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.     

The effect of leukotriene D4 on the isolated stomach and colon of the rat

The effect of synthetic leukotriene D4 (LTD4) was evaluated on isolated gastric longitudinal or circular smooth muscle and distal colon of the rat. The concentrations of LTD4, 2.5 X 10(-10)M to 5 X 10(-7)M, evoked minimal to maximal contractile responses. In addition, selected prostaglandins were used to identify the mediator of LTD4-induced contraction of gastric smooth muscle. FPL 55712 inhibited LTD4-induced contractions of gastric longitudinal or circular muscle. Indomethacin inhibited only LTD4-induced contractions of the longitudinal muscle. A combination of FPL 55712 and indomethacin produced greater inhibition of LTD4-induced contractions of longitudinal muscle than either agent alone. However, the same combination of inhibitors showed no greater effect than FPL 55712 alone on LTD4-induced contractions of circular smooth muscle. Unlike PGI2, PGF2, PGA2, or PGD2, PGE2 evoked contraction of the longitudinal muscle and relaxation of the circular muscle of the stomach. The dissimilar effect of PGE2 in the two smooth muscle layers of the rat stomach may signify that PGE2 is the prostaglandin released by LTD4 from the longitudinal and circular gastric muscle. However, the opposing pharmacologic effects following LTD4-induced release of prostaglandins in the circular muscle of the stomach would preclude the appearance of an inhibitory effect of indomethacin in this tissue. In contrast, PGE2 and other prostaglandins contract gastric longitudinal muscle in response to LTD4. Thus, these studies clearly suggest that LTD4 has both a direct and indirect effect on gastric smooth muscle of the rat. Unlike the stomach, LTD4-induced contraction of the distal colon was not inhibited by indomethacin while FPL 55712 antagonized contractions. Thus, these findings indicate a differential mechanism of stimulation of rat gastrointestinal tissue by LTD4.     

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.     

Expression and function of KCNH2 (HERG) in the human jejunum

Previous studies suggest that ether-a-go-go related gene (ERG) KCNH2 potassium channels contribute to the control of motility patterns in the gastrointestinal tract of animal models. The present study examines whether these results can be translated into a role in human gastrointestinal muscles. Messages for two different variants of the KCNH2 gene were detected: KCNH2 V1 human ERG (HERG) (28) and KCNH2 V2 (HERG(USO)) (13). The amount of V2 message was greater than V1 in both human jejunum and brain. The base-pair sequence that gives rise to domains S3-S5 of the channel was identical to that previously published for human KCNH2 V1 and V2. KCNH2 protein was detected immunohistochemically in circular and longitudinal smooth muscle and enteric neurons but not in interstitial cells of Cajal. In the presence of TTX (10(-6) M), atropine (10(-6) M). and l-nitroarginine (10(-4) M) human jejunal circular muscle strips contracted phasically (9 cycles/min) and generated slow waves with superimposed spikes. Low concentrations of the KCNH2 blockers E-4031 (10(-8) M) and MK-499 (3 x 10(-8) M) increased phasic contractile amplitude and the number of spikes per slow wave. The highest concentration of E-4031 (10(-6) M) produced a 10-20 mV depolarization, eliminated slow waves, and replaced phasic contractions with a small tonic contracture. E-4031 (10(-6) M) did not affect [(14)C]ACh release from enteric neurons. We conclude that KCNH2 channels play a fundamental role in the control of motility patterns in human jejunum through their ability to modulate the electrical behavior of smooth muscle cells.     

The influence of prostaglandin E2 (PGE2) on the acetylcholine-initiated contractions of isolated gastric muscularis muscle of Bufo marinus

Acetylcholine (ACh) (1.5 X 10(-5) M) elicited three different types of tonic and phasic contraction of muscularis muscle from different parts (cardiac, middle and pyloric) of the stomach of Bufo marinus. Prostaglandin E2 (PGE2) (10(-9)-10(-6) M) induced a concentration-dependent relaxation of tonic contractions elicited by ACh (1.5 x 10(-5) M) of strips from the cardiac part while potentiating the phasic contractions from the middle part of the stomach. PGE2 (10(-7) M) relaxed tonic contraction and potentiated phasic contraction concomitantly in preparations in which tonic and phasic contractions were elicited by ACh (1.5 x 10(-5) M). The effects of PGE2 on the preparation are related to the part of the stomach from where the strips are prepared and the muscle tone of the preparation.     

An in vitro study on spontaneous myometrial contractility in the cow during estrus and diestrus

Myometrial smooth muscle strips were collected from slaughtered cows in estrus and diestrus. Longitudinal and circular smooth muscle strips were mounted in organ baths and after equilibration time and 2 g preload, their physiologic contractility was recorded for 3 h. Area under the curve (AUC), mean amplitude (MA) and frequency of contractions (F) were studied. Differences between cycle phases, between muscle layers and over the recorded time period were statistically evaluated. In the cow, physiologic contractility patterns (measured as AUC and MA) of circular versus longitudinal myometrial strips are always different during the 3 h recording. Significant differences between estrus versus diestrus are only found for circular layers, but not for longitudinal layers. Significant differences over time are only found for longitudinal layers.     

Inhibitor effect of omeprazole in isolated human myometrial smooth muscle.

The aim of the present study was to investigate the effect of omeprazole, an H+-K+-ATPase inhibitor, in myometrial smooth muscle strips from women undergoing elective caesarean section at term. Isolated myometrial strips taken with informed consent were obtained from eight pregnant women undergoing elective caesarean section at term (not in labour) and mounted in organ baths for recording of isometric tension. We recorded the effect of increasing concentrations of omeprazole on spontaneous and Ca2+-induced contractions of myometrial smooth muscle and on contractions of myometrial smooth muscle pretreated with indomethacin (3 x 10(-6) M) and L-NAME (3 x 10(-5) M). Omeprazole (10(-4)-10(-3) M) decreased the amplitude and frequency of spontaneous contractions in a time- and concentration-dependent manner in all myometrial smooth muscle isolated from pregnant women. The decrease in amplitude of contractions in myometrial smooth muscle reached statistical significance beginning from the concentration of 3 x 10(-4) M. Addition of indomethacin (3 x 10(-6) M) and L-NAME (3 x 10(-5) M) in to the organ baths 30 min before did not change relaxation responses to omeprazole. When 8 mM Ca2+-precontracted in Ca2+-free medium myometrial smooth muscle were exposed to increasing concentrations of omeprazole (10(-5)-10(-3) M), omeprazole produced relaxation responses in a time- and concentration-dependent manner, reaching statistical significance at 10(-4) M. These results show: (1) omeprazole time- and concentration-dependently decreased spontaneous contractile activity in myometrial smooth muscle isolated from pregnant women, (2) omeprazole-induced relaxations was not influenced by indomethacin and N(G)-nitro-L-arginine methyl ester (L-NAME), suggesting that it is not mediated by cyclooxygenase products and nitric oxide, and (3) omeprazole brought about time- and concentration-dependently relaxation of myometrial smooth muscle precontracted by 8 mM Ca2+ in Ca2+-free medium. This effect of omeprazole may be due to blockade of the calcium channels.     

Oxytocin-induced phasic and tonic contractions are modulated by the contractile machinery rather than the quantity of oxytocin receptor

To investigate the relationship between the oxytocin (OT) receptor (OTR) quantity and the contractile features systematically, we measured the mRNA expression levels of OTR and L-type Ca(2+) channel alpha(1C)-subunit (alpha(1C)) and examined the regulatory mechanisms of OT-induced phasic or tonic contractions of the longitudinal smooth muscles in mouse uteri. The mRNA expression of OTR in 19.0 G (19.0 days of gestation) was greater than those in nonpregnant phases, and that of alpha(1C) in estrus and 19.0 G was higher than in diestrus. OT-induced contractions sparsely occurred in diestrus. The OT-induced all-or-none-type phasic contractions at low concentrations were abolished by verapamil in both estrus and 19.0 G. OT-induced tonic contractions had similar pD(2) values in both estrus and 19.0 G. However, the magnitude in 19.0 G was much greater than that in estrus. The large tonic contractions also occurred in PGF(2alpha) receptor (FP) knockout mice in 19.0 G despite a small amount of OTR. Verapamil and Y-27632 partially inhibited the tonic contractions in 19.0 G. Cyclopiazonic acid-induced tonic contractions were reciprocally decreased with the increase in the OT-induced ones in 19.0 G. These results indicate that the phasic contractions are dependent on alpha(1C). The tonic contractions in 19.0 G are dependent on both Ca(2+) influxes via L-type Ca(2+) channels and store-operated Ca(2+) channels, and the force is augmented by the Rho signal pathway, which increases the Ca(2+) sensitivity. Thus the uterine contractions are mainly controlled by the modification of contractile signal machinery rather than simply by the OTR quantity.     

Mechanism of the effect of dibasol on the contractile and electric activities of the smooth muscle of the portal vein

The effects of dibasol on spontaneous electrical and contractile activities as well as on the reactions evoked by hyperkalemic solution and noradrenaline were studied in smooth muscle of rabbit portal vein. It was shown that dibasol blocked the potential-operated influx Ca2+ into smooth muscle cells. The noninactivating calcium channels were found to be more sensitive to dibasol than inactivating ones. Significant part of the tonic contraction induced by noradrenaline was resistant to dibasol suggesting its weak effect on Ca2+ influx through calcium channels operated by alpha 1-adrenoceptors. It is supposed that vasodilative effect of dibasol is associated with blocking the influx Ca2+ through potential-operated noninactivating calcium channels into smooth muscle cells.     

Functional and molecular characterization of voltage-gated sodium channels in uteri from nonpregnant rats

We investigated the function and expression of voltage-gated Na(+) channels (VGSC) in the uteri of nonpregnant rats using organ bath techniques, intracellular [Ca(2+)] fluorescence measurements, and RT-PCR. In longitudinally arranged whole-tissue uterine strips, veratridine, a VGSC activator, caused the rapid appearance of phasic contractions of irregular frequency and amplitude. After 50-60 min in the continuous presence of veratridine, rhythmic contractions of very regular frequency and slightly increasing amplitude occurred and were sustained for up to 12 h. Both the early and late components of the contractile response to veratridine were inhibited in a concentration-dependent manner by tetrodotoxin (TTX). In small strips dissected from the uterine longitudinal smooth muscle layer and loaded with Fura-2, veratridine also caused rhythmic contractions, accompanied by transient increases in [Ca(2+)](i), which were abolished by treatment with 0.1 microM TTX. Using end-point and real-time quantitative RT-PCR, we detected the presence of the VGSC alpha subunits Scn2a1, Scn3a, Scn5a, and Scn8a in the cDNA from longitudinal muscle. The mRNAs of the auxiliary beta subunits Scbn1b, Scbn2b, Scbn4b, and traces of Scn3b were also present. These data show for the first time that Scn2a1, Scn3a, Scn5a, and Scn8a, as well as all VGSC beta subunits are expressed in the longitudinal smooth muscle layer of the rat myometrium. In addition, our data show that TTX-sensitive VGSC are able to mediate phasic contractions maintained over long periods of time in the uteri of nonpregnant rats.     

Mechanisms of alpha-adrenoceptor mediated contractions of rat mesenteric artery

The nature of the calcium channels associated with the activation of alpha-adrenoceptors in vascular smooth muscle has been investigated. The inhibitory effects of nitrendipine, a calcium antagonist, were studied on the contractions elicited by alpha-adrenoceptor agonists in rat superior mesenteric artery. Responses to equieffective concentrations of phenylephrine (alpha 1-adrenoceptor agonist), clonidine and BHT-920, (alpha 2-adrenoceptor agonists), and noradrenaline (nonselective agonist) were inhibited differentially by the calcium antagonist, with the sensitivity order being as follows: BHT-920 = clonidine greater than phenylephrine greater than noradrenaline. When the contractions to two doses of noradrenaline were compared, the low dose response was more sensitive to nitrendipine inhibition than the high dose response. This differential inhibition was not seen for noradrenaline in the presence of verapamil or for phenylephrine in the presence of nitrendipine. The contractions of the vessel to the agonists in zero calcium conditions were not significantly different from each other. The sensitivity differences among the agonists to nitrendipine may arise from differences in the postreceptor mechanisms of activation. The differential sensitivity of noradrenaline responses suggests a greater heterogeneity of calcium channels than those available for the other agonists.     

CyPPA, a positive modulator of small-conductance Ca(2+)-activated K(+) channels, inhibits phasic uterine contractions and delays preterm birth in mice

Organized uterine contractions, including those necessary for parturition, are dependent on calcium entry through voltage-gated calcium channels in myometrial smooth muscle cells. Recent evidence suggests that small-conductance Ca(2+)-activated potassium channels (K(Ca)2), specifically isoforms K(Ca)2.2 and 2.3, may control these contractions through negative feedback regulation of Ca(2+) entry. We tested whether selective pharmacologic activation of K(Ca)2.2/2.3 channels might depress uterine contractions, providing a new strategy for preterm labor intervention. Western blot analysis and immunofluorescence microscopy revealed expression of both K(Ca)2.2 and K(Ca)2.3 in the myometrium of nonpregnant (NP) and pregnant (gestation day 10 and 16; D10 and D16, respectively) mice. Spontaneous phasic contractions of isolated NP, D10, and D16 uterine strips were all suppressed by the K(Ca)2.2/2.3-selective activator CyPPA in a concentration-dependent manner. This effect was antagonized by the selective K(Ca)2 inhibitor apamin. Whereas CyPPA sensitivity was reduced in D10 and D16 versus NP strips (pIC(50) 5.33 ± 0.09, 4.64 ± 0.03, 4.72 ± 0.10, respectively), all contractions were abolished between 30 and 60 μM. Blunted contractions were associated with CyPPA depression of spontaneous Ca(2+) events in myometrial smooth muscle bundles. Augmentation of uterine contractions with oxytocin or prostaglandin F(2α) did not reduce CyPPA sensitivity or efficacy. Finally, in an RU486-induced preterm labor model, CyPPA significantly delayed time to delivery by 3.4 h and caused a 2.5-fold increase in pup retention. These data indicate that pharmacologic stimulation of myometrial K(Ca)2.2/2.3 channels effectively suppresses Ca(2+)-mediated uterine contractions and delays preterm birth in mice, supporting the potential utility of this approach in tocolytic therapies.     

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.     

EDRF-release and Ca+(+)-channel blockade by magnolol, an antiplatelet agent isolated from Chinese herb Magnolia officinalis, in rat thoracic aorta

Magnolol is an antiplatelet agent isolated from Chinese herb Magnolia officinalis. It inhibited norepinephrine (NE, 3 microM)-induced phasic and tonic contractions in rat thoracic aorta. At the plateau of the NE-induced tonic contraction, addition of magnolol caused two phases (fast and slow) of relaxation. These two relaxations were concentration-dependent (10-100 micrograms/ml), and were not inhibited by indomethacin (20 microM). The fast relaxation was completely antagonized by hemoglobin (10 microM) and methylene blue (50 microM), and disappeared in de-endothelialized aorta while the slow relaxation was not affected by the above treatments. Magnolol also inhibited high potassium (60 mM)-induced, calcium-dependent (0.03 to 3 mM) contraction of rat aorta in a concentration-dependent manner. 45Ca(+)+ influx induced by high potassium or NE was markedly inhibited by magnolol. Cyclic GMP, but not PGI2, was increased by magnolol in intact, but not in de-endothelialized aorta. It is concluded that magnolol relaxed vascular smooth muscle by releasing endothelium-derived relaxing factor (EDRF) and by inhibiting calcium influx through voltage-gated calcium channels.     

Effect of papaverine on the longitudinal and circular smooth muscle of the guinea-pig caecum.

Electrical and mechanical activity of smooth muscle of the guinea-pig caecum was recorded by means of the sucrose-gap technique. The responses of longitudinal and circular smooth muscle to acetycholine were differently affected by changes of extracellular calcium concentration (0.8, 2.5 and 7.5 mM). The contractions of both preparations were depressed at high Ca++ concentrations, whereas at low Ca++ concentrations only contractions of the circular smooth muscle were augmented. The stimulatory effect of acetylcholine was decreased by papaverine in both preparations at all three concentrations of Ca++. This inhibition was the greater the lower the concentration of extracellular Ca++ and this process was more pronounced in the circular muscle. The ability of papaverine to counteract the effect of lowered concentrations of extracellular Ca++ on membrane excitability may well explain its inhibitory effect upon intestinal smooth muscle.     

Sensitization does not alter phasic and tonic responses to carbachol in isolated epithelium-free guinea-pig trachea

An alteration in the handling of Ca2+ has been proposed as the pathogenic mechanism underlying the airway smooth muscle hyperresponsiveness of asthma. The present study tested the hypothesis that the altered responsiveness of receptor operated contraction to carbachol in allergic asthma results from a change in the phasic or tonic components. Using a kinetic approach, the phasic and tonic responses to 10 microM carbachol were quantitated in isolated epithelium-free trachea 21 days after guinea-pigs were sensitized with ovalbumin and aluminum hydroxide (as adjuvant) to generate preferentially IgE-like antibodies. Sensitization was confirmed by challenge of the isolated trachea with ovalbumin. The steady-state and kinetic characteristics of the phasic and tonic responses were the same from sensitized animals and animals treated with saline and aluminum hydroxide (control) and before and after challenge of the trachea from both groups of animals. The present results demonstrate that immunologic sensitization and challenge do not appear to elicit a defect in the phasic or tonic responses of receptor mediated contractions in airway smooth muscle and suggest there is no alteration in the handling of Ca2+ in smooth muscle from sensitized and challenged guinea-pig trachea.     

Inhibitory and excitatory mechanisms of neurotensin action in canine intestinal circular muscle in vitro.

The effect of neurotensin on canine ileal circular muscle devoid of myenteric plexus was investigated using single and double sucrose gap techniques. Similar results were obtained with microelectrode techniques. Neurotensin caused a temperature-sensitive and dose-dependent biphasic response, an initial hyperpolarization associated with inhibition of contractile activity, followed by an excitatory phase, usually consisting of spike discharge and tonic and phasic contractions, for which depolarization was not required. Neither response was affected by tetrodotoxin, phentolamine, propranolol, or atropine. The hyperpolarization was associated with decreased membrane resistance, blocked by 10(-7) M apamin, and converted to tonic depolarization by apamin (10(-6) M). Tachyphylaxis to neurotensin occurred when the stimulation interval was less than 20 min. After Ca2+ depletion, depolarization was observed instead of the hyperpolarization; this depolarization was not affected by nitrendipine and was gradually abolished with repetitive stimulation at 20-min intervals. When Ca2+ was present, nifedipine did not alter the hyperpolarizing phase of the response but inhibited spiking and blocked all contractions. The excitatory phase of the response was enhanced by Bay K-8644. Neuromedin N elicited a response identical with that of neurotensin. The responses of the two peptides were completely cross tachyphylactic. Inhibitory junction potentials were not affected by neurotensin tachyphylaxis. It is concluded that neurotensin and neuromedin N activate apamin-sensitive, calcium-dependent potassium channels in circular muscle, causing membrane hyperpolarization and inhibition of muscle contraction. Release of intracellular calcium is involved in the activation of these potassium channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bombesin-induced changes in membrane potential-dependent phasic contractions of cat gastric muscle

Electrical and contractile activities of smooth muscle strips isolated from the circular muscle layer of cat gastric antrum were studied using the sucrose gap technique. Bombesin (10(-8) mol/l) depolarized the gastric muscle; this was accompanied by an increase in the strip tone, in the plateau action potential frequency and in both the frequency and the amplitude of the spike potentials as well as by a shortening of the plateau action potential duration. Both the frequency and the amplitude of the phasic contractions increased thereafter. The changes in the frequency of the plateau action potentials and contractions were not influenced either by antagonists of cholinergic and adrenergic receptors or by TTX. In the presence of the Ca antagonists D600 (10(-6) mol/l) and nifedipine (10(-7) mol/l) or in Ca-free medium containing EGTA the effect of bombesin on the frequency of the plateau action potentials and phasic contractions remained unchanged; however, spike potentials were not observed and no increase in the amplitude of phasic contractions occurred. UV-light inactivation of nifedipine restored the typical bombesin effect on the electrical and contractile activities of the gastric smooth muscle. The present data suggest that the effect of bombesin on the frequency of both plateau action potentials and phasic contractions is not linked with Ca2+ influx.     

The role of intra- and extracellular Ca in the activation of contraction of pulmonary artery smooth muscles induced by serotonin

In Ca-free EGTA-containing solution serotonin induced a transient contraction of rabbit pulmonary artery smooth muscle which decayed to nearly steady-state level accounted for 17.7 +/- 1.6% of original contraction in Krebs solution. Both phasic and tonic components of this contraction were effectively inhibited by verapamil and Cd2+. Caffeine induced no contraction of muscle strips if it was applied after withdrawal of serotonin. But when the sequence of these drugs application was reversed, serotonin still evoked contraction with reduced phasic component. The results obtained in these experiments suggest, that serotonin-induced contraction of pulmonary artery smooth muscle is partly (less than 20%) due to mobilization of bound calcium from at least two stores located on the opposite sides of the cell membrane. Calcium released from external store site enters the cell via receptor-operated calcium channels.