Effects of ginsenoside Re on rat jejunal contractility

Ginsenoside Re (GRe) exerts diverse effects. Based on our observations, the present study was designed to investigate GRe-exerted bidirectional regulation (BR) on the contractility of isolated jejunal segment. Six pairs of different low and high contractile states of rat jejunal segment were established and used in the study. Stimulatory effects on the contractility of jejunal segment were exerted by GRe (10.0 μM) in all 6 low contractile states, and inhibitory effects were exerted in all 6 high contractile states, indicating that GRe exerted BR on the contractility of jejunal segment. The effects of GRe on the phosphorylation of 20 kDa myosin light chain, protein contents of myosin light chain kinase (MLCK) and MLCK mRNA expression in jejunal segment in low and high contractile states were also bidirectional. GRe-exerted BR was abolished in the presence of neurotoxin tetrodotoxin or Ca²⁺ channel blocker verapamil or c-Kit receptor tyrosine kinase inhibitor imatinib. Atropine blocked the stimulatory effects of GRe on jejunal contractility in low-Ca²⁺-induced low contractile state; phentolamine, propranolol and l-NG-nitro-arginine blocked the inhibitory effects in high-Ca²⁺-induced high contractile state, respectively. In summary, GRe-exerted BR depends on jejunal contractile state and requires the presence of enteric nervous system, Ca²⁺, and interstitial cells of Cajal; the stimulatory effects of GRe on jejunal contractility are related to cholinergic stimulation and inhibitory effects are related to adrenergic activation and nitric oxide relaxing mechanisms.     

The characteristics of genistin-induced inhibitory effects on intestinal motility

Genistin belongs to isoflavones. Based on the facts that genistin exerts inhibitory effects on the contractility of vascular smooth muscle,the present study was designed to characterize the effects of genistin on intestinal contractility and evaluate its potential clinical implication. Ex vivo [isolated jejunal segment (IJS) of rat], in vitro, and in vivo assays were used in the study. The results indicated that genistin (5–80 μmol/L) inhibited the contraction of IJS in a dose-dependent manner and inhibited the increased-contractility of IJS induced by acetylcholine (ACh), histamine, high Ca²⁺, and erythromycin, respectively. The inhibitory effects of genistin were correlated with the stimulation of alpha adrenergic and beta adrenergic receptors since these inhibitory effects were significantly blocked in the presence of phentolamine and propranolol respectively. No further inhibitory effects of genistin were observed in the presence of verapamil or in Ca²⁺-free condition, indicating genistin-induced inhibitory effects are Ca²⁺-dependent. Genistin decreased myosin light chain kinase (MLCK) protein contents and MLCK mRNA expression in IJS, and inhibited both phosphorylation and Mg²⁺-ATPase activity of purified myosin, implicating that the decrease of MLCK contents and inhibition of MLCK activity are involved in the genistin-induced inhibitory effects. The study suggests the potential clinical implications of genistin in relieving intestinal hypercontractility.     

Characteristics of Diprophylline-Induced Bidirectional Modulation on Rat Jejunal Contractility

In this study, we propose that diprophylline exerts bidirectional modulation (BM) on the isolated rat jejunal segment depending on its contractile state. The results supported the hypothesis. Diprophylline (20 µM) exerted stimulatory effects on the contractility of jejunal segment in six low contractile states while inhibitory effects in six high contractile states, showing the characteristics of BM. Diprophylline-induced stimulatory effect was significantly blocked by atropine, indicating the correlation with cholinergic activation. Diprophylline-induced inhibitory effect was partially blocked by phentolamine, propranolol, and L-N-Nitro-Arginine respectively, indicating their correlation with sympathetic activation and nitric oxide-mediated relaxing mechanisms. Diprophylline-induced BM was abolished by tetrodotoxin or in a Ca²⁺ free condition or pretreated with tyrosine kinase inhibitor imatinib, suggesting that diprophylline-induced BM is Ca²⁺ dependent, and that it requires the presence of enteric nervous system as well as pacemaker activity of interstitial cells of Cajal. Diprophylline significantly increased the reduced MLCK expression and myosin extent in constipation-prominent rats and significantly decreased the increased MLCK expression and myosin extent in diarrhea-prominent rats, suggesting that the change of MLCK expression may also be involved in diprophylline-induced BM on rat jejunal contractility. In summary, diprophylline-exerted BM depends on the contractile states of the jejunal segments, requires the presence of Ca²⁺, enteric nervous system, pacemaker activity of interstitial cells of Cajal, and MLCK-correlated myosin phosphorylation. The results suggest the potential implication of diprophylline in relieving alternative hypo/hyper intestinal motility.     

Inhibitory effects of daidzein on intestinal motility in normal and high contractile states

Context: Daidzein is a naturally occurring compound and has various health benefits. However, its effects on intestinal smooth muscle contractility remain unknown.

Aims: The present study was to characterize the effects of daidzein on the contractility of isolated jejunal smooth muscle and its underlying mechanisms.

Methods: Ex vivo assay was selected as the major method to determine the effects of daidzein on the contractility of isolated jejunal smooth muscle fragment (JSMF).

Results: Daidzein (5–160 µmol/L) inhibited the contractility of JSMF in normal contractile state and in a dose-dependent manner. Daidzein also inhibited the contractility of JSMF induced by ACh, histamine, erythromycin and high Ca²⁺, respectively, and decreased charcoal propulsion in the small intestine in vivo. The inhibitory effects of daidzein were partially blocked by phentolamine or propranolol and were abolished in the presence of varapamil or at Ca²⁺-free assay condition. However, the inhibitory effects of daidzein on jejunal contraction were not significantly influenced by nitric oxide (NO) synthase inhibitor L-NG-nitro-arginine (l-NNA). Daidzein was also found to directly inhibit the phosphorylation and Mg²⁺-ATPase activity of smooth muscle myosin.

Discussion and Conclusion: The results implicated that α- and β-adrenergic receptors were involved in the inhibitory effects produced by daidzein rather than via NO pathway. As a phytoestrogen, daidzein has shown its potential value in relieving the hypercontractility of small intestine.     

Relaxant effect induced by wogonin from Scutellaria baicalensis on rat isolated uterine smooth muscle

Context: Wogonin is a flavone derivative isolated from Scutellaria baicalensis Georgi (Labiatae) root, which is a traditional Chinese drug used as an anti-inflammatory and for management of dysmenorrhea.

Objective: The effect of wogonin on the uterus has not yet been examined. We investigated the relaxant effects of wogonin on contractile activity of isolated uterine strips of rats.

Materials and methods: The effect of wogonin on spontaneous uterine contraction, and uterine contraction induced by agonists, K⁺-depolarization and oxytocin in Ca²⁺-free solution was observed. To clarify the type of potassium channel, we tested the effects of 4-aminopyridine, tetraethylammonium and glibenclamide.

Results: Wogonin reduced the contractile amplitude of uterine strip smooth muscle of rats in a dose-dependent manner. The concentration of wogonin for reducing the contraction amplitude by 50% (IC₅₀) on spontaneous contractions was 60.5 μM. Wogonin also inhibited the contraction induced by three agonists (oxytocin, prostaglandin F_2α and acetylcholine). For the uterine strips pretreated with oxytocin in Ca²⁺-free solution or K⁺-depolarization, wogonin showed relaxant effect on the induced uterine contractions. In addition, whereas the inhibitive effect of wogonin on the contraction of uterine smooth muscle in rats could be partly blocked by 4-aminopyridine and tetraethylammonium, it was not influenced by glibenclamide.

Discussion and conclusion: Wogonin significantly inhibited the contraction of rat uterine smooth muscle probably through the inhibition of the inflow of extracellular calcium into cells via cell membrane, and intracellular release of calcium ions. In addition, the relaxant effect induced by wogonin might be due in part to the opening of voltage-dependent and large conductance Ca²⁺-activated K⁺ channels.     

Pharmacological basis for the medicinal use of Holarrhena antidysenterica in gut motility disorders

Context: Holarrhena antidysenterica Wall. (Apocynaceae) is widely used in traditional medical system for treatment of constipation, colic, and diarrhea.

Aim: This study was carried out to provide pharmacological basis for medicinal use of Holarrhena antidysenterica in gastrointestinal disorders.

Materials and methods: Hydro-ethanolic crude extract of Holarrhena antidysenterica (HaCE) and its fractions were studied in various gastrointestinal isolated tissue preparations.

Results: In guinea pig ileum tissues, HaCE at 0.3-10?mg/mL caused pyrilamine-sensitive spasmogenic effect. When tested in spontaneously contracting rabbit jejunum preparations, HaCE (0.01-3.0?mg/mL) caused moderate stimulation, followed by a relaxant effect at next higher concentrations. In presence of pyrilamine, the contractile effect was blocked and the relaxation was observed at lower concentrations (0.01-0.3?mg/mL). HaCE inhibited the high K⁺ (80?mM)-induced contractions at concentration range of 0.01-1.0?mg/mL and shifted Ca⁺⁺ concentration response curves to the right, like that caused by verapamil. Activity-directed fractionation revealed that the spasmogenic component was concentrated in the aqueous fraction, while the spasmolytic component was concentrated in the organic fraction.

Discussion and conclusion: These results indicate that the gut stimulant and relaxant activities of Holarrhena antidysenterica are mediated possibly through activation of histamine receptors and Ca⁺⁺ channel blockade, respectively and this study provides sound mechanistic background for its usefulness in gut motility disorders such as constipation, colic, and possibly diarrhea.     

Thyroid hormone and chronically unloaded hearts

The heart is subjected to chronic mechanical unloading during prolonged spaceflight and microgravity. The heart in patients with end-stage heart failure is also unloaded in prolonged duration after left ventricular assist devices (LVAD) are implanted. Heterotopic heart transplantation in rats is an established model of chronic cardiac unloading, and has been used to investigate the effects of chronic cardiac unloading on the heart. Observations that have been found using this experimental model are as follow. Chronic cardiac unloading induces time-dependent depressions of Ca²⁺ handling and myocyte contractility, which are associated with the shift of myosin heavy chain (MHC) isozymes and altered expressions of Ca²⁺ cycling-related proteins. Treatment with the physiological treatment dose of thyroid hormone restores the expression levels of Ca²⁺ cycling-related proteins, Ca²⁺ handling, and contractile function of cardiac myocytes in chronically unloaded hearts. Although future studies are required to determine precise mechanisms of the beneficial effects of thyroid hormone on chronically unloaded hearts, these observations may have clinical implications in the future for chronic cardiac unloading in the space industry as well as in the treatment of patients with end-stage heart failure supported by LVAD.     

Effect of a novel inhibitor of cyclic AMP phosphodiesterase,E-1020, on cytosolic Ca++ level and contraction in vascular smooth muscle

Summary 1. The effects of a novel cyclic AMP phosphodiesterase inhibitor, E-1020 (1,2-dihydro-6-methyl-2-oxo5-(imidazo[1,2-a]pyridin-6-yl)-3-pyridine carbonitrile hydrochloride monohydrate), on cytosolic Ca⁺⁺ level ([Ca⁺⁺]_cyt) and muscle tension were examined in rat aorta using a fluorescent Ca⁺⁺ indicator, fura-2. 2. The sustained contraction induced by norepinephrine was more strongly inhibited by E-1020 than that induced by high K⁺. The contraction induced by a higher concentration of the stimulant was less sensitive to E-1020 than that due to a lower concentration. 3. Contractions induced by high K⁺ and norepinephrine followed the increase in [Ca⁺⁺]_cyt. E-1020 inhibited the increments in [Ca⁺⁺]_cyt and muscle tension. A Ca⁺⁺ channel blocker, verapamil, inhibited the norepinephrine-stimulated [Ca⁺⁺]_cyt more strongly than the contraction. E-1020 inhibited the verapamil-insensitive portion of the norepinephrine-stimulated [Ca⁺⁺]_cyt and contraction. 4. Norepinephrine transiently increased [Ca⁺⁺]_cyt and muscle tension in Ca⁺⁺-free solution. E-1020 inhibited the transient contraction but not the stimulated [Ca⁺⁺]_cyt. 5. E-1020 increased the cyclic AMP content of the muscle. The effects of E-1020 on cyclic AMP content and contraction were potentiated by an activator of adenylate cyclase, forskolin. 6. These results suggest that E-1020 inhibits the vascular contractility by the decrease in [Ca⁺⁺]_cyt and decrease in Ca⁺⁺ sensitivity of contractile elements. These effects may be mediated by the increase in cyclic AMP content of the muscle. Send offprint requests to M. Täjimi at the above address     

Mechanism of potentiation by tea epigallocatechin of contraction in porcine coronary artery: the role of protein kinase Cδ-mediated CPI-17 phosphorylation

The effects of green tea catechins, (+)- and (−)-catechins (C), (−)-epicatechin (EC), (−)-epicatechin gallate (ECG), (−)-epigallocatechin (EGC), and (−)-epigallocatechin gallate (EGCG), on vascular contractility were investigated in porcine coronary artery. At the concentration of 200 μM, only EGC, but not other catechins, potentiated high K⁺-induced contraction in a concentration-dependent manner, although EGC by itself did not produce contraction. The potentiator effect of EGC was still observed in endothelium-denuded preparations. Moreover, EGC increased the translocation of protein kinase Cδ (PKCδ) from the cytosol to the plasma membrane and increased phosphorylations of 17-kDa PKC-potentiated protein phosphatase inhibitor protein (CPI-17) and myosin light chain (MLC₂₀). These effects of EGC were inhibited by the PKCδ inhibitor rottlerin, but not by the conventional PKC inhibitor Gö6976. These results suggest that EGC activates PKCδ, leading to the phosphorylation of CPI-17, which in turn inhibits myosin light chain phosphatase and increases MLC₂₀ phosphorylation. The series of events would increase Ca²⁺ sensitivity of contractile elements, thereby augmenting high K⁺-induced vascular contraction.     

Myoplasmic [Ca2+], Crossbridge Phosphorylation and Latch in Rabbit Bladder Smooth Muscle

Tonic smooth muscle exhibit the latch phenomenon: high force at low myosin regulatory light chains (MRLC) phosphorylation, shortening velocity (Vo), and energy consumption. However, the kinetics of MRLC phosphorylation and cellular activation in phasic smooth muscle are unknown. The present study was to determine whether Ca²⁺-stimulated MRLC phosphorylation could suffice to explain the agonist- or high K⁺-induced contraction in a fast, phasic smooth muscle. We measured myoplasmic [Ca²⁺], MRLC phosphorylation, half-time after step-shortening (a measure of Vo) and contractile stress in rabbit urinary bladder strips. High K⁺-induced contractions were phasic at both 22℃ and 37℃: myoplasmic [Ca²⁺], MRLC phosphorylation, 1/half-time, and contractile stress increased transiently and then all decreased to intermediate values. Carbachol (CCh)-induced contractions exhibited latch at 37℃: stress was maintained at high levels despite decreasing myoplasmic [Ca²⁺], MRLC phosphorylation, and 1/half-time. At 22℃ CCh induced sustained elevations in all parameters. 1/half-time depended on both myoplasmic [Ca²⁺] and MRLC phosphorylation. The steady-state dependence of stress on MRLC phosphorylation was very steep at 37℃ in the CCh- or K⁺-depolarized tissue and reduced temperature flattend the dependence of stress on MRLC phosphorylation compared to 37℃. These data suggest that phasic smooth muscle also exhibits latch behavior and latch is less prominent at lower temperature.     

Possible mechanisms of action of the aqueous extract of Artocarpus altilis (breadfruit) leaves in producing hypotension in normotensive Sprague–Dawley rats

Context and objectives: Artocarpus altilis (Parkinson) Fosberg (Moraceae) (breadfruit) leaves are used as an antihypertensive remedy. We investigated the possible mechanisms of action of its aqueous extract and its effect on cytochromes P450 (CYP) enzyme activities.

Materials and methods: Intravenous administration of an aqueous leaf extract (20.88–146.18 mg/kg) of A. altilis on mean arterial pressure and heart rate were recorded via cannulation of the carotid artery on anaesthetized normotensive Sprague–Dawley rats. Recordings of the contractile activity of the aortic rings to the extract (0.71–4.26 mg/mL) were studied using standard organ bath techniques. Inhibitions of human CYP3A4 and CYP2D6 enzyme activities were evaluated by means of a fluorometric assay in 96 well plates using heterologously expressed microsomes.

Results: A. altilis caused significant (p < 0.05) hypotensive and bradycardiac responses unaffected by atropine (2 mg/kg) and mepyramine (5 mg/kg), but attenuated by propranolol (1 mg/kg) and N(G)-nitro-l-arginine methyl ester (5 mg/kg). The extract (0.71–4.26 mg/mL) significantly (p < 0.05) relaxed phenylephrine (10^?9–10^?4 M) and 80 mM KCl-induced contractions in endothelium intact and denuded aortic rings; and caused a significant (p < 0.05) rightward shift of the Ca²⁺ dose-response curves in Ca²⁺-free Kreb’s solution. Moderate inhibitions of cytochrome P450s (CYP3A4 and CYP2D6) enzyme activities with IC₅₀ values of 0.695 ± 0.187 and 0.512 ± 0.131 mg/mL, respectively, were produced.

Conclusion: A. altilis exhibits negative chronotropic and hypotensive effects through α-adrenoceptor and Ca²⁺ channel antagonism. Drug adversity effects are unlikely if the aqueous leaf extract is consumed with other medications reliant on CYP3A4 and CYP2D6 metabolism. This study thus provides scientific evidence for the use of the breadfruit in the treatment of hypertension.     

Emodin inhibits ATP-induced IL-1β secretion,ROS production and phagocytosis attenuation in rat peritoneal macrophages via antagonizing P2X7 receptor

Context: Previous in vitro studies have demonstrated that emodin (1,3,8-trihydroxy-6-methyl-anthraquinone), an anthraquinone derivative from the rhizome of Rheum palmatum L., can inhibit the activation of P2X₇ receptors (P2X₇R) as a potential antagonist. However, the effects of emodin on P2X₇R-related inflammatory processes remain unclear.

Objective: This study aimed to investigate the effects of emodin on different inflammation responses of macrophages induced by ATP, the natural ligand of P2X₇R.

Materials and methods: Rat peritoneal macrophages were treated with millimolar ATP and emodin (0.1, 0.3,?1,?3,?10?µM) or brilliant blue G (BBG, 0.1,?1,?10?µM). Cytosolic Ca²⁺ concentration ([Ca²⁺]_c) was detected by fluorescent Ca²⁺ imaging. Interleukin-1β (IL-1β) release was measured by rat IL-1β ELISA kits. Reactive oxygen species (ROS) generation was examined by dihydroethidium (DHE) fluorescent staining. Phagocytic activity was tested by neutral red uptake assay.

Results: We found that the [Ca²⁺]_c increase evoked by ATP (5?mM) was inhibited by emodin, in a dose-dependent manner with IC₅₀ of 0.5?μM. Furthermore, emodin reduced the IL-1β release induced by ATP (2?mM) in lipopolysaccharide (LPS)-activated macrophages, with an IC₅₀ of 1.6?μM. Emodin also strongly suppressed the ROS production and phagocytosis attenuation triggered by ATP (1?mM), with IC₅₀ values of 1?μM and 0.7?μM, respectively. Besides, BBG, a specific antagonist of P2X₇R, exhibited similar suppressive effects on these inflammation responses.

Conclusion: These results showed the inhibitory effects of emodin on ATP-induced [Ca²⁺]_c increase, IL-1β release, ROS production and phagocytosis attenuation in rat peritoneal macrophages, by inhibiting the activation of P2X₇R.     

Vascular KCa-channels as therapeutic targets in hypertension and restenosis disease

Importance of the field: Cardiovascular disease is a leading cause of death in modern societies. Hyperpolarizing Ca²⁺-activated K⁺ channels (K_Ca) are important membrane proteins in the control of arterial tone and pathological vascular remodelling and thus could serve as new drug targets.

Areas covered in this review: We summarize recent advances in the field of vascular K_Ca and their roles in cardiovascular pathologies such as hypertension and restenosis disease and draw attention to novel small-molecule channel modulators and their possible therapeutic utility. This review focuses on literature from the last four to five years.

What the reader will gain: Pharmacological opening of endothelial KCa3.1/KCa2.3 channels stimulates endothelium-derived-hyperpolarizing-factor-mediated arteriolar dilation and lowers blood pressure. Inhibition of smooth muscle KCa3.1 channels has beneficial effects in restenosis disease and atherosclerosis. We consider the therapeutic potential of KCa3.1/KCa2.3 openers as novel endothelium-specific antihypertensive drugs as well as of KCa3.1-blockers for the treatment of pathological vascular remodelling and discuss advantages and disadvantages of the pharmacotherapeutic approaches.

Take home message: Pharmacological manipulation of vascular K_Ca channels by novel small-molecule modulators offers new venues for alternative treatments of hypertension, restenosis and atherosclerosis. Additional efforts are required to optimize these compounds and to validate them as cardiovascular-protective drugs.     

Inhibitory effects of fucoidan on NMDA receptors and l-type Ca2+ channels regulating the Ca2+ responses in rat neurons

Context: Fucoidan, a sulphated polysaccharide extracted from brown algae [Fucus vesiculosus Linn. (Fucaceae)], has multiple biological activities.

Objective: The effects of fucoidan on Ca²⁺ responses of rat neurons and its probable mechanisms with focus on glutamate receptors were examined.

Materials and methods: The neurons isolated from the cortex and hippocampi of Wistar rats in postnatal day 1 were employed. The intracellular Ca²⁺ responses triggered by various stimuli were measured in vitro by Fura-2/AM. Fucoidan at 0.5?mg/mL or 1.5?mg/mL was applied for 3?min to determine its effects on Ca²⁺ responses. RT-PCR was used to determine the mRNA expression of neuron receptors treated with fucoidan at 0.5?mg/mL for 3?h.

Results: The Ca²⁺ responses induced by NMDA were 100% suppressed by fucoidan, and those induced by Bay K8644 90% in the cortical neurons. However, fucoidan has no significant effect on the Ca²⁺ responses of cortical neurons induced by AMPA or quisqualate. Meanwhile, the Ca²⁺ responses of hippocampal neurons induced by glutamate, ACPD or adrenaline, showed only a slight decrease following fucoidan treatment. RT-PCR assays of cortical and hippocampal neurons showed that fucoidan treatment significantly decreased the mRNA expression of NMDA-NR1 receptor and the primer pair for l-type Ca²⁺ channels, PR1/PR2.

Discussion and conclusions: Our data indicate that fucoidan suppresses the intracellular Ca²⁺ responses by selectively inhibiting NMDA receptors in cortical neurons and l-type Ca²⁺ channels in hippocampal neurons. A wide spectrum of fucoidan binding to cell membrane may be useful for designing a general purpose drug in future.     

Genetic polymorphisms and drug interactions modulating CYP2D6 and CYP3A activities have a major effect on oxycodone analgesic efficacy and safety

Background and purpose:

Thrombus formation is commonly associated with pulmonary arterial hypertension (PAH). Thrombin may thus play an important role in the pathogenesis and pathophysiology of PAH. Hence, we investigated the contractile effects of thrombin and its mechanism in pulmonary artery.

Experimental approach:

The cytosolic Ca²⁺ concentrations ([Ca²⁺]_i), 20 kDa myosin light chain (MLC20) phosphorylation and tension development were evaluated using the isolated porcine pulmonary artery.

Key results:

Thrombin induced a sustained contraction in endothelium-denuded strips obtained from different sites of a pulmonary artery, ranging from the main pulmonary artery to the intrapulmonary artery. In the presence of endothelium, thrombin induced a transient relaxation. The contractile effect of thrombin was abolished by either a protease inhibitor or a proteinase-activated receptor 1 (PAR₁) antagonist, while it was mimicked by PAR₁-activating peptide (PAR₁AP), but not PAR₄AP. The thrombin-induced contraction was associated with a small elevation of [Ca²⁺]_i and an increase in MLC20 phosphorylation. Thrombin and PAR₁AP induced a greater increase in tension for a given [Ca²⁺]_i elevation than that obtained with high K⁺-depolarization. They also induced a contraction at a fixed Ca²⁺ concentration in α-toxin-permeabilized preparations.

Conclusions and implications:

The present study revealed a unique property of the pulmonary artery. In contrast to normal arteries of the systemic circulation, thrombin induces a sustained contraction in the normal pulmonary artery, by activating PAR₁ and thereby increasing the sensitivity of the myofilament to Ca²⁺. This responsiveness of the pulmonary artery to thrombin may therefore contribute to the pathogenesis and pathophysiology of PAH.     

Modulation of mouse gastrointestinal motility by allyl isothiocyanate, a constituent of cruciferous vegetables (Brassicaceae): evidence for TRPA1-independent effects

BACKGROUND AND PURPOSE

Allyl isothiocyanate (AITC, mustard oil), a constituent of many common cruciferous vegetables (Brassicaceae), activates transient receptor potential of ankyrin type-1 (TRPA1) channels, claimed to regulate gastrointestinal contractility. In this study, we have investigated the effect of AITC on intestinal motility.

EXPERIMENTAL APPROACH

Effects of AITC were investigated in vivo on upper gastrointestinal transit in mice and in mouse isolated ileum [contractions induced by electrical field stimulation (EFS), acetylcholine and spontaneous contractility]. The contractor activity of AITC was studied in mouse isolated colon. The ability of TRPA1 channel antagonists to block AITC-induced elevation of intracellular Ca²⁺[Ca²⁺]_i was assessed in HEK293 cells transfected with rat TRPA1 channels.

KEY RESULTS

AITC increased [Ca²⁺]_i in HEK293 cells, reduced ileal contractility (acetylcholine-, EFS-induced contractions and spontaneous contractility), but contracted the isolated colon. Gentamicin and camphor (non-selective TRPA1 channel antagonists), HC-030031 and AP18 (selective TRPA1 channel agonists) inhibited AITC-induced effects in HEK293 cells but not in the ileum or colon. AITC-induced contractions were reduced by tetrodotoxin and strongly reduced by nifedipine, cyclopiazonic acid and ryanodine. In vivo, AITC reduced (following i.p. administration) or increased (following intragastric administration) upper gastrointestinal transit in mice These effects were not affected by HC-030031.

CONCLUSION AND IMPLICATIONS

AITC, depending, in vitro, on the regions of gut examined and, in vivo, on the route of administration, exerted both stimulatory and inhibitory effects on intestinal motility, which were not sensitive to TRPA1 channel antagonists. The proposition that TRPA1 channels are the primary targets for AITC to induce contraction should be revised.     

Gastrointestinal effect of methanol extract of Radix Aucklandiae and selected active substances on the transit activity of rat isolated intestinal strips

Context: Radix Aucklandiae, the dry rhizome of Aucklandia lappa Decne (Asteraceae), enjoyed traditional popularity for its antidiarrheal effects. Although there are many investigations on its chemical constituents and pharmacologic actions, few studies explaining its activity and mechanism in gastrointestinal disorders are available.

Objective: In this paper, we focused on the effects of the methanol extract of R. Aucklandiae (RA ext) on gastrointestinal tract, so as to assess some of the possible mechanisms involved in the clinical treatment.

Materials and methods: In vivo, in neostigmine-induced mice and normal mice, after intragastric administration, RA ext (100, 200, 300, and 400?mg/kg) was studied on gastrointestinal transit including gastric emptying and small intestinal motility. Meanwhile, in vitro, the effect of it (0.1, 0.2, 0.3, and 0.4?mg/mL) on the isolated tissue preparations of rat jejunum was also investigated, as well as costunolide and dehydrocostuslactone which were the main constituents.

Results: In vivo, the gastric emptying increased and intestinal transit decreased after the administration of RA ext in normal mice. However, RA ext inhibited the gastric emptying and the intestinal transit throughout the concentrations in neostigmine-induced mice. In vitro, RA ext caused inhibitory effect on the spontaneous contraction of rat-isolated jejunum in a dose-dependent manner ranging from 0.1 to 0.4?mg/mL, and it also relaxed the acetylcholine chloride (Ach, 10^?5?M), 5-hydroxytryptamine (5-HT, 200?μM)-induced, and K⁺ (60?mM)-induced contractions. RA ext shifted the Ca²⁺ concentration–response curves to right, similar to that caused by verapamil (0.025?mM). The Ca²⁺ concentration–response curves were shifted by costunolide (CO) (5.4, 8.1, and 10.8?μg/mL), dehydrocostuslactone (DE) (4.6, 6.9, and 9.2?μg/mL), costunolide–dehydrocostuslactone (CO–DE) (5.4–4.6, 8.1–6.9, and 10.8–9.2?μg/mL) to the right, similar to that caused by verapamil (0.01?mM).

Discussion and conclusion: These results indicate that RA ext played a spasmolytic role in gastrointestinal motility, which is probably mediated through the inhibition of muscarinic receptors, 5-HT receptors, and calcium influx. The presence of cholinergic and calcium antagonist constituents may be the compatibility of CO and DE. All these results provide a pharmacological basis for its clinical use in the gastrointestinal tract.     

Unrepeatable extracellular Ca-dependent contractile effects of cyclopiazonic acid in rat vascular smooth muscle

Cyclopiazonic acid (CPA), a specific reversible inhibitor of Ca²⁺-pumps in sarcoplasmic reticulum, causes a slowly developing and subsequently diminishing characteristic contraction in endothelium-denuded rat vascular smooth muscle. We recently found that CPA-induced contractions were not completely repeatable in endothelium-denuded rat aorta and superior mesenteric artery. 10 µM CPA-induced contractions expressed as a percentage of 80 mM KCl-induced contraction were significantly decreased from 51.4 ± 5.7% to 11.8 ± 2.6% (P < 0.0001) upon the second application in endothelium-denuded rat aorta, and this was not due to any irreversible cytotoxic effects of CPA. The decrease of CPA-induced contractile responses upon the second application was dependent on both types of blood vessels and doses of CPA upon the first application. CPA upon the second application in Ca²⁺-containing solutions did induce its characteristic contractions in the rings pretreated with Ca²⁺-free solutions or Ca²⁺ entry blockers before and during its first application, suggesting that capacitative mode of Ca²⁺ influx during the application of CPA might be responsible for the diminishment of contractions upon the second application. These data suggest that CPA by inducing a transient rise in cytosolic Ca²⁺ level might cause a long-lasting upregulation of Ca²⁺ extrusion across the plasma membrane in vascular smooth muscle cells and thus accelerate Ca²⁺ efflux over a prolonged period, leading to unrepeatable contractile effects of CPA. Such long-lasting upregulation of Ca²⁺ extrusion may contribute to the regulation of excitability of vascular smooth muscle cells and protect the cells against excitotoxic injury.     

Influence of kaempferol,a flavonoid compound,on membrane-bound ATPases in streptozotocin-induced diabetic rats

Abstract

Context: Kaempferol is a flavonoid found in many edible plants (e.g. tea, cabbage, beans, tomato, strawberries, and grapes) and in plants or botanical products commonly used in traditional medicine. Numerous preclinical studies have shown that kaempferol have a wide range of pharmacological activities, including antioxidant, anti-inflammatory, anticancer, cardioprotective, neuroprotective, and antidiabetic activities.

Objective: The present study investigates the effect of kaempferol on membrane-bound ATPases in erythrocytes and in liver, kidney, and heart of streptozotocin (STZ)-induced diabetic rats.

Materials and methods: Diabetes was induced into adult male albino rats of the Wistar strain, by intraperitoneal administration of STZ (40?mg/kg body weight (BW)). Kaempferol (100?mg/kg BW) or glibenclamide (600?µg/kg BW) was administered orally once daily for 45?d to normal and STZ-induced diabetic rats. The effects of kaempferol on membrane-bound ATPases (total ATPase, Na⁺/K⁺-ATPase, Ca²⁺-ATPase, and Mg²⁺-ATPase) activity in erythrocytes and in liver, kidney, and heart were determined.

Results: In our study, diabetic rats had significantly (p?<?0.05) decreased activities of total ATPases, Na⁺/K⁺-ATPase, Ca²⁺-ATPase, and Mg²⁺-ATPase in erythrocytes and tissues. Oral administration of kaempferol (100?mg/kg BW) or glibenclamide (600?µg/kg BW) for a period of 45?d resulted in significant (p?<?0.05) reversal of these enzymes' activities to near normal in erythrocytes and tissues when compared with diabetic control rats.

Discussion and conclusion: Thus, obtained results indicate that administration of kaempferol has the potential to restore deranged activity of membrane-bound ATPases in STZ-induced diabetic rats. Further detailed investigation is necessary to discover kaempferol’s action mechanism.     

Methanol-Induced Contraction of Canine Cerebral Artery and Its Possible Mechanism of Action

In the present report, we investigated the effects of methanol on canine basilar cerebral arterial rings. Our data indicate that acute methanol exposure (5–675 mM) induces potent contractile responses of cerebral arteries in a concentration-dependent manner. Pharmacological antagonists, such as propranolol, phentolamine, haloperidol, methysergide, naloxone, diphenhydramine, and cimetidine, did not exert any effects on these methanol-induced contractions. Likewise, a potent antagonist of cyclo-oxygenase, and subsequent synthesis of prostanoids (i.e., indomethacin), failed to exert any effect on methanol-induced contractions. No differences in responsiveness to methanol in canine cerebral arteries were found in vessel segments with or without endothelial cells. Removal of extracellular Ca²⁺([Ca²⁺]_o) partially attenuated methanol-induced contractions, while withdrawal of extracellular Mg²⁺([Mg²⁺]_o) potentiated the contractions. In the complete absence of [Ca²⁺]_o, 10 mM caffeine and 400 mM methanol induced similar, transient contractions followed by relaxation in K⁺-depolarized cerebral vascular tissues. Methanol-induced contractions were, however, completely abolished by pretreatment of tissue with 10 mM caffeine. Our results indicate that (1) methanol causes contractile responses of cerebral arterial smooth muscle (independent of amine, prostanoid, or opioid mediation; (2) in addition to a need for [Ca²⁺]_o, an intracellular release of Ca²⁺is required for methanol-induced contractions; and (3) Mg deficiency potentiates the contractile responses of methanol on these brain vessels. The data presented in the study suggest that methanol-induced contractions occur via an sarcoplasmic reticulum-releasable store of [Ca²⁺]_i; via mediation of either ryanodine–caffeine type receptors or a caffeine-releasable intracellular store of Ca²⁺.