Local carbachol application induces oral microvascular recruitment and improves gastric tissue oxygenation during hemorrhagic shock in dogs.

Introduction: Hemorrhagic shock is characterized by derangements of the gastrointestinal microcirculation. Topical therapy with nitroglycerine or iloprost improves gastric tissue oxygenation but not regional perfusion, probably due to precapillary adrenergic innervation. Therefore, this study was designed to investigate the local effect of the parasympathomimetic carbachol alone and in combination with either nitroglycerine or iloprost on gastric and oral microcirculation during hemorrhagic shock. Methods: In a cross-over design five female foxhounds were repeatedly randomized into six experimental groups. Carbachol, or carbachol in combination with either nitroglycerine or iloprost were applied topically to the oral and gastric mucosa. Saline, nitroglycerine, or iloprost application alone served as control groups. Then, a fixed-volume hemorrhage was induced by arterial blood withdrawal followed by blood retransfusion after 1h of shock. Gastric and oral microcirculation was determined using reflectance spectrophotometry and laser Doppler flowmetry. Oral microcirculation was visualized with videomicroscopy. Statistics: 2-way-ANOVA for repeated measurements and Bonferroni post-hoc analysis (mean ± SEM; p < 0.05). Results: The induction of hemorrhage led to a decrease of gastric and oral tissue oxygenation, that was ameliorated by local carbachol and nitroglycerine application at the gastric mucosa. The sole use of local iloprost did not improve gastric tissue oxygenation but could be supplemented by local carbachol treatment. Adding carbachol to nitroglycerine did not further increase gastric tissue oxygenation. Gastric microvascular blood flow remained unchanged in all experimental groups. Oral microvascular blood flow, microvascular flow index and total vessel density decreased during shock. Local carbachol supply improved oral vessel density during shock and oral microvascular flow index in the late course of hemorrhage. Conclusion: The specific effect of shifting the autonomous balance by local carbachol treatment on microcirculatory variables varies between parts of the gastrointestinal tract. Contrary to our expectations, the improvement of gastric tissue oxygenation by local carbachol or nitroglycerine application was not related to increased microvascular perfusion. When carbachol is used in combination with local vasodilators, the additional effect on gastric tissue oxygenation depends on the specific drug combination. Therefore, modulation of tissue oxygen consumption, mitochondrial function or alterations in regional blood flow distribution should be investigated.

Antidiarrheal, antisecretory and intestinal smooth muscle relaxant effects of Platanus orientalis in mice.

Platanus orientalis is traditionally used to treat diarrhea and spasm. However, studies are lacking on its mechanism of action in diarrhea and spasm. Pharmacological in-vivo activities were performed. In-vitro activities were carried out to explore the underlying mechanism(s) of action in isolated tissue preparations of mice jejunum and ileum. Crude extract of Platanus orientalis, loperamide and verapamil were used. The crude extract provided dose-dependent protection in castor oil diarrhea like verapamil and reduced the intestinal fluid accumulation and charcoal meal transit distance. In-vitro studies produced spasmolytic effect on the spontaneous (EC50 value=0.21mg/mL), high K+ (EC50 value=0.37mg/mL) and carbachol (CCh)-induced contractions 5.35mg/mL (3.88-6.85) respectively. The quiescent ileum responded well to the high K+ and carbachol (CCh)-induced contractions when tested against crude extract. It caused inhibition of the induced contraction with EC50 values of 0.20mg/mL (0.10-0.30) and 3.25mg/mL (2-4.5) respectively and showed potent effect against CCh-induced contractions. Calcium response curves produced a similar effect to verapamil. The crude extract of Platanus orientalis remained safe up to 5g/kg dose.

Investigations of plausible pharmacodynamics supporting the antispasmodic, bronchodilator, and antidiarrheal activities of Berberis lycium Royle. Via in silico, in vitro, and in vivo studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Berberis lycium Royle, a member of the Berberidaceae family, is a high-value medicinal plant with a documented history of usage in traditional medicine and has demonstrated significant therapeutic results among local populations throughout the globe. It is used traditionally in many parts of Pakistan to treat diarrhea, abdominal spasms, coughs, and chest problems. AIM OF THE STUDY: To investigate the antispasmodic, bronchodilator, and antidiarrheal effects of B. lycium and its possible underlying mechanisms through in silico, in vitro, and in vivo studies. MATERIALS AND METHODS: LC ESI-MS/MS analysis was used to identify bioactive components within the hydromethanolic extract of B. lycium. In silico studies, including network pharmacology and molecular docking, were utilized to investigate the antispasmodic and bronchodilator properties of the extract's bioactive components. In vitro pharmacological studies were conducted using isolated rabbit jejunum, trachea, urinary bladder, and rat ileum preparations. In vivo antidiarrheal activities were conducted in mice, including castor oil-induced diarrhea, intestinal transit, and castor oil-induced enteropooling. RESULTS: The LC ESI-MS/MS analysis of the hydromethanolic extract of B. lycium identified 38 bioactive compounds. Network pharmacology study demonstrated that the mechanism of BLR for the treatment of diarrhea might involve IL1B, TLR4, PIK3R1, TNF, PTPRC, IL2, PIK3CD, and ABCB1, whereas, for respiratory ailments, it may involve PIK3CG, TRPV1, STAT3, ICAM1, ACE, PTGER2, PTGS2, TNF, MMP9, NOS2, IL2, CCR5, HRH1, and VDR. Molecular docking research revealed that chlorogenic acid, epigallocatechin, isorhamnetin, quinic acid, gallic acid, camptothecin, formononetin-7-O-glucoside, velutin, caffeic acid, and (S)-luteanine exhibited a higher docking score than dicyclomine with validated proteins of smooth muscle contractions such as CACB2_HUMAN, ACM3_HUMAN, MYLK_HUMAN, and PLCG1_HUMAN. In vitro investigations demonstrated that Blr.Cr, Blr.EtOAc, and Blr.Aq relaxed spontaneously contracting jejunum preparations; carbachol (1 µM)-induced and K+ (80 mM)-induced jejunum, trachea, and urinary bladder contractions in a concentration-dependent manner, similar to dicyclomine. Moreover, Blr.Cr, Blr.EtOAc, and Blr.Aq exhibited a rightward shift in Ca+2 and carbachol cumulative response curves, similar to dicyclomine, demonstrating the coexistence of antimuscarinic and Ca+2 antagonistic mechanisms due to the presence of alkaloids and flavonoids. In vivo antidiarrheal activities showed that the hydromethanolic extract was significantly effective against castor oil-induced diarrhea and castor oil-induced enteropooling, similar to loperamide, and charcoal meal intestinal transit, similar to atropine, in mice at doses of 50, 100, and 200 mg/kg body weight, which supports its traditional use in diarrhea. CONCLUSION: The dual blocking mechanism of muscarinic receptors and Ca+2 channels behind the smooth muscle relaxing activity reveals the therapeutic relevance of B. lycium in diarrhea, abdominal spasms, coughs, and chest problems.

Pharmacological effects (gastrointestinal, respiratory and vascular) of Calligonum polygonoides: Animal model based studies.

The aim of this study was to analyze gastrointestinal, respiratory and vascular pharmacological effects of 70% hydro-alcoholic extract of Calligonum polygonoides (Cp. Cr) in animal models. All the procedures were carried-out as per previous literature with slight modification where necessary. It was found that Cp. Cr affected significant relaxation of spontaneous and K+ (80 mM) induced contractions. The results showed a corresponding shift of calcium concentration response curves. Similarly Cp. Cr showed relaxant effect on trachea in carbachol (Cch) induced tracheal contractions. Moreover, contractions induced by phenylephrine (1µM) in quarantine rabbit aortic preparations causes Cp. Cr induced relaxation of aortal contractions. Verapamil was used as a standard calcium channel blocker. The findings of this study suggested vasodilator, bronchodilator and spasmolytic effects of Cp. Cr.

Chinese Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycota), Cold-Water Extract Relaxes the Rat Isolated Bladder.

Ophiocordyceps sinensis is a popular medicinal mushroom used for various health conditions, including alleviation of frequent urination, which is a major symptom of overactive bladder (OAB) syndrome. This study aimed to investigate the effect of O. sinensis (OCS02 cultivar) cold-water extract (CWE) against bladder contractility using the organ bath technique. The bladder was removed from male Sprague-Dawley rats and cut into longitudinal strips of 2 mm × 8 mm. In some experiments, the urothelium was removed to study its role in CWE-induced responses. CWE elicited a biphasic response consisting of an immediate, transient contraction that was followed by a sustained relaxation in bladder strips precontracted with carbachol, a muscarinic agonist. Removal of urothelium did not alter the magnitude of the contractile response but significantly attenuated the relaxation response. In the presence of L-NAME (nitric oxide synthase inhibitor) and sodium nitroprusside (nitric oxide donor), CWE-induced transient contraction was enhanced, whereas the relaxation response was significantly reduced. Following preincubation with CWE, the amplitude and the frequency of the spontaneous myogenic contractions induced by carbachol, as well as the contractile response toward calcium, were significantly suppressed. Findings from this study show that the urothelium plays a role in the relaxant effect of CWE. Its mechanisms of action include the regulation of nitric oxide and inhibition of calcium influx.

Lippia origanoides essential oil induces tocolytic effect in virgin rat uterus and inhibits writhing in a dysmenorrhea mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: The species Lippia origanoides Kunth, popularly known as "salva-de-marajó", is used in Brazilian traditional "quilombola" communities to treat menstrual cramps and uterine inflammation. AIM OF THE STUDY: Evaluate the spasmolytic activity of Lippia origanoides essential oil (LOO) on experimental models of uterine conditions related to menstrual cramps and investigate its mechanism of action. MATERIALS AND METHODS: Virgin rat-isolated uterus was mounted in the organ bath apparatus to evaluate the spasmolytic effect of LOO on basal tonus and contractions induced by carbachol, KCl, or oxytocin. We used pharmacological agents to verify the relaxation mechanism of LOO. The evaluation of uterine contractility in virgin rats, after treatment with LOO for three consecutive days, was carried out by the construction of a concentration-response curve with oxytocin or carbachol. The primary dysmenorrhea animal model was replicated with an injection of estradiol cypionate in female mice for three consecutive days, followed by intraperitoneal application of oxytocin. RESULTS: LOO relaxed the rat uterus precontracted with 10-2 IU/mL oxytocin (logEC50 = 1.98 ± 0.07), 1 µM carbachol (logEC50 = 1.42 ± 0.07) or 60 mM KCl (logEC50 = 1.53 ± 0.05). It was also able relax uterus on spontaneous contractions (logEC50 = 0.41 ± 0.05). Preincubation with glibenclamide, propranolol, phentolamine or L-NAME in contractions induced by carbachol did not alter significantly the relaxing effect of LOO. However, in the presence of 4-aminopyridine, CsCl or tetraethylammonium there was a reduction of LOO potency, whereas the blockers methylene blue, ODQ, aminophylline and heparin potentiated the LOO relaxing effect. Preincubation with LOO in a Ca2+ free medium at concentrations of 27 µg/mL or 81 µg/mL reduced the contraction induced by carbachol. The administration of LOO for 3 days did not alter uterus contractility. The treatment with LOO at 30 or 100 mg/kg intraperitoneally, or 100 mg/kg orally, inhibited writhing in female mice. The association of LOO at 10 mg/kg with nifedipine or mefenamic acid potentiated writhing inhibition in mice. CONCLUSIONS: The essential oil of L. origanoides has tocolytic activity in rat isolated uterus pre-contracted with KCl, oxytocin, or carbachol. This effect is possibly related to the opening of potassium channels (Kir, KV, and KCa), cAMP increase, and diminution of intracellular Ca2+. This relaxant effect, probably, contributed to reduce the number of writhings in an animal model of dysmenorrhea being potentiated by nifedipine or mefenamic acid. Taken together, the results here presented indicate that this species has a pharmacological potential for the treatment of primary dysmenorrhea, supporting its use in folk medicine.

Antispasmodic activity and mechanism of action of polyherbal formulation DCD-684 on rabbit jejunum.

Digas colic drops (DCD-684) is a polyherbal formulation containing decoctions of five medicinal plants namely Carum carvi L., Foeniculum vulgare Mill, Mentha arvensis L., Mentha piperita L. and Zingiber officinale Roscoe. These plants have been extensively used in traditional medicine for the treatment of various gastrointestinal diseases including abdominal colic. This study was conducted to determine the spasmolytic effect of DCD-684 (100% v/v) and its individual plant components on isolated rabbit jejunum (in vitro) and their possible mechanism of action. The effects were evaluated on spontaneous and pre-contracted tissues using KCl (80mM) and other contractile agonists including acetylcholine (0.3µM), carbamylcholine (0.3µM), serotonin (10 µM) and histamine (100µM) in the presence and absence of DCD-684. The various concentrations of DCD-684 (0.1-3% v/v) demonstrated spasmolytic effects on both spontaneous (IC50=0.75%) and KCl-induced contractions (IC50=1.6%), respectively. It also inhibited the contractions induced by acetylcholine (IC50=0.45%), carbamylcholine (IC50=0.95%), serotonin (IC50=0.95%) and histamine (IC50=0.87%). The DCD-684 exhibited synergistic effect due to its five plant components suggesting that spasmolytic cascade is probably governed by muscarinic and/or nicotinic receptors, serotonergic histaminergic, as well as calcium channel blocking mechanisms. Thereby, providing the pharmacological basis of its therapeutic use in the gastrointestinal motility disorders and related inflammatory ailments.

Pharmacological screening of the phenolic compound caffeic acid using rat aorta, uterus and ileum smooth muscle.

Caffeic acid is a phenolic compound widely found in commonly consumed foods such as pears, apples and coffee, and is pharmacologically known for its antioxidant, anti-inflammatory and anti-asthmatic properties. However, its relaxant activity in the aorta, uterus and ileum smooth muscle has not been investigated. This study aimed to comparatively evaluate the effect of caffeic acid on smooth muscle from different organs (aorta, uterus and ileum), and the contractions of this different organ were induced by different agonists. The organ bath technique was used, where the organs were placed in different cuvettes with 10 mL of Tyrode solution for 1 h to stabilize, then, myometrial, intestinal strip and aortic ring contractions were evoked using different contractile agonists (KCl 60 mM, PHE 0.1 µM, OT 10-2 IU/mL, CCh 10-6 M and BaCl2 0.1-30 mM); increasing concentrations of caffeic acid (0.03-7 mM) were administered in the experimental preparations. In the presence of KCl (60 mM), caffeic acid caused relaxations with the following EC50 values: 2.7 ± 0.26 mM/mL (aorta), 5.7 ± 0.71 mM/mL (uterus) and 2.1 ± 0.39 mM/mL (ileum). When in the presence of different agonists, PHE (0.1 µM) for the aorta, OT (10-2 IU/mL) for the uterus and CCh (10-6 M) for the ileum, caffeic acid caused relaxations with EC50 values of: 2.7 ± 0.31 mM/mL; 2.2 ± 0.34 mM/mL and 2.0 ± 0.28 mM/mL, respectively. The inhibitory effect of caffeic acid on serotonergic (aorta and uterus) and muscarinic receptors (uterus and ileum), as well as its possible involvement with L-type Ca2+ channels, was also observed. This study reports the pharmacological characterization of caffeic acid on smooth muscle from different organs, for which caffeic acid was more potent in the ileum. A diverse understanding of its performance as a possible therapeutic product is attributed to its relaxant effect.

Hyperbaric oxygen therapy prevents subarachnoid hemorrhage-induced apoptosis and impaired contractility of the rabbit bladder.

AIM: To explore the effects of experimental subarachnoid hemorrhage (SAH) on rabbit urinary bladder and to assess the potential protective effects of hyperbaric oxygen therapy (HBOT). METHODS: A total of 15 male New Zealand white rabbits were divided randomly to one of three groups: group I was spared as the control group (n = 5), group II was exposed to SAH, received no treatment, and acted as the SAH group (n = 5) and group III was exposed to SAH and received five sessions of HBOT (started 12 hours after SAH induction and was given twice daily for the first 2 days and once on the third day) and acted as the treatment group (n = 5). At 72 hours after the SAH induction, bladders from all animals were removed for in vitro organ bath experiments and biochemical analyses. RESULTS: Isometric tension studies revealed that compared to group I, the contractile responses of the strips to carbachol in group II were significantly decreased whereas HBOT restored the contractile responses (P < .05). Caspase-3 and nitric oxide synthase (NOS) activities of bladder tissues were significantly increased in group II when compared with group I, whereas caspase-3 and NOS activities were significantly decreased in the tissues of group III (P < .01). CONCLUSIONS: Subarachnoid hemorrhage stimulates apoptosis of the rabbit bladder and impairs the contractile response of the rabbit bladder to carbachol. HBOT creates a protective effect in rabbit bladder tissues and restores SAH-induced changes.

Acetylcholine acts on songbird premotor circuitry to invigorate vocal output.

Acetylcholine is well-understood to enhance cortical sensory responses and perceptual sensitivity in aroused or attentive states. Yet little is known about cholinergic influences on motor cortical regions. Here we use the quantifiable nature of birdsong to investigate how acetylcholine modulates the cortical (pallial) premotor nucleus HVC and shapes vocal output. We found that dialyzing the cholinergic agonist carbachol into HVC increased the pitch, amplitude, tempo and stereotypy of song, similar to the natural invigoration of song that occurs when males direct their songs to females. These carbachol-induced effects were associated with increased neural activity in HVC and occurred independently of basal ganglia circuitry. Moreover, we discovered that the normal invigoration of female-directed song was also accompanied by increased HVC activity and was attenuated by blocking muscarinic acetylcholine receptors. These results indicate that, analogous to its influence on sensory systems, acetylcholine can act directly on cortical premotor circuitry to adaptively shape behavior.

In Silico, Ex Vivo and In Vivo Studies of Roflumilast as a Potential Antidiarrheal and Antispasmodic agent: Inhibition of the PDE-4 Enzyme and Voltage-gated Ca++ ion Channels.

The aim of the present study was to evaluate the possible gut inhibitory role of the phosphodiesterase (PDE) inhibitor roflumilast. Increasing doses of roflumilast were tested against castor oil-induced diarrhea in mice, whereas the pharmacodynamics of the same effect was determined in isolated rabbit jejunum tissues. For in silico analysis, the identified PDE protein was docked with roflumilast and papaverine using the Autodock vina program from the PyRx virtual screening tool. Roflumilast protected against diarrhea significantly at 0.5 and 1.5 mg/kg doses, with 40% and 80% protection. Ex vivo findings from jejunum tissues show that roflumilast possesses an antispasmodic effect by inhibiting spontaneous contractions in a concentration-dependent manner. Roflumilast reversed carbachol (CCh, 1 µM)-mediated and potassium (K+, 80 mM)-mediated contractile responses with comparable efficacies but different potencies. The observed potency against K+ was significantly higher in comparison to CCh, similar to verapamil. Experiments were extended to further confirm the inhibitory effect on Ca++ channels. Interestingly, roflumilast deflected Ca++ concentration-response curves (CRCs) to the right with suppression of the maximum peak at both tested doses (0.001-0.003 mg/mL), similar to verapamil. The PDE-inhibitory effect was authenticated when pre-incubation of jejunum tissues with roflumilast (0.03-0.1 mg/mL) produced a leftward deflection of isoprenaline-mediated inhibitory CRCs and increased the tissue level of cAMP, similar to papaverine. This idea was further strengthened by molecular docking studies, where roflumilast exhibited a better binding affinity (-9.4 kcal/mol) with the PDE protein than the standard papaverine (-8.3 kcal/mol). In conclusion, inhibition of Ca++ channels and the PDE-4 enzyme explains the pharmacodynamics of the gut inhibitory effect of roflumilast.

Lippia alnifolia essential oil induces relaxation on Guinea-pig trachea by multiple pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Lippia alnifolia Mart. & Schauer, known as "alecrim-do-mato", "alecrim-de-vaqueiro" and "pedrécio", is used in folk medicine as antiseptic and to treat diseases that affect respiratory system, like bronchitis and asthma. AIM OF THE STUDY: The aim of this work was to investigate the spasmolytic activity and relaxant mechanism of the Lippia alnifolia essential oil (EOLA) on isolated guinea-pig trachea and to correlate with its use in folk medicine. MATERIALS AND METHODS: Leaves from L. alnifolia were collected in Pico das Almas, Chapada Diamantina, situated in the city of Rio de Contas, Bahia, Brazil. EOLA was extracted by hydrodistillation, analyzed by GC/FID and GC/MS and the volatile constituents were identified. Spasmolytic activity was assayed in isolated guinea-pig trachea pre-contracted with carbachol 1 µM or histamine 10 µM. Relaxant mechanism of EOLA was determined comparing concentration-response curves in the presence or absence of different blockers. RESULTS: Chemical analysis revealed the presence of carvone (60 ±â€¯0.8%) as major constituent. EOLA (1-243 µg/mL) relaxed isolated guinea-pig trachea pre-contracted with carbachol 1 µM [EC50 = 53.36 (44.75-63.51) µg/mL] or histamine 10 µM [EC50 = 5.42 (4.42-6.65) µg/mL]. The pre-incubation of 4-aminopyridine in histamine-induced contractions did not alter significantly the relaxant effect of EOLA. However, the presence of cesium chloride, glibenclamide, tetraethylammonium, propranolol, indomethacin, dexamethasone, hexamethonium, atropine, L-NAME, methylene blue or ODQ reduced EOLA relaxant effect. EOLA 18 µg/mL pre-incubation in calcium-free medium reduced histamine-evoked contractions, but did not alter histamine contractions in the presence of nifedipine. CONCLUSIONS: Lippia alnifolia essential oil has spasmolytic activity on isolated guinea-pig trachea and its mechanism of action possibly involves the activation of multiple signal transduction pathways, which culminate in potassium channels activation and cytosolic calcium reduction.

The interaction of gamma radiation with drugs used in cholinergic medications.

Purpose: Pharmacological medications can reduce the radiation damage in the organism when applied in the stage before or after exposure to radiation. Cholinergic drugs are a category of pharmaceutical agents acting on the neurotransmitter acetylcholine, the primary neurotransmitter in the parasympathetic nervous system. In this investigation, some gamma radiation interaction parameters namely mass attenuation coefficients (µρ), effective atomic number (Zeff) and electron densities (Nel) of 12 cholinergic system drugs have been calculated in the energy range 1 KeV-100 GeV. In addition, gamma-ray energy absorption (EABF) and exposure (EBF) of buildup factors have been computed using the five-parameter geometric progression (G-P) fitting formula for investigated drugs in the energy range 0.015-15 MeV, and for penetration depths up to 40 mean free path (mfp).Materials and methods: In order to perform these calculations, data obtained from WinXCom computer program were used. The computed µρ values were then used to calculate the effective atomic numbers and electron density of the investigated drugs. To compute the buildup factors, the G-P fitting parameters were determined by the method of interpolation from the equivalent atomic number, 'Zeq'Results and Conclusions: It has been concluded that effective atomic number and electron density of malathion is bigger than the other drugs and the variations in values of Zeff and Nel for all drugs depend on chemical compositions and photon energy where the K-absorption edge of elements may affect the energy dependence of Zeff and Nel. It should also be noted that the buildup of photons is less in malathion and carbachol and is more in tabun and parathion compared with other drugs. Photon interaction parameters evaluated in the present study may be beneficial in radiation dosimetry and therapy.

Acetaminophen is both bronchodilatory and bronchoprotective in human precision cut lung slice airways.

Epidemiologic studies have demonstrated an association between acetaminophen (APAP) use and the development of asthma symptoms. However, few studies have examined relationships between APAP-induced signaling pathways associated with the development of asthma symptoms. We tested the hypothesis that acute APAP exposure causes airway hyper-responsiveness (AHR) in human airways. Precision cut lung slice (PCLS) airways from humans and mice were used to determine the effects of APAP on airway bronchoconstriction and bronchodilation and to assess APAP metabolism in lungs. APAP did not promote AHR in normal or asthmatic human airways ex vivo. Rather, high concentrations mildly bronchodilated airways pre-constricted with carbachol (CCh), histamine (His), or immunoglobulin E (IgE) cross-linking. Further, the addition of APAP prior to bronchoconstrictors protected the airways from constriction. Similarly, in vivo treatment of mice with APAP (200 mg/kg IP) resulted in reduced bronchoconstrictor responses in PCLS airways ex vivo. Finally, in both mouse and human PCLS airways, exposure to APAP generated only low amounts of APAP-protein adducts, indicating minimal drug metabolic activity in the tissues. These findings indicate that acute exposure to APAP does not initiate AHR, that high-dose APAP is protective against bronchoconstriction, and that APAP is a mild bronchodilator.

Intestinal, urinary and uterine antispasmodic effects of isoespintanol, metabolite from Oxandra xylopioides leaves.

BACKGROUND: Isoespintanol is a monoterpene isolated from the leaves of Oxandra xylopioides Diels. (Annonaceae) with antioxidant and antiinflammatory effects. It was of interest to know whether it has antispasmodic effects such as other known drugs, phloroglucinol and trimethoxybenzene, used in therapeutics for treating biliary, urinary and uterine spasms. PURPOSE: To assess whether isoespintanol possesses antispasmodic effects on intestine, uterus and bladder. STUDY DESIGN: A preclinical study was performed in which isoespintanol, phloroglucinol and trimethoxybenzene were evaluated with concentration-contractile response curves (CRC) of carbachol in isolated rat intestine and bladder, and with CRC of serotonin (5-HT) in rat uterus. Moreover, it was assessed whether isoespintanol interferes with Ca2+ influx by making CRC of Ca2+ in high-K+ medium in intestine and bladder. RESULTS: Isoespintanol non-competitively inhibited the CRC of carbachol with affinity constant (pK) of 4.78 ±â€¯0.09 in intestine and 4.60 ±â€¯0.09 in bladder. Phloroglucinol and trimethoxybenzene were also non-competitive antagonists, but isoespintanol was 8 times more potent than trimethoxybenzene and similarly potent than phloroglucinol in intestine. In bladder, isoespintanol resulted 8 times more potent than trimethoxybenzene. The maximal inhibition of contraction followed the order of isoespintanol > trimethoxybenzene > phloroglucinol in intestine, and isoespintanol > trimethoxybenzene in bladder. Moreover, isoespintanol also completely and non-competitively inhibited the CRC of Ca2+, with a pK of 5.1 ±â€¯0.1 in intestine, and 4.32 ±â€¯0.07 in bladder. In uterus isoespintanol reduced, completely and non-competitively, the contraction produced by 5-HT with pK of 5.05 ±â€¯0.07. CONCLUSION: Results demonstrate that isoespintanol is a very good intestinal, urinary and uterine antispasmodic, with higher potency than the other drugs used in therapeutics. The mechanism of action of isoespintanol is the interference with Ca2+ influx, at a difference of trimethoxybenzene and phloroglucinol.

Bronchodilator effects of Lignosus rhinocerotis extract on rat isolated airways is linked to the blockage of calcium entry.

BACKGROUND: Lignosus rhinocerotis (Cooke) Ryvarden is a popular medicinal mushroom used for centuries in Southeast Asia to treat asthma and chronic cough. The present study aimed to investigate the effect of this mushroom on airways patency. MATERIALS AND METHODS: The composition of L. rhinocerotis TM02 cultivar was analyzed. Organ bath experiment was employed to study the bronchodilator effect of Lignosus rhinocerotis cold water extract (CWE) on rat isolated airways. Trachea and bronchus were removed from male Sprague-Dawley rats, cut into rings of 2 mm, pre-contracted with carbachol before adding CWE into the bath in increasing concentrations. To investigate the influence of incubation time, tissues were exposed to intervals of 5, 15 and 30 min between CWE concentrations after pre-contraction with carbachol in subsequent protocol. Next, tissues were pre-incubated with CWE before the addition of different contractile agents, carbachol and 5-hydroxytrptamine (5-HT). The bronchodilator effect of CWE was compared with salmeterol and ipratropium. In order to uncover the mechanism of action of CWE, the role of beta-adrenoceptor, potassium and calcium channels was investigated. RESULTS: Composition analysis of TM02 cultivar revealed the presence of ß-glucans and derivatives of adenosine. The extract fully relaxed the trachea at 3.75 mg/ml (p < 0.0001) and bronchus at 2.5 mg/ml (p < 0.0001). It was observed that lower concentrations of CWE were able to fully relax both trachea and bronchus but at a longer incubation interval between concentrations. CWE pre-incubation significantly reduced the maximum responses of carbachol-induced contractions (in both trachea, p = 0.0012 and bronchus, p = 0.001), and 5-HT-induced contractions (in trachea, p = 0.0048 and bronchus, p = 0.0014). Ipratropium has demonstrated a significant relaxation effect in both trachea (p = 0.0004) and bronchus (p = 0.0031), whereas salmeterol has only affected the bronchus (p = 0.0104). The involvement of ß2-adrenoceptor and potassium channel in CWE-mediated airway relaxation is ruled out, but the bronchodilator effect was unequivocally affected by influx of calcium. CONCLUSIONS: The bronchodilator effect of L. rhinocerotis on airways is mediated by calcium signalling pathway downstream of Gαq-coupled protein receptors. The airway relaxation effect is both concentration- and incubation time-dependent. Our findings provide unequivocal evidence to support its traditional use to relieve asthma and cough.

Effects of chlorogenic acid on carbachol-induced contraction of mouse urinary bladder.

Chlorogenic acid (CGA) is a polyphenol found in coffee and medicinal herbs such as Lonicera japonica. In this study, the effect of CGA-induced relaxation on carbachol (CCh)-induced contraction of mouse urinary bladder was investigated. CGA (30-300 µg/ml) inhibited CCh- or U46619-induced contraction in a concentration-dependent manner. SQ22536 (adenylyl cyclase inhibitor) recovered CGA-induced relaxation of CCh-induced contraction; however, ODQ (guanylyl cyclase inhibitor) did not have the same effect. In addition, 3-isobutyl-1-methylxanthine (IBMX) enhanced CGA-induced relaxation; however, forskolin or sodium nitroprusside did not have the same effect. Moreover, Ro 20-1724, a selective phosphodiesterase (PDE) 4 inhibitor, enhanced CGA-induced relaxation, but vardenafil, a selective PDE5 inhibitor, did not have the same effect. In the presence of CCh, CGA increased cyclic adenosine monophosphate (cAMP) level, whereas SQ22536 inhibited the increase of cAMP levels. Moreover, higher cAMP levels were obtained with CGA plus IBMX treatment than the total cAMP levels obtained with separate CGA and IBMX treatments. In conclusion, these results suggest that CGA inhibited CCh-induced contraction of mouse urinary bladder by partly increasing cAMP levels via adenylyl cyclase activation.

Tissue-Level Cardiac Electrophysiology Studied in Murine Myocardium Using a Microelectrode Array: Autonomic and Thermal Modulation.

Cardiac electrophysiology is regulated by the autonomic nervous system, and this has both pathophysiological, and possibly therapeutic importance. Furthermore, chamber differences in electrophysiology exist between atria and ventricles, yet there have been few direct comparisons. There is substantial literature on ion channel modulation at the single-cell level but less work on how this affects tissue-level parameters. We used a microelectrode array system to explore these issues using murine atrial and ventricular tissue slices. Activation time, conduction velocity and repolarisation were measured, and their modulation by temperature and pharmacological autonomic agonists were assessed. The system recorded reliable measurements under control conditions in the absence of drug/thermal challenge, and significant baseline differences were found in chamber electrophysiology. The sodium channel blocker mexiletine, produced large magnitude changes in all three measured parameters. Carbachol and isoprenaline induced differing effects in atria and ventricles, whereas temperature produced similar effects on activation and repolarisation.

Phase-amplitude coupled persistent theta and gamma oscillations in rat primary motor cortex in vitro.

In vivo, theta (4-7 Hz) and gamma (30-80 Hz) neuronal network oscillations are known to coexist and display phase-amplitude coupling (PAC). However, in vitro, these oscillations have for many years been studied in isolation. Using an improved brain slice preparation technique we have, using co-application of carbachol (10 µM) and kainic acid (150 nM), elicited simultaneous theta (6.6 ± 0.1 Hz) and gamma (36.6 ± 0.4 Hz) oscillations in rodent primary motor cortex (M1). Each oscillation showed greatest power in layer V. Using a variety of time series analyses we detected significant cross-frequency coupling in 74% of slice preparations. Differences were observed in the pharmacological profile of each oscillation. Thus, gamma oscillations were reduced by the GABAA receptor antagonists, gabazine (250 nM and 2 µM), and picrotoxin (50 µM) and augmented by AMPA receptor antagonism with SYM2206 (20 µM). In contrast, theta oscillatory power was increased by gabazine, picrotoxin and SYM2206. GABAB receptor blockade with CGP55845 (5 µM) increased both theta and gamma power, and similar effects were seen with diazepam, zolpidem, MK801 and a series of metabotropic glutamate receptor antagonists. Oscillatory activity at both frequencies was reduced by the gap junction blocker carbenoxolone (200 µM) and by atropine (5 µM). These data show theta and gamma oscillations in layer V of rat M1 in vitro are cross-frequency coupled, and are mechanistically distinct. The development of an in vitro model of phase-amplitude coupled oscillations will facilitate further mechanistic investigation of the generation and modulation of coupled activity in mammalian cortex.

Nucleus accumbens dopamine receptors mediate hypothalamus-induced antinociception in the rat formalin test.

BACKGROUND: Lateral hypothalamus (LH) involves in modulation of tonic pain. Regarding the direct and indirect neural connections between the LH and nucleus accumbens (NAc), we aimed to examine the pain modulatory role of NAc dopamine receptors in modulation of LH-induced analgesia in the formalin test. METHODS: Vehicle-control groups received saline or DMSO into the NAc and saline into the LH. Carbachol-control groups received carbachol (250 nmol/L) into the LH, 5 min after saline or DMSO injection into the NAc. In treatment groups, intra-NAc administration of SCH-23390 or sulpiride (D1-and D2-like dopamine receptor antagonists, respectively) was performed 5 min before carbachol injection. Formalin test was done in all rats 5 min after the second injection. RESULTS: The blockade of NAc dopamine receptors reduced carbachol-induced antinociception during both phases of formalin test and reduction in LH-induced analgesia during the late phase was more than that during the early phase. Furthermore, contribution of D2-like dopamine receptors to mediation of anti-hyperalgesic effect of carbachol was greater than that of D1-like dopamine receptors during the late phase. CONCLUSIONS: The findings suggest that LH-VTA-NAc circuit is contributed to the modulation of formalin-induced pain. These findings demonstrate that transmission at D1- and D2-like dopamine receptors mediates the LH-induced analgesia. SIGNIFICANCE: Blockade of accumbal dopamine receptors attenuated analgesia induced by carbachol injection into the lateral hypothalamus during both phases of formalin test. Effect of blockade of D1- and D2-like dopamine receptors on reduction in antinociception was more during the late phase. Contribution of D2-like dopamine receptors to mediation of antinociception during the late phase was greater than the early phase.