nM) (Fig. 1). At a concentration of 1 µM, [Met]enkephalin decreased the velocity of propulsion by 74 ± 4% (control velocity 1.6 ± 0.2 mm/s; velocity in the presence of [Met]enkephalin 0.4 ± 0.1 mm/s, n = 6, P < 0.01). Dynorphin-13 and [Leu]enkephalin decreased the velocity of propulsion by 63 ± 7% (n = 8, P < 0.01) and 59 ± 1% (n = 5, P < 0.01), respectively.


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Fig. 1.   Decrease in velocity of colonic propulsion by opioid peptides. Three clay pellets were inserted at 5-min intervals into the orad end of an isolated guinea pig colonic segment, and a control velocity was measured from the time taken to traverse a fixed 3-cm length. After 30 min an opioid agonist was added to the serosal medium bathing the segment, and the measurement of velocity was repeated using 3 successive pellets. Only one concentration of a given agonist was used with each colonic segment. Results are expressed as percent of control velocity. Each point represents mean ± SE of 4-9 experiments (i.e., separate colonic segments or agonist concentrations). Dyn-13, dynorphin-13; [Leu]enk, [Leu]enkephalin; [Met]enk, [Met]enkephalin.

Synthetic, selective opioid receptor agonists also caused a concentration-dependent decrease in the velocity of pellet propulsion with an order of potency of DPDPE (delta -receptor agonist, EC50 12 ± 7 nM) >=  U-69593 (kappa -receptor agonist, EC50 15 ± 12 nM) > DAMGO (µ-receptor agonist, EC50 750 ± 14 nM) (Fig. 2). At a concentration of 1 µM, DPDPE decreased the velocity of propulsion by 60 ± 7% (control velocity 2.6 ± 0.2 mm/s, velocity in the presence of DPDPE 1.1 ± 0.1 mm/s, n = 8, P < 0.001). U-69593 and DAMGO decreased the velocity of propulsion by 58 ± 5% (n = 4, P < 0.01) and 52 ± 1% (n = 4, P < 0.01), respectively.


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Fig. 2.   Decrease in velocity of colonic propulsion by selective opioid-receptor agonists. Control velocity of pellet propulsion and velocity on addition of various selective agonists were measured as described in legend to Fig. 1. Results are expressed as percent of control velocity. DAMGO, µ-receptor agonist; U-69593, kappa -receptor agonist; DPDPE, delta -receptor agonist. Each point represents mean ± SE of 4-8 experiments (i.e., separate colonic segments or agonist concentrations).

Increase in velocity of propulsion induced by opioid receptor antagonists. Addition of selective opioid receptor antagonists to the bathing medium had the opposite effect, causing a concentration-dependent increase in the velocity of pellet propulsion with an order of potency of naltrindole (delta -receptor antagonist, EC50 2.8 ± 3.5 µM) > nor-binaltorphimine (kappa -receptor antagonist) >=  CTOP (µ-receptor antagonist) (Fig. 3). At the highest concentration used (10 µM), naltrindole increased the velocity of propulsion by 110 ± 16% (n = 8, P < 0.01), whereas nor-binaltorphimine and CTOP increased velocity by 43 ± 8% (n = 8, P < 0.01) and 39 ± 5% (n = 4, P < 0.01), respectively. The increase in velocity induced by the delta -receptor antagonist was significantly greater than that induced by the kappa -receptor antagonist (P < 0.01) or the µ-receptor antagonist (P < 0.01).

Synergistic increase in propulsion velocity induced by a combination of delta -receptor antagonist and 5-HT4 agonists. Previous studies (17) have shown that intraluminal perfusion with the selective 5-HT4 agonists HTF-919 or R-093877 causes a concentration-dependent increase in the velocity of pellet propulsion. To examine whether the effects of a combination of a 5-HT4 agonist and a delta -receptor antagonist were synergistic, a threshold concentration (10 nM) of the delta -receptor antagonist naltrindole was perfused intraluminally with various concentrations of HTF-919. Whether added to the intraluminal perfusate or the serosal medium, naltrindole at a concentration of 10 nM had no significant effect on the velocity of propulsion (2 ± 5 and 2 ± 3%, not significant, Fig. 5). The combination of a threshold concentration of naltrindole with various concentrations of HTF-919 in the intraluminal perfusate caused a significant increase in the velocity of propulsion, shifting the concentration-response curve to the left (EC50 0.13 ± 0.12 nM in the presence of 10 nM naltrindole vs. 9.3 ± 0.3 nM in the absence of naltrindole) (Fig. 4). In particular, intraluminal perfusion with a combination of naltrindole (10 nM) and threshold concentrations of HTF-919 (0.1 and 1 nM) increased the velocity of propulsion by 37 ± 3% (n = 4, P < 0.001) and 77 ± 8% (n = 4, P < 0.001), respectively (Figs. 4 and 5). Addition of 10 nM naltrindole to the serosal bathing medium in combination with intraluminal perfusion of 1 nM HTF-919 resulted in a similar increase in the velocity of propulsion to 72 ± 12% (n = 3, P < 0.005) above control velocity (Fig. 5). A similar synergistic increase in the velocity of propulsion was observed for a combination of a threshold concentration of naltrindole (10 nM) and a threshold concentration of R-093877 (10 nM): 50 ± 7 and 45 ± 5% increase in velocity for intraluminal and serosal application of natrindole, respectively, n = 3-4, P < 0.01 for each combination (Fig. 5).


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Fig. 3.   Increase in velocity of colonic propulsion by selective opioid-receptor antagonists. Control velocity of pellet propulsion and velocity on addition of various selective antagonists were measured as described in legend to Fig. 1. Results are expressed as percent of control velocity. CTOP, µ-receptor antagonist; n-BNI, nor-binaltorphimine (kappa -receptor antagonist); NTI, naltrindole (delta -receptor antagonist). Each point represents mean ± SE of 4-8 experiments (i.e., separate colonic segments or antagonist concentrations).


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Fig. 4.   Synergistic increase in velocity of colonic propulsion by combination of an opioid delta -receptor antagonist and 5-HT4 agonist. Three clay pellets were inserted at 5-min intervals into the orad end of an isolated guinea pig colonic segment perfused with Krebs medium at an optimal rate of 0.25 ml/min, and control velocity was measured from the time taken to traverse a fixed 3-cm length. After 30 min, one concentration of the 5-HT4 agonist HTF-919 was added to the perfusate alone or in combination with a threshold concentration of naltrindole (NTI) (10 nM) and the measurement of velocity was repeated using 3 successive pellets. A marked increase in velocity of propulsion was observed in the presence of naltrindole. Each point represents mean ± SE of 3-7 experiments (i.e., separate colonic segments or HTF-919 concentrations).


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Fig. 5.   Synergistic increase in velocity of colonic propulsion by a combination of threshold concentrations of opioid delta -receptor antagonist and 5-HT4 agonists. Velocity of propulsion was measured as described in legend to Fig. 4. Solid bars, effects of threshold concentrations of naltrindole (NTI), HTF-919, and R-093877 perfused intraluminally. Hatched bars, effect of combinations of naltrindole with either HTF-919 or R-093877 perfused intraluminally. Open bars, effect of a threshold concentration of naltrindole added to serosal medium alone or in combination with intraluminal perfusion with threshold concentration of HTF-919 or R-093877. Each bar represents mean ± SE of 3-7 experiments.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

This study shows that opioid receptor antagonists, particularly antagonists with high selectivity for delta -receptors, facilitate propulsive activity in the guinea pig colon and act synergistically with 5-HT4 receptor agonists. Recent studies (17) using the same preparation have shown that 5-HT4 receptor agonists also facilitate propulsive activity. The delta -receptor antagonists and the 5-HT4 receptor agonists act at different neural locations consistent with the roles of endogenous opioid peptides and 5-HT in the regulation of the peristaltic reflex. After release from enterochromaffin cells, 5-HT acts on 5-HT4 receptors (rat and human) or on both 5-HT4 and 5-HT3 receptors (guinea pig) located on sensory CGRP nerve terminals (8, 12). Activation of CGRP nerves triggers the ascending and descending phases of the peristaltic reflex underlying propulsive activity.

In contrast, opioid peptides, chiefly the delta -receptor agonist, [Met]enkephalin, are released from modulatory neurons to act predominantly on inhibitory neurons. The selective involvement of delta -receptors in regulating the activity of inhibitory neurons is reflected in the ability of opioid peptides to inhibit VIP release (11, 14), induce phasic contractile activity (1, 2, 14), and suppress inhibitory junction potentials (1, 2, 16). The release of [Met]enkephalin decreases during the initial phase of peristalsis (4, 11, 13), relieving the restraint normally exerted by opioid neurons on inhibitory VIP/pituitary adenylate cyclase-activating polypeptide (PACAP)/NOS neurons (11, 14). This effect also is preferentially mediated by delta -receptors and leads to increases in VIP/PACAP/NO release and relaxation of circular muscle and reciprocal contraction of longitudinal muscle. The effect is amplified by opioid receptor antagonists that augment the release of inhibitory neurotransmitters and enhance descending relaxation of circular muscle and contraction of longitudinal muscle (11, 13).

The effectiveness of the delta -receptor antagonist naltrindole in enhancing propulsive activity confirms that endogenous opioid peptides exert their effect predominantly by acting on delta -receptors located on inhibitory neurons. The low potency of CTOP and nor-binaltorphimine implied that endogenous opioids do not regulate propulsive activity by acting on kappa - and µ-receptors located on cholinergic/tachykinin neurons (3, 19, 23).

The effectiveness of delta -receptor antagonists is underscored by the fact that even threshold concentrations of naltrindole are effective when applied in combination with threshold concentrations of the 5-HT4 agonists, HTF-919, and R-093877. The combination of threshold concentrations elicited an increase in the velocity of propulsion that was about 70% of the maximal increase elicited by naltrindole alone and closely similar to the maximal increase in velocity elicited by HTF-919 alone. The marked synergism between naltrindole and 5-HT4 agonists is a reflection of the sites and modes of action of the two agents. 5-HT acts in an excitatory capacity at the point of origin of the peristaltic reflex (8, 12), whereas opioid antagonists act to facilitate the descending phase of the reflex by eliminating the restraint exerted by endogenous opioids on inhibitory interneurons and motoneurons (1, 2, 11, 13, 16). Synergism might also be anticipated from a combination of CGRP and delta -receptor antagonists. A test of this notion awaits the development of active, permeable analogs of CGRP.

Thus far selective 5-HT4-receptor agonists and delta -receptor antagonists are the only two types of agents that enhance propulsive activity and whose actions reflect the roles of endogenous regulators of peristalsis. Other agents, such as VIP, NO, or substance P, which normally act as motor neurotransmitters, cause indiscriminate relaxation (VIP and NO) or contraction (substance P) of intestinal smooth muscle when applied exogenously and thus inhibit propulsive activity. The NOS inhibitor NG-nitro-L-arginine, VIP receptor antagonist VIP-10---28, and substance P receptor antagonist MEN-10376, also inhibit propulsive activity, particularly when applied in combination (22).

    ACKNOWLEDGEMENTS

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-34153.

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: J. R. Grider, PO Box 980551, Medical College of Virginia, Richmond, VA 23298.

Received 25 March 1998; accepted in final form 27 July 1998.

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Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 275(5):G979-G983
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society