E. Goñalons

Effects of cholecystokinin and gastrin on gastroduodenal motility and coordination in chickens.

Cholecystokinin (CCK) has not been isolated from chicken gut yet and there has been no study on the effects of chicken gastrin (CG), the only gastrin/CCK peptide isolated from avian gut, on gastrointestinal motility. The main objective was to study the effects of CCK and CG on gastroduodenal motility and coordination in chickens. Electrodes for electromyography were implanted in the stomach and proximal and distal duodenum of 4 wk old chickens. Sulphated CCK-octapeptide (CCK8) (10(-10) to 10(-8) moles/kg), CCK-tetrapeptide (CCK4) (2 x 10(-10) to 2 x 10(-8) moles/kg) and CG (3 x 10(-10) to 10(-8) moles/kg) were given in a 10 min i.v. infusion. All these peptides induced a dose-dependent inhibition of gastric motility. CCK8 induced a duodenal hyperactivity whereas CCK4 and CG induced a duodenal inhibition. Neither the CCK-A receptor antagonist L364,718 nor the CCK-B receptor antagonist L365,260 (10(-9)-10(-7) moles/kg) antagonized CCK8 actions. From these results we suggest that the receptors mediating CCK effects are different from those of mammals. The site of action for these peptides is the same in the stomach whereas in the duodenum there are two different ones, one mediating excitation and the other inhibition. These results suggest a physiological role for CCK regulating gastroduodenal motility in birds.

Effect of different calcium channel blockers on inhibitory junction potentials and slow waves in porcine ileum

The effect of several calcium channel blockers was evaluated: (i) on spontaneous electrical and mechanical activities and (ii) on the response to electrical field stimulation. The study was carried out on whole-thickness preparation of porcine ileum. Glass microelectrodes were used to record membrane potential from smooth muscle cells. Resting membrane potential was -60 +/- 2mV (n = 18) and preparations generated spontaneous slow waves. Electrical field stimulation (EFS) was applied using different parameters. The amplitude and duration of inhibitory junction potentials (IJPs) increased with EFS strength. IJPs were abolished by tetrodotoxin (1 microM). Nifedipine (1 microM) did not modify the amplitude or duration of IJPs. The frequency of slow waves was not modified, however a slight but significant (p < 0.001) reduction in slow wave duration was observed. Mechanical activity was abolished in presence of nifedipine within approximately 6 min. omega-agatoxin IVA (50 nM) or omega-conotoxin MVIIC (100 nM), respectively a P-type and a Q-type calcium channel blockers, did not modify slow wave and IJP characteristics. In contrast, in presence of omega-conotoxin GVIA (100 nM), a N-type calcium channel blocker, or omega-conotoxin MVIIC (1 microM), IJPs were completely abolished. These data suggest that, in porcine ileum, N-type but not P-,Q- or L-type calcium channels are involved in the release of the non-adrenergic non-cholinergic neurotransmitters mediating IJPs. L-type calcium channels underlie electrical mechanical coupling but are not involved in slow wave generation.

Receptors implicated in the actions of serotonin on chicken ileum longitudinal smooth muscle

The presence of serotonin (5-HT) in the chicken gastrointestinal tract has been previously reported, but its motor effects have been poorly described. The aims of this work were: A) to define the effects of 5-HT on chicken longitudinal ileum; B) to explore the mechanisms by which such effects occur and C) to identify the subtype(s) of 5-HT-ergic receptors implicated. The motor responses to 5-HT were assayed in vitro using ileal strips taken from male White Leghorn chickens 7-9 week old. 5-HT elicited ileal contraction (EC50 9.6 x 10(-8) M), which was markedly decreased in the presence of tetrodotoxin (TTX). Repeated exposure of the tissue to supramaximal concentrations of 5-HT did not however lead to desensitization. Atropine (10(-6) M), ketanserin (10(-5) M), methysergide (10(-5) M) and methiothepine (10(-6) M) attenuated the response to 5-HT. Ketanserin was an effective inhibitor of the residual response to 5-HT obtained even in the presence of TTX. Several serotonergic agonists were assayed to further analyse the type of receptors involved in the response to 5-HT. 5-methoxytryptamine (5-MOT), a mixed 5-HT1, 5-HT2 and 5-HT4 agonist, reproduced all the effects of 5-HT. 8-OH-DPAT, a selective 5-HT1A agonist, trifluoromethylphenylpiperazine, a mixed 5-HT1B/C agonist, and m-chlorophenylbiguanide, a 5-HT3 agonist, did not induce any consistent contractile effects. Sumatriptan, a 5-HT1D agonist, exerted a slight agonistic effect which was blocked by methiothepine and decreased by TTX but not by atropine. Cisapride, a 5-HT4 partial agonist in mammals, decreased the effects of both 5-HT and 5-MOT. These results indicate that chicken ileum contains 5-HT1 receptors similar to the 5-HT1D mammalian subtype but not the 5-HT1A, 5-HT1B, 5-HT1C or 5-HT3 subtypes. 5-HT2 receptors are also present and would appear to be located on smooth muscle.

Effect of cholecystokinin receptor antagonists on voluntary food intake in chickens

The effect of two cholecystokinin (CCK) receptor antagonists (L-364718 and L-365260) and of diazepam on voluntary food intake in chicken was studied. L-365260 significantly increased cumulative food intake at 30 and 60 min, whereas L-364718 had no effect. Diazepam also increased food intake at 30 minutes. It was therefore concluded that anxiolytic effects could account for an increase in food intake in the conditions under which the study was carried out. Consequently, the effect of L-365260 could either be due to its anxiolytic properties or to its direct effects on food intake control.

Central and peripheral cholecystokinin receptors in chickens differ from those in mammals.

Specific binding for the radioligand [3H]CCK-8s has been identified in chicken brain, hypothalamus, pancreas, gallbladder and caecum membranes. This binding was found to be of high affinity, low capacity and saturable, suggesting the presence of specific CCK receptors in these tissues. Scatchard analysis indicated the existence of a single binding site for each tissue. Dissociation constant (kd) values were 0.63 +/- 0.18, 0.73 +/- 0.13, 0.85 +/- 0.12, 1.47 +/- 0.21 and 0.96 nM for brain, hypothalamus, pancreas, caecum and gallbladder, respectively. Binding densities (Bmax) were higher for brain, pancreas and caecum (32.60 +/- 10.70, 30.33 +/- 2.40 and 35.83 +/- 5.10 fmol/mg protein, respectively) than for the other two tissues (9.75 +/- 1.90 and 6.31 fmol/mg protein for hypothalamus and gallbladder, respectively). As in mammals, CCK-4 shows high affinity for CCK receptors located in chicken brain and hypothalamus, and very low affinity for those located in peripheral structures. L-364,718 (a CCK-A antagonist) showed a relative selectivity and a high affinity for those receptors located in central tissues, whereas L-365,260 (a CCK-B antagonist) is almost inactive in all studied tissues. These results give support for the existence of at least two distinct CCK receptors in birds and that these receptors are relatively different from those described in mammals.

Effects of cholecystokinin on chicken cecal motility: Mechanisms involved

The aims of this work are to characterize the effects of cholecystokinin (CCK) on chicken ceca and to study in vitro the mechanisms through which such actions are mediated. Longitudinal and circular cecal strips kept in vitro in organ baths were responsive to CCK sulphated octapeptide (CCK-8s). On longitudinal strips the response consisted of a fast phasic contraction followed by a sustained increase in tone which was dose dependent and decreased markedly in the presence of tetrodotoxin (TTX). Ketanserin (10(-5) M) also caused a decrease in the CCK-8s response. CCK tetrapeptide (CCK-4) and CCK unsulphated octapeptide (CCK-8ns) induced slightly less contractile effects at concentrations of 2 x 10(-6) M only. L365,260 and L364,718 decreased the response of longitudinal strips to CCK-8s with similar efficacy. On circular strips CCK-8s caused rhythmic phasic contractions of dose dependent amplitude and frequency, and both effects were resistant to TTX. The EC50 for the amplitude was about 4 times higher than that for the frequency. CCK-8ns (2x 10(-6) M) also caused phasic contractions, whereas the same concentrations of CCK-4 did not elicit any motor effects. L365,260 and L364,718 showed different efficacy in decreasing amplitude or frequency of contraction. These results suggest that 1) Both muscularly and neurally located CCK receptors are present on the longitudinal layer of chicken ceca whereas only muscular receptors are present on the circular muscle. 2) 5HT2 receptors seem to be involved in the neurally mediated CCK-8s response observed in the longitudinal layer. 3) The different potency of CCK-8s, CCK-8ns and CCK4 to induce contractile effects and of the CCK-A and CCK-B antagonists to block such effects suggests the existence of two different CCK receptors on the circular layer.

Mechanisms mediating the effects of cholecystokinin on avian small intestine longitudinal smooth muscle.

The aims of this study were (1) to define the effects of CCK-8s and related peptides on chicken ileum longitudinal smooth muscle and (2) to explore the mechanisms by which such effects occur. The effects of CCK-8s were assayed in vitro on chicken longitudinal ileal strips. CCK-8s produced contraction of ileal strips (EC50 8.8.10(-9) M). CCK-8ns and CCK-4 did not have remarkable contractile effects even when added at concentrations 200-times higher than the EC50 for CCK-8s. L365,260 slightly inhibited the effects of CCK-8s whereas L364,718 was ineffective. Tetrodotoxin (10(-6) M) markedly decreased the effects of CCK-8s. Atropine (10(-6) M) did not modify the neurally mediated effects of CCK-8s, whereas ketanserin (10(-5) M) decreased the response to CCK-8s. Substance P-desensitized preparations exhibited reduced responses to CCK-8s. Our results indicate that CCK receptors present in chicken ileum behave similarly but not identically to the CCK-A receptor described in mammals. Most of these CCK receptors are neurally located but a minor proportion is also present on smooth muscle. The neurally mediated response to CCK-8s does not involve cholinergic mechanisms, but serotonin and substance P releasing neurons.

Cecocolonic motility in the chicken. Effects of cholecstokinin

The aims of this work were 1) to define electromyographically the motility pattern of chicken ceca and colon; 2) to study the changes induced by photoperiod and food intake on the motility of this area and 3) to characterize the motor effects of intravenous (i.v.) cholecystokinin in vivo, measuring changes in electrical activity and intracecal pressure. Electromyographical studies show that in ceca, the spike burst frequency is higher during the day than during the night and in the fed than in the fasted state; about 90% of the bursts propagate towards the apex and corresponde to filling movements. In the colon the spike burst frequency during the day is 2.9 bursts/min in animals fed ad libitum. Nocturnal recordings in animals fed ad libitum and diurnal recordings in fasted animals show a significantly decreased electrical activity. Both CCK-8s and CCK-4 induce a dose-dependent decrease of colonic electrical activity and a dose-dependent increase in the number of colonic defecations. CCK-4 also causes a slight inhibition in the cecum, whereas CCK-8s induces an increase in cecal electrical activity. Intracecal pressure recordings performed in anaesthetized animals provide similar results. In conclusion, the cecocolonic motility of the chicken displays a circadian pattern and undergoes substantial modifications in the fed compared to the fasted state. CCK-8s is not mediating the increased colonic activity that follows food intake, as its effects on colonic motility are inhibitory. In contrast, i.v. CCK-8s induces defecation and a dose dependent increase in cecal electrical activity, intraluminal pressure and colonic defecation. Intravenous CCK-4 induces inhibitory effects both on ceca and colon.

In vivo modulation of gastrointestinal motor activity by Met-enkephalin, morphine and enkephalin analogs in chickens

The main objectives of this work have been to study (1) whether or not opioid effects on gastrointestinal motility in chicken are mediated through central or peripheral pathways; (2) the receptors involved; (3) the correlation of the motor response to the distribution of Met-enkephalin in the gastrointestinal tract and (4) to evaluate the physiological role of endogenous opioids in spontaneous MMCs. Intravenous infusion of Met-enkephalin and morphine (5 x 10(-7) mol/kg) induced gastric inhibition and a migrating intestinal hyperactivity. Induced intestinal activity was faster in vagotomized chickens. In the stomach there was a correlation between the duration of the inhibitory response and the affinity of the agonists for mu-receptors ([D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAGO) > morphine > Met-enkephalin > Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE)). DPDPE induced duodenal hyperactivity which was not propagated. Immunohistochemistry showed that Met-ENK like material is mainly located at the myenteric plexus and the outer circular muscle in stomach. In the intestine, it was found in the myenteric and the deep muscular plexuses. When endogenous MMCs were studied, i.v. infusion of naloxone lengthened significantly their duration. In conclusion, the fact that Met-enkephalin and their analogs induced a migrating activity in the intestine and the lengthening of MMC by naloxone, suggest a physiological role for opioids on induction of MMC in birds, acting at peripheral level.

Opioid-induction of migrating motor activity in chickens

Enkephalin and morphine initiation of phase III of MMC has been reported in dog and humans. In chickens, a similar migrating activity initiated at the duodenum occurs 7-9 times a day while the gastric activity ceases. The main objective was to determine whether this migrating activity could be induced by opioids. Electrodes for electromyography were implanted in the stomach, proximal and distal duodenum, jejunum and proximal and distal ileum of 4 wk old chickens. Met-enkephalin, morphine and beta-casomorphin-5 (5 x 10(-7) moles/Kg) were infused i.v.. All these substances initiated an intestinal migrating activity concurrent with gastric inhibition. The mean duration of gastric inhibition depended on the substance, lasting from 5 min (met-enkephalin) to 27 min (beta-casomorphin-5). The migrating activity started in the distal duodenum and propagated to the ileum in about 18 min. These effects were partially blocked by naloxone at equimolar doses. In conclusion, in chickens, as in dogs and humans, migrating myoelectrical activity can be initiated by opioids.