Effects of Cinnamaldehyde on Smooth Muscle Preparations.

The effects of cinnamaldehyde (CNA), known as a transient receptor potential ankyrin 1 (TRPA1) agonist, on guinea-pig ileum and urinary bladder were studied in isolated organ experiments. Contractile effects were found to be present on both preparations. In the ileum, both cholinergic and purinergic (pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid tetrasodium salt-sensitive) mechanisms are involved; the TRPA1 antagonist A967079 (1 µmol/L) significantly reduced the response. The contractile response to CNA in the bladder, but not in the ileum, was significantly reduced by in vitro capsaicin desensitization. In the bladder A967079 or the TRPV1 antagonist, BCTC failed to reduce the response. A direct relaxation on the smooth muscle was detected in the precontracted ileum. In the precontracted urinary bladder, CNA also caused relaxation that was insensitive to capsaicin pretreatment. It is suggested that CNA excites the muscles of the bladder via activation of capsaicin-sensitive nerves; in the ileum, it may interact with TRPA1 located on tissue elements that initiate both purinergic and cholinergic mechanisms. The relaxant effects of CNA may be due to the direct inhibition of the smooth muscles.

Effects of the venom of the brown bullhead catfish (Ameiurus nebulosus) on isolated smooth muscles.

Aqueous extract of the spines of the brown bullhead catfish (Ameiurus nebulosus Lesueur, 1819) caused contraction of the guinea-pig small intestine in vitro, a widely-used preparation in pharmacology. The action is dependent on extracellular Ca2+, and probably takes place on the smooth muscle cells. Mouse gastrointestinal preparations were also contracted by the extract. Stings by the spines of this fish species causes a painful sensation in man. We tested the effect of an extract of spines in isolated organ experiments on innervated smooth muscle preparations. In the guinea-pig ileum, the response to the extract was abolished by the Ca2+-channel blocker nifedipine, but only slightly reduced by atropine (a muscarine receptor antagonist) or tetrodotoxin (TTX; a blocker axonal conduction) or antagonists for P2X purinoceptors. Blocking of serotonin or histamine H1 receptors, tachykinin NK1 receptors, functional impairment of capsaicin-sensitive sensory nerve endings or inhibition of cyclo-oxygenases failed to influence the contractile effect of the extract. No inhibitory action of the extract was detected on the ileum subject to electrical motor nerve stimulation.

Evidence Supports Tradition: The in Vitro Effects of Roman Chamomile on Smooth Muscles.

The dried flowers of Chamaemelum nobile (L.) All. have been used in traditional medicine for different conditions related to the spasm of the gastrointestinal system. However, there have been no experimental studies to support the smooth muscle relaxant effect of this plant. The aim of our research was to assess the effects of the hydroethanolic extract of Roman chamomile, its fractions, four of its flavonoids (apigenin, luteolin, hispidulin, and eupafolin), and its essential oil on smooth muscles. The phytochemical compositions of the extract and its fractions were characterized and quantified by HPLC-DAD, the essential oil was characterized by GC and GC-MS. Neuronally mediated and smooth muscle effects were tested in isolated organ bath experiments on guinea pig, rat, and human smooth muscle preparations. The crude herbal extract induced an immediate, moderate, and transient contraction of guinea pig ileum via the activation of cholinergic neurons of the gut wall. Purinoceptor and serotonin receptor antagonists did not influence this effect. The more sustained relaxant effect of the extract, measured after pre-contraction of the preparations, was remarkable and was not affected by an adrenergic beta receptor antagonist. The smooth muscle-relaxant activity was found to be associated with the flavonoid content of the fractions. The essential oil showed only the relaxant effect, but no contracting activity. The smooth muscle-relaxant effect was also detected on rat gastrointestinal tissues, as well as on strip preparations of human small intestine. These results suggest that Roman chamomile extract has a direct and prolonged smooth muscle-relaxant effect on guinea pig ileum which is related to its flavonoid content. In some preparations, a transient stimulation of enteric cholinergic motoneurons was also detected. The essential oil also had a remarkable smooth muscle relaxant effect in this setting. Similar relaxant effects were also detected on other visceral preparations, including human jejunum. This is the first report on the activity of Roman chamomile on smooth muscles that may reassure the rationale of the traditional use of this plant in spasmodic gastrointestinal disorders.

Serotonin or the Mucosa Do Not Mediate the Motor Effect of Allyl Isothiocyanate in the Guinea-Pig Small Intestine.

BACKGROUND: Serotonin (5-hydroxytryptamine, 5-HT), originating from the enterochromaffin cells has been reported to mediate the contractile effect of the sensory stimulant and TRPA1 activator allyl isothiocyanate (AITC) in the guinea-pig small intestine [Nozawa et al: Proc Natl Acad Sci U S A 2009;106:3408-3413]. SUMMARY: In the present experiments, the nerve-mediated contraction of this preparation due to AITC was not inhibited by a combination of methysergide (broad-spectrum 5-HT antagonist; 0.3 µmol/l), Y 25130 (azasetron, 5-HT3 receptor antagonist; 1 µmol/l) and SB 204070 (5-HT4 receptor antagonist; 2 µmol/l) or by 5-HT receptor desensitization, that is, pretreatments that practically abolished contractions of similar size in response to exogenous 5-HT, without causing nonspecific effects. AITC also contracted longitudinal muscle-myenteric plexus preparations, an effect also fully resistant to the combination of 5-HT receptor antagonists. The pharmacology of AITC in strip preparations matched that in the whole ileum. Key Messages: It is concluded that neither endogenous 5-HT nor the gut mucosa contributes to the excitatory effect of AITC in the guinea-pig small intestine. The combination of 5-HT antagonists elaborated is suitable for studying the possible involvement of 5-HT in motor responses of the guinea-pig intestine.

The TRPA1 Activator Allyl Isothiocyanate (AITC) Contracts Human Jejunal Muscle: Pharmacological Analysis.

The contractile effect of AITC (300 µM) on human jejunal longitudinal strips was inhibited by the TRPA1 antagonist HC 030031 and atropine or scopolamine, but was insensitive to tetrodotoxin, purinoceptor antagonists or capsaicin desensitization. It is concluded that TRPA1 activation stimulates a cholinergic mechanism in a tetrodotoxin-resistant manner.

Combined Inhibition of Histamine H1 Receptors and Leukotrienes Reduces Compound 48/80-Induced Contraction of the Human Bronchus in vitro.

BACKGROUND/AIMS: Bronchial asthma continues to be a big challenge to therapy. Mast cells play an important role in allergic asthma. Histamine and leukotrienes are established mast cell mediators, but antihistamines currently play no role in asthma therapy. METHODS: Human bronchial strips were exposed to the mast cell activator compound 48/80 (200 µg/ml) in isolated organ experiments. RESULTS: The contractile response was not inhibited by the H1 receptor antagonist antihistamine chloropyramine (0.3 µmol/l), the leukotriene cys-LT1 receptor antagonist MK 571 (3 µmol/l), the 5-lipoxygenase inhibitor MK 886 (5 µmol/l), the cyclo-oxygenase inhibitor indomethacin (5 µmol/l), tetrodotoxin, or atropine. Chloropyramine, combined with either MK 571 or MK 886 significantly reduced the response. CONCLUSION: A supra-additive effect is proposed for the antihistamine and the anti-leukotrienes, which might have relevance to human asthma therapy as well; such a combination deserves a large-scale clinical study. These data also indicate that substances like compound 48/80 should be denoted as mast cell activators rather than 'histamine liberators'.

High-Concentration Piperine: Capsaicin-Sensitive and -Insensitive Effects on Isolated Organs.

Piperine (P), a sensory stimulant in black pepper, is an agonist on TRPV1 receptors. Earlier work has showed capsaicin-sensitive and -insensitive mechanisms of the contractile action of P on the intestine. The current isolated organ study in the guinea-pig ileum, urinary bladder and trachea (a) confirms the presence of such components of effect (ileum and bladder); (b) indicates TRPV1 involvement in the effect of 5 or 30 µmol/l of P on the basis of an inhibitory action of the antagonist BCTC (ileum); (c) indicates that HC 030031-sensitive TRPA1 receptors and nifedipine-sensitive Ca(2+) channels contribute to the capsaicin-resistant contraction to 30 µmol/l P (ileum) and (d) shows that the contractile effect of P up to 100 µmol/l (guinea-pig trachea) or 30 µmol/l (guinea-pig urinary bladder) is capsaicin-sensitive and mediated by TRPV1 receptors/channels.

Dual excitatory and smooth muscle-relaxing effect of Sideritis montana extract on guinea-pig ileum.

The neuronal and smooth muscle effects of a methanol extract prepared from the air-dried flowering aerial parts of Sideritis montana L. (SME) was tested in vitro on Guinea-pig ileum. The chemical composition of the investigated extract was analysed by HPLC-MS, and chrysoeriol, chlorogenic acid and caffeic acid were detected as main constituents. The isolated organ assay showed that S. montana extract caused an immediate contraction and a more slowly developing inhibitory response in the ileum. The SME-induced contractions were strongly inhibited by the acetylcholine muscarinic receptor antagonist atropine (0.5 µM), but not by either the Na+ channel blocker tetrodotoxin (TTX; 0.5 µM) or the histamine H1 receptor antagonist chloropyramine (0.5 µM). Selective desensitization of capsaicin-sensitive neurons by the sensory neuron stimulant and blocker capsaicin did not influence the contractile effect of SME. As to the spasmolytic effect, SME inhibited the effects of electrical field stimulation, exogenous acetylcholine, and histamine. These smooth muscle-relaxing effects were reversible in 40 min by repeated renewals of the bathing solution.

Effects of some natural carotenoids on TRPA1- and TRPV1-induced neurogenic inflammatory processes in vivo in the mouse skin.

Mechanisms of the potent anti-inflammatory actions of carotenoids are unknown. Since carotenoids are incorporated into membranes, they might modulate transient receptor potential ankyrin 1 and vanilloid 1 (TRPA1 and TRPV1) activation predominantly on peptidergic sensory nerves. We therefore investigated the effects of three carotenoids (ß-carotene, lutein and lycopene) on cutaneous neurogenic inflammation. Acute neurogenic edema and inflammatory cell recruitment were induced by smearing the TRPA1 agonist mustard oil (5%) or the TRPV1 activator capsaicin (2.5%) on the mouse ear. Ear thickness was then determined by micrometry, microcirculation by laser Doppler imaging and neutrophil accumulation by histopathology and spectrophotometric determination of myeloperoxidase activity. The effects of lutein on the stimulatory action of the TRPA1 agonist mustard oil were also tested on the guinea-pig small intestine, in isolated organ experiments. Mustard oil evoked 50-55% ear edema and granulocyte influx, as shown by histology and myeloperoxidase activity. Swelling was significantly reduced between 2 and 4 h after administration of lutein or ß-carotene (100 mg/kg subcutane three times during 24 h). Lutein also decreased neutrophil accumulation induced by TRPA1 activation, but did not affect mustard oil-evoked intestinal contraction. Lycopene had no effect on any of these parameters. None of the three carotenoids altered capsaicin-evoked inflammation. It is proposed that the dihydroxycarotenoid lutein selectively inhibits TRPA1 activation and consequent neurogenic inflammation, possibly by modulating lipid rafts.

Thermal spring water drinking attenuates dextran-sulfate-sodium-induced colitis in mice.

INTRODUCTION: The present study investigates the effect of oral consumption of hydrogen sulfide-containing Harkány thermal spring water, as well as sodium hydrogen sulfide (NaHS) solution on experimental colitis. METHODS: Colitis was induced by 2% dextran sulfate sodium (DSS) in the drinking water of C57BL/6 mice for 7 days. Some animal groups drank Harkány thermal spring water or water supplemented with 21.68 mg/L NaHS. General signs of colitis, myeloperoxidase (MPO) enzyme activity of colon samples, histological features of colitis and function of the enteric nervous system were assessed. RESULTS: Oral administration of Harkány thermal spring water significantly attenuated general signs of colitis, MPO enzyme activity of colon samples and detrimental effect of colitis on the function of the enteric nervous system, but not histological signs of colitis. These findings could be reproduced using NaHS solution with additional significantly diminished histological damage. CONCLUSIONS: We conclude that oral treatment with Harkány thermal spring water relieves various aspects of DSS-evoked colitis in mice. This effect is most likely to be mediated by hydrogen sulfide content of the Harkány water. Our data might promote complementary utilization of sulfurous thermal spring water in the therapy of inflammatory bowel disease.

Smooth muscle-depressant activity of AP-18, a putative TRPA1 antagonist in the guinea pig intestine.

AP-18, a putative antagonist at TRPA1 receptor/ion channel, caused smooth muscle relaxation at 10-100 µmol/l. It inhibited cholinergic twitch responses evoked by electrical field stimulation of cholinergic nerves as well as contractions in response to acetylcholine and histamine in the guinea pig small intestine. AP-18 (30 µmol/l) blocked spontaneous contractions of longitudinal strips of human jejunum. It is concluded that AP-18 may have limited value in studying TRPA1-mediated responses in smooth muscles and should probably be used with care in other preparations because of possible nonspecific effects.

Phytochemical evaluation of Lythrum salicaria extracts and their effects on guinea-pig ileum.

n-Hexane, chloroform, ethyl acetate and 50% ethanol in water extracts prepared from the air-dried flowering parts of Lythrum salicaria L. were tested for in vitro pharmacological properties on Guinea-pig ileum, which is suitable for detecting a whole range of neuronal and smooth muscle effects. UHPLC-MS was used to evaluate polyphenol components of the extracts. In the ileum, the most prominent response (46.4% related to 0.5 microM histamine) of the extracts causing smooth muscle contractions were triggered by the 50% ethanol in water extract in a concentration-dependent manner. Atropine, indomethacin and PPADS plus suramin significantly reduced the contractile response caused by this extract. The strongest inhibition was due to atropine. The results suggest that L. salicaria extracts have a moderate muscarinic receptor agonist effect in Guinea-pig ileum and that prostanoids and purinoceptor mechanisms are involved to some extent. Therefore diluted extracts of L. salicaria p.o. could be used as a mild stimulant of gastrointestinal motility. The 50% ethanol in water extract was rich in polyphenols. n-Hexane, chloroform and ethyl acetate extracts failed to contain catechin, caffeic acid, quercetin-3-D-galactoside and rutin, but they all showed spasmogenic effects, and, therefore we do not think that these compounds could be involved in the spasmogenic activity.

Effects of reference analgesics and psychoactive drugs on the noxious heat threshold of mice measured by an increasing-temperature water bath.

The study aimed at validating an increasing-temperature water bath suitable for determining the noxious heat threshold for use in mice. The noxious heat threshold was determined by immersing the tail of the gently held awake mouse into a water container whose temperature was near-linearly increased at a rate of 24°C/min. until the animal withdrew its tail, that is, heating attained the noxious threshold. The effects of standard analgesic, neuroleptic and anxiolytic drugs were investigated in a parallel way on both the noxious heat threshold and the psychomotor activity assessed by the open field test. Morphine, diclofenac and metamizol (dipyrone) elevated the heat threshold of mice with minimum effective doses of 6, 30 and 1000 mg/kg i.p., respectively. These doses of morphine and diclofenac failed to induce any remarkable effect on psychomotor activity in the open field test while that of metamizol exerted a profound inhibition. The anxiolytic diazepam and the neuroleptic droperidol at doses evoking a mild and moderate, respectively, psychomotor inhibition failed to alter the heat threshold. Combination of a subliminal dose of morphine (regarding both antinociceptive and psychomotor inhibitory action) with diclofenac, metamizol, diazepam or droperidol at doses also subliminal regarding the thermal antinociceptive effect elevated the noxious heat threshold without major additional effects in the open field test. It is concluded that the increasing-temperature water bath is suitable for studying the thermal antinociceptive effects of morphine and diclofenac as well as the morphine-sparing action of diclofenac, metamizol, droperidol and diazepam. Behavioural testing is recommended when testing analgesics.

Evidence that adrenergic and cholinergic but not capsaicin-sensitive nerves are involved in the nerve-mediated contraction of the guinea pig seminal vesicle.

BACKGROUND/AIMS: The neurotransmitters participating in the nerve-mediated contraction of the guinea pig seminal vesicle (GPSV) have not been firmly established. There is debate as to the mediating role of norepinephrine and acetylcholine. METHODS: We have used longitudinally and circularly oriented strips of GPSV and activated their intramural nerves by electrical field stimulation (5 and 10 Hz for 30 s). RESULTS: Contractile responses to stimulation were enhanced by a cholinesterase inhibitor and reduced by the adrenergic α-receptor antagonist phentolamine (2.5 µmol/l). Atropine (1 µmol/l) significantly reduced responses in longitudinal preparations; a less consistent inhibition was found in circular preparations. The sensory neuron stimulant and blocker capsaicin was without effect. CONCLUSIONS: It is concluded that adrenergic nerves and also acetylcholine mediate the contractile response of the GPSV.

Purinoceptor-mediated, capsaicin-resistant excitatory effect of allyl isothiocyanate on neurons of the guinea-pig small intestine.

Allyl isothiocyanate (AITC; 200 µM) caused atropine- and tetrodotoxin-sensitive longitudinal muscle contraction on the guinea-pig small intestine. The response was not influenced by hexamethonium, a functional blockade of capsaicin-sensitive neurons or by antagonists acting at TRPV1 or TRPA1, but was abolished by the P2 purinoceptor antagonist PPADS (50 µM). It is concluded that cholinergic motoneurons are activated by a purinergic mechanism in the course of the AITC response, independently of capsaicin-sensitive processes or even TRPA1.

Should antihistamines be re-considered as antiasthmatic drugs as adjuvants to anti-leukotrienes?

In spite of histamine mimicking the symptoms of allergic bronchoconstriction and severe anaphylaxis, histamine antagonists most probably represent no effective treatment for these conditions. Anti-leukotrienes proved effective for preventing attacks of allergic asthma. In vitro evidence supports a supra-additive effect of histamine H1 receptor antagonists and anti-leukotrienes in vitro, in asthma models utilizing human bronchi. The same seems to hold true for human allergen provocation tests in vivo. We conclude that combinations of second-generation antihistamines and anti-leukotrienes deserve a large-scale clinical trial for preventing and/or treating attacks of allergic asthma. If useful, these drugs could provide a cost-effective alternative to some recent antiasthmatics. Given that redundant mechanisms may be included in asthma pathophysiology, other combinations (including thromboxane or platelet activating factor antagonists) could also be considered.

The contractile effect of anandamide in the guinea-pig small intestine is mediated by prostanoids but not TRPV1 receptors or capsaicin-sensitive nerves.

Although exogenous and endogenous cannabinoid receptor agonists have well-documented inhibitory effects on gastrointestinal motility, a TRPV1 receptor-mediated excitatory action of anandamide (arachidonoyl ethanolamide, AEA) in the guinea-pig ileum strip has also been described. We used in vitro capsaicin desensitization for assessing the possible participation of sensory neurons in the contractile effect of anandamide on the guinea-pig whole ileum, as well as autonomic drugs and a cyclooxygenase inhibitor for characterizing this response. Isolated organ experiments were used with isotonic recording. Contractions induced by anandamide (1 or 10 µM) were strongly inhibited by tetrodotoxin, indomethacin or atropine plus a tachykinin NK(1) receptor antagonist, but weakly to moderately reduced by atropine alone and partly diminished by the fatty acid amide hydrolase inhibitor URB 597. Neither capsaicin pre-treatment nor the TRPV1 receptor antagonist BCTC, the ganglionic blocking drug hexamethonium or cannabinoid (CB1 or CB2 ) receptor antagonists, influenced the effect of anandamide. It is concluded that the capsaicin-insensitive, neuronal excitatory effect of anandamide in the intestine is most probably mediated by cyclooxygenase products. Such a mechanism may also play a role at other sites in the mammalian body.

The putative somatostatin antagonist cyclo-somatostatin has opioid agonist effects in gastrointestinal preparations.

AIMS: Specificity of receptor antagonists used is crucial for clarifying physiological/pathophysiological roles of the respective endogenous agonist. We studied the effects (somatostatin antagonist and possibly other actions) of cyclo-somatostatin (CSST), a putative somatostatin receptor antagonist on the guinea-pig small intestine, a preparation where somatostatin causes inhibition of nerve-mediated contractions. MAIN METHODS: In isolated organ experiments, half-maximal cholinergic "twitch" contractions of the guinea-pig small intestine were evoked or tonic contractions of the rat stomach fundus strip (in the presence of physostigmine) were elicited by electrical field stimulation. The effects of somatostatin (somatostatin-14), CSST, naloxone, as well as of direct smooth muscle stimulants were examined. KEY FINDINGS: Somatostatin (10 nM-1 µM) caused transient inhibition of the twitch contraction, in a naloxone-insensitive manner. Surprisingly, CSST (0.3-1 µM) also inhibited twitch contractions (more than 50% reduction at 1 µM). This effect was prevented by the opioid receptor antagonist naloxone. Responses to acetylcholine or histamine were not or only minimally inhibited by CSST (up to 3 µM). CSST (0.3 µM in the absence or 1-10 µM in the presence of naloxone) failed to inhibit the effect of somatostatin. The SST(2) receptor antagonist CYN-154806 (3 µM) attenuated the effect of somatostatin and failed to evoke naloxone-sensitive inhibition of the twitch response. The naloxone-sensitive inhibitory effect of CSST on cholinergic contractions was also confirmed in the rat stomach fundus preparation. SIGNIFICANCE: Cyclo-somatostatin exerts opioid agonist activity in the two preparations tested, while it does not behave as a somatostatin-receptor antagonist in the guinea-pig intestine.

Use and limitations of three TRPV-1 receptor antagonists on smooth muscles of animals and man: a vote for BCTC.

Specificity of the effect is a crucial factor in using antagonists for detecting the physiological/pathophysiological roles of receptors. Here we examined the capsaicin receptor antagonist effects of three commercially-available substances, capsazepine, iodo-resiniferatoxin (I-RTX) and BCTC, on isolated smooth muscle preparations, including the human intestine. Care was taken to observe possible non-specific effects, to find out safe and effective concentrations. Capsazepine appeared to have a low margin of safety. I-RTX (up to 1µM) specifically inhibited capsaicin-induced contractions in the guinea-pig ileum and urinary bladder. I-RTX showed agonist activity on the rat urinary bladder. BCTC (1µM) abolished the contractile effects of capsaicin (1 or 2µM) on all preparations tested (guinea-pig ileum, bladder, trachea, as well as rat and mouse bladder), and on the guinea-pig renal pelvis, where it failed to influence capsaicin-sensitive, sensory neuron-mediated positive inotropy in response to field stimulation. On human intestinal preparations BCTC prevented the relaxant effect of capsaicin. It is concluded that of the three antagonists tested BCTC seems the safest one for inhibiting TRPV-1 receptors. The effect of capsazepine may be complicated by non-specific inhibition of smooth muscle contractility and that of I-RTX by agonist activity. The "local efferent" function of capsaicin-sensitive sensory neurons is not influenced by BCTC, as shown by the results obtained in the renal pelvis. In conclusion, of the TRPV-1 receptor antagonists studied, BCTC (1µM) seems the most reliable in isolated organ experiments. This substance is also effective in the human intestine.