Effects of Met-enkephalin on the mechanical activity and distribution of Met-enkephalin-like immunoreactivity in the cat large intestine.

The effects of Met-enkephalin on the spontaneous and electrically evoked activity were investigated in longitudinal and circular strips isolated from different regions of the large intestine, i.e., proximal colon, distal colon and rectum. Met-enkephalin induced dose-dependent contractile responses which were reversibly blocked by naloxone (10(-6) M). In all longitudinal strips and in the circular strips of the rectum, the effects of Met-enkephalin were prevented by TTX (10(-7) M), demonstrating their neurogenic nature. In the circular strips from the colon, Met-enkephalin induced contractile responses after TTX, proving the existence of smooth muscle opioid receptors. The comparison between the EC50 values of Met-enkephalin showed that the opioid receptors in the different regions have different sensitivity to Met-enkephalin, while the opioid receptors in the longitudinal and circular layers of the same region have equal affinity. Atropine (10(-6) M) and guanethidine (10(-6) M) did not alter significantly the EC50 values, showing that the neurogenic effects of Met-enkephalin on the spontaneous activity involve mainly nonadrenergic, noncholinergic (NANC) neurotransmitter mechanisms. When the preparations were stimulated electrically, Met-enkephalin (10(-9) M) suppressed the cholinergic components of the responses. Met-enkephalin-containing nerve fibers were found in the myenteric plexus of the three intestinal regions. In the colon, where direct smooth muscle effects were observed, fibers containing Met-enkephalin-like immunoreactivity were found to go deep into the circular layer, suggesting that they could supply Met-enkephalin input to the smooth muscle cells.     

Peptide YY induces nerve-mediated responses in the guinea pig intestine.

The actions of peptide YY (PYY) were studied in longitudinal organ-bath preparations of the guinea pig intestine. PYY induced concentration-dependent (10(-9)-5 x 10(-8) M) relaxations of tissue from the duodenum, jejunum, ileum, and colon. These responses were unaffected by adrenergic blockade and atropine treatment but could be prevented by tetrodotoxin. The pharmacology of PYY actions in segments of the small and large intestine indicated the involvement of intrinsic nonadrenergic, noncholinergic inhibitory neurones in the relaxation response to this peptide. All tissues could be made tachyphylactic to PYY without affecting their ability to respond to the direct acting muscle relaxants ATP or papaverine. Moreover, nicotinic ganglion stimulated relaxations and cholinergic nerve-mediated contractions were also unaffected. These results show applied PYY to have potent neurogenic actions in the guinea pig intestine with some similarities to PYY actions in the rat intestine.     

Functional and immunocytochemical evidence that galanin is a physiological regulator of human jejunal motility.

The neuropeptide galanin has species-dependent effects on intestinal motility. It has a contractile effect on rat jejunal muscle while it relaxes guinea-pig ileum by inhibiting cholinergic transmission. Its effect on human gut motility has been unknown. Extensive work led to the discovery of selective galanin analogues such as M15 [galanin(1-12)-Pro-substance-P(5-11)], M35 [galanin(1-12)-Pro-bradykinin(2-9)-amide] that competitively inhibit various actions of galanin in the central nervous system. The present study was designed to examine the effect of galanin, M15 and M35 on longitudinal jejunal smooth muscle strips isolated from humans and rats, and to localize galanin-immunoreactivity in human jejunum. Galanin and ACh were equally effective in stimulating contractions of the isolated jejunal muscle: sigmoid curve fitting showed that maximal contractile response to galanin and ACh were 25.7+/-11.1 mN and 23.7+/-9.7 in humans, while 8.0+/-0.6 and 8.1+/-0.3 mN in rats, respectively. These effects of galanin were not inhibited by either atropine (5 x 10(-6) M) or tetrodotoxin (3 x 10(-6) M). The potency of galanin inducing the contractile actions were similar in humans and rats. Interestingly, neither M15 nor M35 (up to 10(-7) M) were able to inhibit the responses of the smooth muscle to galanin. However, both putative galanin receptor antagonists showed agonist effects in our experimental models. In accordance with the functional studies, both the longitudinal and the circular muscle layers were abundant in nerve fibers and varicosities showing galanin immunoreactivity. Our data suggest that galanin is a potent physiological regulator of jejunal contractions in humans. Its action on the jejunum, however, is mediated by galanin receptors that are different from those located in the central nervous system.     

Enteric opioid neurons modulate the basal tone of the isolated puppy ileum

We studied the role of enteric opioid neurons in the spontaneous motility of the longitudinal muscle in the isolated puppy ileum. Regular fluctuations in tone that rose above and returned to the basal level occurred at an interval of 4.7 +/- 0.3 min. Naloxone (10(-8) and 10(-7) M) reduced the spontaneous tonic contraction by 42.6 +/- 11.6% (p less than 0.02) and 77.0 +/- 3.6% (p less than 0.001), respectively. Tetrodotoxin (3.1 X 10(-7) M) and atropine (10(-7) M) terminated the fluctuations. Met- and Leu-enkephalins (10(-9)-10(-8) M) caused tonic contraction which was abolished by tetrodotoxin and atropine. The contractile response produced by transmural electrical stimulation was reduced by naloxone (10(-7) M). This response was also abolished by atropine and tetrodotoxin. These results suggest that enteric opioid neurons are spontaneously active and might operate, at least in part, to raise the basal tone of the longitudinal muscle in the puppy ileum through a cholinergic excitatory mechanism.     

Interactions of neurogenic responses of longitudinal and circular muscle in the guinea-pig ileum

The relationship between neurogenic responses of longitudinal and circular muscle was studied by measuring contractions and EMG or nonadrenergic, non-cholinergic (NANC) relaxations and NANC inhibitory junction potentials in different preparations of the guinea-pig ileum. NANC relaxation of longitudinal muscle was observed also without any preceding or concomitant circular muscle contraction ruling out the possibility that the latter might be the cause of the NANC relaxation. Circular muscle twitches or powerful contractions were absent if there was no preceding neurogenic or myogenic excitation of longitudinal muscle; in preparations with myenteric plexus-longitudinal muscle layers removed only small residual responses were seen although still under neurogenic influences. Thus excitation of longitudinal muscle seemed a prerequisite for synchronized and powerful contractions of circular muscle to occur. Cholinergic contraction and NANC relaxation of longitudinal muscle evoked by field stimulation were partly inhibited if the submucous plexus was also present suggesting the involvement of a more complex neuronal circuitry in these responses.     

Calcitonin gene-related peptide (CGRP) modulates cholinergic neurotransmission in the small intestine of man, pig and guinea-pig via presynaptic CGRP receptors.

The effect of calcitonin gene-related peptide (CGRP) on the cholinergically mediated twitch contraction in longitudinal muscle strips of the small intestine (duodenum, jejunum, ileum) of guinea-pig, pig and man was investigated. Independently of the anatomical region, CGRP inhibited the twitch response in the different specimens of all three species by about 40% with similar IC50 values (1.5-2.4 nmol/l). Only in the guinea-pig small intestine CGRP induced a contraction of the smooth muscle which was sensitive to scopolamine and tetrodotoxin. The electrically evoked [3H]acetylcholine release from jejunal longitudinal muscle strips with myenteric plexus attached of the guinea-pig, which were incubated with [3H]choline, was concentration-dependently inhibited by CGRP. A direct relaxant effect of CGRP on smooth muscle tone of carbachol precontracted preparations was only observed in specimens of the guinea-pig. In conclusion, presynaptic inhibitory CGRP receptors on cholinergic neurones modulate the release of acetylcholine in different parts of the small intestine.     

Action of FMRFamide-like Peptides on Porcine Gastrointestinal Motility In Vitro

Decker, B., B. Vadokas, U. Kutschenreuter, K. Golenhofen, K. Voigt, G. P. Mcgregor and K. Mandrek. Action of FMRFamide-like peptides on porcine gastrointestinal motility in vitro. Peptides 18(10) 1531–1537, 1997.—Mechanical activity was recorded in circular and longitudinal smooth muscle preparations isolated from extensive regions of the porcine gastrointestinal tract in response to the FMRFamide-like neuropeptides F8Famide and A18Famide. In all preparations, the peptides were about equipotent in producing phasic contractions or enhancing spontaneous activity. The most prominent responses were observed in jejunal longitudinal strips which were on the average 91% (±4% SEM, n = 15; 10⁻⁶ M) of the histamine (10⁻⁵ M) responses. The peptide-induced phasic activity was completely abolished by nifedipine but was unaffected by tetrodotoxin, atropine, phentolamine, yohimbine, phenoxybenzamine, propranolol, methysergide, cimetidine, indomethacin, levallorphane or naloxone. Both peptides enhanced acetylcholine-induced contractions. However, bovine ileum and guinea-pig taenia coli was not affected by these peptides. The results indicate that F8F- and A18F-amide contract porcine gastrointestinal smooth muscle by acting directly via non-opioid receptors on L-type calcium channels. In addition an increase of the sensitivity to cholinergic stimulation occurs.     

Contractile effects of 16-methyl analogues of PGE2 on the circular and longitudinal muscles of the guinea-pig isolated colon.

The mechanical effects of 16-methyl analogues of PGE2, mainly 16,16-dimethyl PGE2, on circular and longitudinal muscles of the guinea-pig isolated proximal colon were investigated. In circular muscle strips, PGE2 100 nM produced an initial contraction followed by relaxation, while 16(R)-methyl PGE2 and 16,16-dimethyl PGE2 (1 nM - 1 microM) produced sustained contractions. In longitudinal muscle strips, PGE2 and 16-methyl analogues of PGE2 produced only contractions. The contractile responses of both muscle strips to 16,16-dimethyl PGE2 were not influenced by atropine or tetrodotoxin, indicating that these analogues act directly on the muscles, but were eliminated by the omission of extracellular Ca ions or in the presence of 1 mM lanthanum ions. However, verapamil, a Ca channel blocker, did not block the contractile response to the methyl analogues in circular muscle strips, although it completely inhibited the contractile response of longitudinal muscle strips. These results suggest that the contractile effect of 16-methyl analogues of PGE2 on the circular muscle may be due to an increased influx of Ca ions mainly via receptor-sensitive and partly voltage-sensitive Ca channels, while the contractile effect of the analogues on the longitudinal muscle may be due to an increase in influx of Ca ions via voltage-sensitive Ca channels.     

Spontaneous electromechanical activity in the rat duodenum in vitro.

Isolated rat duodenum shows spontaneous mechanical and electrical activities. Mechanical activity consists in changes both in endoluminal pressure and in isometric tension. Electrical activity is characterized by slow waves with superimposed bursts. This spontaneous activity is tetrodotoxin (TTX) resistant and therefore it is myogenic in origin. Indeed, TTX pretreatment, even in the presence of atropine and guanethidine, caused an increase in amplitude and in frequency of the electrical and mechanical activities. This finding indicates the presence of tonically active inhibitory intramural non adrenergic, non cholinergic (NANC) nerves. Duodenal longitudinal strips showed a spontaneous mechanical activity resembling that one recorded from isolated segment. Instead, circular strips are quiescent under resting condition and a contractile activity can be detected only after TTX pretreatment suggesting that: i) the circular smooth muscle layer is tonically inhibited by intramural NANC nerves and, ii) the contractions observed in the rat duodenum are due to the activity of the longitudinal one.     

Application of drug receptor theories to the analysis of the myotropic effects of bradykinin.

Cat jejunum and terminal ileum, and rat stomach strip and rat uterus contract to bradykinin, while rat duodenum relaxes. Dose-response curves of classical hyperbolic shape are obtained in the first three preparations, but not in the others. The negative logs of the drug concentrations which give 50% of the maximal response. (pD2) Values were, respectively, 7.68 and 7.77 in the cat jejunum and terminal ileum, 6.78 in the rat stomach strip and 8.64 in the rat uterus in estrus. Theoretical dose-response curves, constructed by using experimental pD2 values in the equation of Clark, (General pharmacology. Verlag Van J. Springer, Berlin, 1937), are superimposed to experimental curves, obtained in the cat jejunum and terminal ileum, but not in the rat stomach strip. This comparison was not made in the rat uterus and duodenum. The myotropic effect of bradykinin appears to be a direct one in the cat jejunum, the terminal ileum and the rat stomach strip, because it is not affected by anticholinergics, antiadrenergics, antihistaminics and indomethacin. pD2 values and the slope of the dose-response curves of the rat uterus were reduced by indomethacin. The results indicate that cat jejunum and terminal ileum are sensitive and specific for bradykinin and appear to be the most reliable preparations for studies on the structure-activity relationships of this peptide.     

The effects of ethylenediamine in the rat small intestine: a powerful relaxant of the muscularis.

The pharmacology of ethylenediamine (EDA) actions in the rat small intestine was examined using isolated gut-bath preparations of proximal segments of the duodenum, jejunum, and ileum. EDA evoked concentration-dependent tetrodotoxin-insensitive relaxations of the intestine, evidently by direct action on the muscularis. Such actions were simultaneous on the longitudinal and circular muscle layers. Investigation of EDA actions on the circular muscle showed that EDA actions were unrelated to any intrinsic GABAergic mechanisms. Moreover, EDA interacted with muscle sites distinct from ATP, histamine, bradykinin, muscarinic, and adrenergic receptors. The ability of EDA to relax the intestinal musculature was generally greater than the smooth muscle relaxant papaverine and substantially better than nicotinic stimulation of the intrinsic inhibitory neurones. It would appear that EDA may be useful as a direct acting smooth muscle relaxant for the study of the physiology-pharmacology of the rodent small intestine.     

Effects of PGD2 and PGF2α on longitudinal and circular muscles of guinea-pig isolated proximal colon

The mechanical effects of PGD₂ and PGF_2α on longitudinal and circular muscles of the guinea-pig isolated proximal colon were investigated. PGD₂ and PGF_2α (1 nM – 10 μM) produced a dose-dependent contraction in longitudinal and circular muscles. The contractile action of PGD₂ was more potent than that of PGF_2α in circular muscle and was less potent in longitudinal muscle.Contractions induced by PGD₂ or PGF_2α(1 μM) were unaffected by atropine (1 μM) in both muscles, but tetrodotoxin (1 μM) slightly inhibited these contractions in longitudinal muscle.The results suggest that in longitudinal muscle PGD₂ and PGF_2α have largely a direct action on the muscle cells and a partial neuronal action on the non-cholinergic intrinsic nerves, whereas in circular muscle these PGs have only a direct action on the muscle cells.     

Galanin: an inhibitory neural peptide of the canine small intestine

Galanin injected intraarterially during phasic activity of the canine small intestine in vivo produced inhibition. Fifty percent inhibition occurred at 1.5 +/- 0.5 X 10(-10) mols lasting for 0.7 min. The inhibitory response was not decreased by treatment with atropine, hexamethonium, yohimbine or naloxone, suggesting that muscarinic, nicotinic, alpha 2 adrenergic or opiate receptors were not being stimulated. Since tetrodotoxin blockade of nerves did not reduce the response and galanin at 10(-10) mols was able to eliminate the smooth muscle response to intraarterial acetylcholine, we suggest that galanin acts to inhibit smooth muscle directly. Galanin 10(-9) M added to the muscle bath also inhibited phasic activity of the canine ileum circular muscle in vitro in the presence of tetrodotoxin. These results suggest that the neural peptide galanin may be a non-adrenergic, non-cholinergic, non-opioid neurotransmitter in the canine small intestine.     

Effects of some autonomic drugs and neuropeptides on the mechanical activity of longitudinal and circular muscle strips isolated from the carp intestinal bulb (Cyprinus carpio)

1. The mechanical responses to some autonomic drugs and neuropeptides of longitudinal muscle (LM) and circular muscle (CM) strips isolated from the carp intestinal bulb were investigated in vitro. 2. Acetylcholine and carbamylcholine caused concentration-dependent transient contraction of both LM and CM strips. Tetrodotoxin had no effect, but atropine selectively decreased the contractile responses to acetylcholine and carbamylcholine. 3. Excitatory alpha-2 and inhibitory beta adrenoceptors were present in both LM and CM strips. 4. 5-Hydroxytryptamine (5-HT) caused concentration-dependent contraction of both LM and CM strips. Tetrodotoxin, atropine and methysergide decreased the contractile responses to 5-HT. 5. Some neuropeptides (angiotensin I, angiotensin II, bombesin, bradykinin, neurotensin, somatostatin and vasoactive intestinal polypeptide) did not cause any mechanical response (contraction or relaxation) in either smooth muscle strip. 6. Substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) caused contraction of both LM and CM strips. However, the time course of the contraction in LM was different from that in CM. The order of potency was NKA greater than SP greater than NKB in LM strips and NKA greater than SP much greater than NKB in CM strips. In LM strips, the contractile responses to tachykinins were unaffected by spantide and methysergide, but partly decreased by tetrodotoxin and atropine. On the other hand, the contractile responses of CM strips were unaffected by tetrodotoxin, atropine, methysergide and spantide. 7. Dynorphin (1-13) (DYN), leucine-enkephalin (L-Enk) and methionine-enkephalin (M-Enk) caused concentration-dependent contraction of both LM and CM strips. The order of potency was DYN greater than M-Enk greater than L-Enk. Naloxone selectively decreased the responses to opiate peptides. 8. The present results indicate that acetylcholine, carbamylcholine, catecholamines, 5-HT, tachykinins (SP, NKA and NKB) and opiate peptides (DYN, L-Enk and M-Enk) affect the mechanical activity of LM and CM strips isolated from the carp intestinal bulb through their specific receptors.     

Effect of prostaglandin F2 alpha on propulsive activity of the isolated segmental colon of the guinea-pig.

The effects of prostaglandin F2alpha (PGF 2alpha) on propulsive activity in segments of isolated colon and on isolated strips of guinea-pig colon were investigated. Using experimental conditions under which spontaneous propulsive activity was negligible, PGF2alpha (5X10(-8)X1X10(-6)M), added to the bathing medium increased propulsive activity in a concentration dependent manner. This increase of propulsive activity was abolished in the presence of atropine or tetrodotoxin (1X10(-7)g/ml). The contractions produced by PGF2alpha (5X10(-7) -1X10(-5)M) in isolated longitudinal and circular smooth muscle strips of guinea-pig colon were unaffected in the presence of atropine or tetrodotoxin (1X10(-7) g/ml). From these results it is concluded that under the conditions employed in this study propulsive activity stimulated by PGF2alpha may depend on the contractions of both muscle layers and stimulation of the peristalic reflex.     

Is there functional cholinergic innervation in the frog duodenum (Rana catesbeiana)?

To determine if functional cholinergic innervation occurs in the frog duodenum or not, the effects of exogenous acetylcholine and electrical transmural stimulation, the contractile activity of an acid extract from the frog duodenum, and the distribution of acetylcholinesterase (AChE) activity in the wall of the frog duodenum were investigated. Acetylcholine caused non-sustained contraction in a dose-dependent manner (100 nM-1 mM). The ED50 value was 17 +/- 2.4 microM. Atropine (500 nM) shifted the dose-response curve for acetylcholine parallel to the right. Transmural stimulation of the frog duodenum caused frequency-dependent (0.5-50 Hz) contraction which was not decreased by atropine (500 nM) at all. The acid extract from the frog duodenum caused contraction of a longitudinal muscle strip of guinea-pig ileum but atropine (500 nM) had no significant effect on the contraction. Only a little AChE activity was found in Auerbach's plexus of the frog duodenum compared with that of the rat ileum. These results suggest that a cholinergic nerve is present in the frog duodenum but its physiological significance is very small.     

Influence of PGF2 alpha on gastrointestinal activity in the conscious piglet.

In 5 conscious piglets with electrodes implanted on the antrum pylori, duodenum, jejunum and ileum, the effect of intravenous infusion of PGF2 alpha, 1 and 10 micrograms/kg/min during 2 h, on gastrointestinal electrical activity was studied. The influence of the PG, 10(-8) to 10(-4) M, on longitudinal tissue strips from the same segments was also examined. The in vitro results demonstrate that PGF2 alpha has only a weak contractile effect on duodenal and jejunal strips. This effect was enhanced in the presence of atropine and indomethacin. In the in vivo part of the study PGF2 alpha induced an inhibition of antral electrical activity as evidenced by a prolongation of the inhibitory phases and a reduction of the frequency of the fast oscillations. In the small intestine only ileal activity was changed significantly. PGF2 alpha provoked an increase in the phase II or irregular spiking activity and an increase in the interval of the migrating myoelectrical complexes in this segment.     

Effects of beta-endorphin, met-enkephalin and somatostatin on the neurotensin-induced neurogenic contraction in the guinea-pig ileum

At maximally effective concentrations, the opiate peptides β-endorphin (240 nm) and Met-enkephalin (1400 nM) virtually abolished the contractions induced by a maximally effective concentration of 60 nM neurotensin (NT), either in the longitudinal smooth muscle strip or in the intact segment of guinea-pig ileum. This inhibitory effect was concentration-dependent and was totally blocked by naloxone at 100 nM. In contrast a maximally effective concentration of somatostatin (60 nM) partially inhibited (50–60%) the contraction induced by 60 nM NT in either smooth muscle preparation. Somatostatin inhibition was concentration-dependent and was not blocked by naloxone at 100 nM. Atropine at 100 nM inhibited by 50% the contractions induced by 60 nM NT in the intact segment of guinea-pig ileum. The remaining contraction was abolished by β-endorphin and Met-enkephalin and partially reduced by somatostatin. Our results confirm that NT-induced contractions in the guinea-pig ileum are neurogenic and involve a cholinergic as well as a non-cholinergic component. Furthermore, we show that the release of mediators from both components     

化学药物对家兔离体小肠平滑肌电生理特性的影响

观察各种化学药物对家兔离体小肠各段平滑肌的作用,采用常规离体灌流的十二指肠、空肠及回肠平滑肌标本作舒缩运动实验,记录用药前后各段小肠平滑肌的收缩活动特征及变化规律.结果显示：不同浓度的乙酰胆碱和磷酸组织胺能增强小肠各段平滑肌的收缩频率与幅度,其幅值变化与用药前有显著性差异（P＜0.01） ,并呈剂量依赖性;而不同浓度的肾上腺素和阿托品则抑制小肠各段平滑肌（P＜0.01） .不同肠段对各种化学药物的作用存在着差异,一般十二指肠作用最强,空肠次之,回肠最差.     

The effects of H2O2 on electromechanical activity of the ileum longitudinal muscle

The effects of H2O2 on electrical and mechanical activity of the longitudinal layer from the guinea-pig ileum were studied using sucrose-gap technique and the influence of H2O2 on ionic current was investigated in single smooth muscle cells by the patch-clamp method. In most of the preparations tested, the spontaneous activity observed was composed of slow waves with superimposed action potentials (APs). Both were resistant to tetrodotoxin and atropine. H2O2 (1 mmol/l) evoked sustained 3-5 mV membrane depolarisation, doubled the amplitude of the slow waves and increased their frequency, augmented the APs and reduced their splitting. These changes were accompanied with significant contraction, which had an amplitude comparable to that of the tonic component of 50 mmol/l K+-induced contraction. Calcium-free solution caused membrane depolarisation, reduction of the slow wave amplitude and frequency, disappearance of APs and decreased the mechanical tension of the preparations. Application of H2O2 (1 mmol/l) into the zero-calcium bath solution recovered the APs, which was accompanied by a low amplitude contraction. H2O2 (up to 1 mmol/l) increased the L-type calcium current (I(Ca)) both under conventional whole-cell patch-clamp configuration and under amphotericin-perforated patches by 16 +/- 3%. These data demonstrated that contractile response of the ileum longitudinal smooth muscle preparation evoked by H2O2 was mainly due to the enhanced electrical activity.