Organ Bath Setup Research Articles

Potassium Ion Channel Modulation as a Potential Remedy for Cadmium-induced Hypertension

Objective: Hypertension remains a chief non-communicable lifestyle disease that is negatively associated with dietary cadmium (Cd). The exact mechanism of Cd toxicity and the mitigating role of potassium supplementation are not fully known, hence, this study aimed to investigate their effects on the aorta. Hypothesis: We hypothesized that potassium supplementation can modulate Cd-induced vascular dysfunction. Method: Six cohorts of control, Cd-exposed, and potassium supplemented male Sprague-Dawley rats were selected. Hypertension was induced using cadmium chloride (2.5 or 5 mg/kg b.w.). Blood pressure was measured twice weekly. Reactivity to pharmacological agents with and without potassium ion channel modulation was assessed in thoracic aortic rings after eight weeks, using an organ bath set-up. Data: Statistical analysis was done using one-way analysis of variance and the results were reported as mean ± standard error of the mean. The Games-Howell or Bonferroni post hoc test was used for multiple comparisons. Summary of Results: Cadmium significantly (p<0.05) elevated systolic, diastolic, mean arterial, and pulse pressures, reduced (p<0.001) phenylephrine-induced contraction mostly via BKCa channels, and augmented (p<0.05) sodium nitroprusside-induced relaxation via K-ATP, KV, and KIR channels, without affecting acetylcholine-induced relaxation. Potassium supplementation ameliorated these effects via KIR and BKCa channels. Conclusions: The results indicate that modulation of potassium ion channels in the vasculature might provide therapeutic management for environmentally-induced hypertension and should be further explored. Funding source: The Mona Campus Committee for Research and Publications and Graduate Awards, School for Graduate Studies and Research, University of the West Indies. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of Chinese ginger Alpinia officinarum-based phytopharmaceutical compound on gastric motility

BackgroundAlpinia officinarum, better known as Chinese ginger, is used for the treatment of gastrointestinal disorders in traditional Chinese and Ayurveda medicine. However, scientific evidence of an effect on gastrointestinal motility is missing. PurposeThis study aimed to estimate the effect of a phytopharmaceutical compound (PPC) based on Alpinia officinarum on gastric motility in vitro in order to assess its potential use in the treatment of functional dyspepsia. Study DesignWe investigated to which extent the compound affects the activity of gastric smooth muscle and we identified involved mediating cellular mechanisms. MethodsBasal and stimulated muscle activity were recorded from muscle-myenteric plexus preparations from three different regions of the guinea pig stomach before and after incubation with different concentrations of the PPC in an organ bath setup. Effect-mediating mechanisms were investigated by pharmacology. ResultsPPC exhibited a smooth muscle relaxation in a concentration-dependent manner. This was region- and muscle type specific as it was greater in fundus and corpus than in antrum and more pronounced in the circular than in the longitudinal muscle layer. The effect was primarily myogenic and at least partly mediated by TRPA1. ConclusionThe investigated PPC reduces basal tone particularly of fundus smooth muscle in a non-neuronal manner by inhibiting calcium influx via TRPA1, without affecting antral motility. Based on these data, Alpinia officinarum could be considered for phytopharmaceutical treatment of functional dyspepsia.

Vasorelaxant effect of a phenylethylamine analogue based on schwarzinicine A an alkaloid isolated from the leaves of Ficus schwarzii

N-Phenethyl-1-phenyl-pentan-3-amine (1) is a new compound synthesised as a simplified analogue of schwarzinicine A (2), a natural compound extracted from Ficus schwarzii. Compound 1 differs from compound 2 due to its structural simplification, featuring two phenyl rings without methoxy substitution, as opposed to compound 2, which possesses three 3,4-dimethoxy aromatic rings. Our previous research findings highlighted the calcium-inhibitory effects of compound 2, but the mechanism of action for compound 1 remains unexplored, serving as the primary focus of this study. Building upon our earlier research, this study aimed to elucidate compound 1's calcium-modulating potential by using rat-isolated aortae in an organ bath set-up and HEK cells expressing hTRPC channels with the fluorometric assay to measure calcium influx. Compound 1 elicited a vasorelaxation response (Emax 111.4%) similar to its parent compound 2 (Emax 123.1%), and inhibited hTRPC3-, hTRPC4-, hTRPC5-, and hTRPC6-mediated calcium influx into HEK cells with IC50 values of 6, 2, 2, 5 µM, respectively. Compound 1 has a similar pharmacological profile as its parent compound 2, whereby it exerts a vasorelaxant effect by attenuating calcium influx and inhibits multiple TRPC channels.

The Detailed Pharmacodynamics of the Gut Relaxant Effect and GC-MS Analysis of the Grewia tenax Fruit Extract: In Vivo and Ex Vivo Approach.

The study was performed to assess and rationalize the traditional utilization of the fruit part of Grewia tenax (G. tenax). The phytoconstituents present in the methanolic extract were analyzed using Gas-Chromatography-Mass Spectroscopy (GC-MS), while the anti-diarrheal activity was investigated in the Swiss albino mice against castor oil-provoked diarrhea in vivo. The antispasmodic effect and the possible pharmacodynamics of the observed antispasmodic effect were determined in an isolated rat ileum using the organ bath setup as an ex vivo model. GC-MS findings indicate that G. tenax is rich in alcohol (6,6-dideutero-nonen-1-ol-3) as the main constituent (20.98%), while 3-Deoxy-d-mannoic lactone (15.36%) was detected as the second major constituents whereas methyl furfural, pyranone, carboxylic acid, vitamin E, fatty acid ester, hydrocarbon, steroids, sesquiterpenes, phytosterols, and ketones were verified as added constituents in the methanolic extract. In mice, the orally administered G. tenax inhibited the diarrheal episodes significantly (p < 0.05) at 200 mg/kg (40% protection), and this protection was escalated to 80% with the next higher dose of 400 mg/kg. Loperamide (10 mg/kg), a positive control drug, imparted 100% protection, whereas no protection was shown by saline. In isolated rat ileum, G. tenax completely inhibited the carbamylcholine (CCh; 1 µM) and KCl (high K+; 80 mM)-evoked spasms in a concentrations-mediated manner (0.03 to 3 mg/mL) by expressing equal potencies (p > 0.05) against both types of evoked spasms, similar to papaverine, having dual inhibitory actions at phosphodiesterase enzyme (PDE) and Ca2+ channels (CCB). Similar to papaverine, the inhibitory effect of G. tenax on PDE was further confirmed indirectly when G. tenax (0.1 and 0.3 mg/mL) preincubated ileal tissues shifted the isoprenaline-relaxation curve towards the left. Whereas, pre-incubating the tissue with 0.3 and 1 mg/mL of G. tenax established the CCB-like effect by non-specific inhibition of CaCl2−mediated concentration-response curves towards the right with suppression of the maximum peaks, similar to verapamil, a standard CCB. Thus, the present investigation revealed the phytochemical constituents and explored the detailed pharmacodynamic basis for the curative use of G. tenax in diarrhea and hyperactive gut motility disorders.

Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses.

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of vasoconstriction and vasorelaxation. After isolating the IPA and VSMCs, the characteristics of vascular responses in physiological and pathological conditions can be assessed in the absence of extrinsic factors such as nerve signals, hormones, cytokines, etc. Thus, the IPA and VSMCs serve as excellent models for studying vascular physiology/pathophysiology, along with various experimental investigations, such as modulation by pharmacological agents, patch-clamp electrophysiological analysis, calcium imaging, etc. Here, we have used a technique for isolating the IPA to investigate vascular responses in an organ bath setup. IPA segments were mounted on the organ bath chamber via intraluminal wires and stimulated by various pharmacological agents. The changes in IPA vascular tone (i.e., vasoconstriction and vasorelaxation), were recorded using an isometric force transducer and physiological data analysis software program. We implemented several experimental protocols, which can be adapted to investigate the mechanisms of vasorelaxation/vasoconstriction for studying the pharmacological activities of phytochemical or synthetic drugs. The protocols can also be used to evaluate drugs' roles in modulating various diseases, including pulmonary arterial hypertension. The IPA model allows us to investigate the concentration-response curve, which is crucial in assessing drugs' pharmacodynamic parameters.

Novel Oxime Synthesized from a Natural Product of Senecio nutans SCh. Bip. (Asteraceae) Enhances Vascular Relaxation in Rats by an Endothelium-Independent Mechanism.

Senecio nutans Sch. Bip. and its constituents are reported to have antihypertensive effects. We isolated metabolite–1, a natural compound from S. nutans (4-hydroxy-3-(isopenten-2-yl)-acetophenone), and synthesized novel oxime – 1 (4-hydroxy-3-(isopenten-2-yl)-acetophenoxime) to evaluate their effect on vascular reactivity. Compounds were purified (metabolite–1) or synthetized (oxime–1) and characterized using IR and NMR spectroscopy and Heteronuclear Multiple Quantum Coherence (HMQC). Using pharmacological agents such as phenylephrine (PE) and KCl (enhancing contraction), acetylcholine (ACh), L-NAME (nitric oxide (NO) and endothelial function), Bay K8644-induced CaV1.2 channel (calcium channel modulator), and isolated aortic rings in an organ bath setup, the possible mechanisms of vascular action were determined. Pre-incubation of aortic rings with 10−5 M oxime–1 significantly (p < 0.001) decreased the contractile response to 30 mM KCl. EC50 to KCl significantly (p < 0.01) increased in the presence of oxime–1 (37.72 ± 2.10 mM) compared to that obtained under control conditions (22.37 ± 1.40 mM). Oxime–1 significantly reduced (p < 0.001) the contractile response to different concentrations of PE (10−7 to 10−5 M) by a mechanism that decreases Cav1.2-mediated Ca2+ influx from the extracellular space and reduces Ca2+ release from intracellular stores. At a submaximal concentration (10−5 M), oxime–1 caused a significant relaxation in rat aorta even without vascular endothelium or after pre-incubate the tissue with L-NAME. Oxime–1 decreases the contractile response to PE by blunting the release of Ca2+ from intracellular stores and blocking of Ca2+ influx by channels. Metabolite–1 reduces the contractile response to KCl, apparently by reducing the plasma membrane depolarization and Ca2+ influx from the extracellular space. These acetophenone derivates from S. nutans (metabolite–1 and oxime–1) cause vasorelaxation through pathways involving an increase of the endothelial NO generation or a higher bioavailability, further highlighting that structural modification of naturally occurring metabolites can enhance their intended pharmacological functions.

Endothelial Contribution to Warfarin-Induced Arterial Media Calcification in Mice.

Arterial media calcification (AMC) is predominantly regulated by vascular smooth muscle cells (VSMCs), which transdifferentiate into pro-calcifying cells. In contrast, there is little evidence for endothelial cells playing a role in the disease. The current study investigates cellular functioning and molecular pathways underlying AMC, respectively by, an ex vivo isometric organ bath set-up to explore the interaction between VSMCs and ECs and quantitative proteomics followed by functional pathway interpretation. AMC development, which was induced in mice by dietary warfarin administration, was proved by positive Von Kossa staining and a significantly increased calcium content in the aorta compared to that of control mice. The ex vivo organ bath set-up showed calcified aortic segments to be significantly more sensitive to phenylephrine induced contraction, compared to control segments. This, together with the fact that calcified segments as compared to control segments, showed a significantly smaller contraction in the absence of extracellular calcium, argues for a reduced basal NO production in the calcified segments. Moreover, proteomic data revealed a reduced eNOS activation to be part of the vascular calcification process. In summary, this study identifies a poor endothelial function, next to classic pro-calcifying stimuli, as a possible initiator of arterial calcification.

Human Myometrial Contractility Assays.

Traditional contractility assays using an organ bath setup consist of several chambers (or baths) perfused with temperature-controlled, oxygenated physiological saline. Strips or rings of tissue (usually smooth or cardiac muscle) are mounted within the organ bath between a fixed hook and an isometric force transducer. The contraction force is recorded by the transducer and different parameters of contraction are analyzed. Different experimental protocols can be performed to investigate the effect of drugs and reagents on tissue contractility to investigate tissue physiology or determine the in vivo potential of novel pharmaceutical compounds. Here, the application of a modified organ bath to measure ex vivo contractions of small strips of human uterine smooth muscle (myometrium) is described, as well as protocols to study the effect of oxytocin and uterine relaxants on contraction.

Red Yeast Rice Mitigates High-Fat Diet Induced-Obesity Related Vascular Dysfunction in Wistar Albino Rats

The red yeast rice methanolic extract (RYR) was suggested to have promising therapeutic effects against vascular endothelial dysfunction. In the present study, the protective effects of RYR at 200 and 400 mg/kg/day were investigated in a specific animal model that exhibits a high-fat diet (HFD)-induced dyslipidemia and vascular endothelium dysfunction. Vascular endothelial reactivity experiments were evaluated using aorta obtained from adult rats in an ex-vivo organ bath setup. Three consecutive weeks of RYR treatment exhibited a significant reduction in body weight (BW), liver weight (LW), and retroperitoneal fat pad weight (RFPW)/BW ratios as compared to HFD only treated rats where a significant body weight gain was observed. RYR treatment also significantly decreased the average daily food intake, waist, lee index, and body mass index compared to rats treated with HFD only. RYR treatment significantly reduced total cholesterol, triglyceride, low-density lipoprotein, and very-low-density lipoprotein levels compared to the HFD group. HFD-induced endothelium dysfunction in the aorta of animals was reversed by RYR treatment, comparable to the HFD group. RYR treatment, specifically at 400 mg/kg/day dose, revealed a significant protective effect on vascular endothelium and an improvement in the lipid panel, hence justifying therapeutic involvement of RYR in dyslipidemia and endothelial dysfunctions.

Combination drug therapy against OAB normalizes micturition parameters and increases the release of nitric oxide during chemically induced cystitis.

Today, monotherapy is the most common pharmacological treatment option for patients suffering from overactive bladder (OAB). Recent reports have indicated potential benefits of combination therapy, using a muscarinic antagonist and a β3‐adrenoceptor agonist. This may be of particular interest for therapy‐resistant patients with OAB and concomitant cystitis. The objective of the current study was to assess how combination therapy affects bladder parameters in health and cystitis and if the efficacy of the drugs can be linked to altered release of nitric oxide (NO). Rats were pretreated with either a combination of the muscarinic antagonist tolterodine and β3‐selective adrenoceptor agonist mirabegron or saline for 10 days. Forty‐eight hours prior to assessing micturition parameters in a metabolic cage, the rats were intraperitoneally injected with cyclophosphamide, causing cystitis, or saline. Urine samples were collected and analyzed for NO content. Bladder contractile properties were assessed in an organ bath setup. Induction of cystitis led to bladder overactivity. Combination therapy normalized bladder parameters. Both induction of cystitis and drug treatment increased the release of NO. The innate contractile properties of the bladder were unaffected by combination therapy. This study demonstrates positive effects of combination drug therapy on symptoms of OAB, possibly indicating it to be a good option for treatment of OAB during concomitant cystitis. It remains to be determined if increased release of NO is crucial for successful pharmacological treatment of bladder overactivity during cystitis.

Morin attenuates high-fat diet induced-obesity related vascular endothelial dysfunction in Wistar albino rats

Vascular endothelial dysfunction is caused by dyslipidemia, hypertension, and deficiency of antioxidant systems. In this study, the protective effect of a flavonol, morin was investigated in high-fat diet (HFD)-induced dyslipidemia and vascular endothelium dysfunction. The dose-dependent attenuating effect of morin was tested at doses of 50 and 100 mg/kg/day in an in-vivo model of HFD-induced dyslipidemia using rats whereas vascular endothelial reactivity was assessed in isolated rat aorta using ex-vivo organ bath setup. Morin administration in HFD-induced dyslipidemic rats for three weeks, resulted in a significant decrease in the body weight, LW/BW ratio as compared to rats treated with HFD only where the increase in body weight was observed. Significant reduction in the waist, BMI and lee index was also observed after morin treatment in HFD-induced dyslipidemic rats. In the lipid profile studies, HFD group showed a significant increase in the total cholesterol, triglyceride, LDL, and VLDL levels while HDL levels were decreased significantly, whereas morin treatment reversed all these parameters which were comparable to standard diet (SD) group. In the ex-vivo isolated aorta studies, HFD-induced endothelium dysfunction was observed, whereas it was reversed in the aorta of animals treated with morin at doses of 50 and 100 mg/kg/day, comparable to SD group. Morin treatment produced dose-dependent improvement in lipid profile and vascular endothelium protection, thus rationalizing its medicinal use in dyslipidemia and cardiovascular-related endothelial disorders.

Effects of caveolae depletion and urothelial denudation on purinergic and cholinergic signaling in healthy and cyclophosphamide-induced cystitis in the rat bladder.

Cholesterol rich membrane invaginations, caveolae, have important roles in various cellular activities, one of them being signal transduction. This signaling pathway seems to be affected during various bladder disorders and the current study aimed to elucidate the plausible involvement of caveolae mediated signal transduction during cyclophosphamide induced cystitis. Furthermore, the urothelial cholinergic part of ATP-evoked contractions and its possible link to caveolae were investigated. Cholinergic, as well as purinergic, contractile responses in rat urinary bladders were examined using a classic organ bath set-up with full-thickness strip preparations or a whole bladder model that enabled luminal administration of substances. Furthermore, sub groups with and without urothelium were examined. The expression of caveolin-1 was also tested using western blot and immunofluorescence. Caveolae cholesterol depletion by methyl-β-cyclodextrin entailed a significant decrease of ATP-evoked bladder contractility. Interestingly, after muscarinic blockade the ATP induced contractions were significantly reduced in the same manner. Furthermore, this atropine-sensitive part of ATP-evoked responses was absent in denuded as well as inflamed bladders. A tendency towards a reduced expression of caveolin-1 was observed in rats with experimental cystitis. The cholinergic part of ATP-induced contractile responses seemed to be affected by urothelium denudation as well as caveolae depletion. Removing one of these structures nullifies the effect of the other, suggesting an important interaction between the urothelium and the caveolar structures. These effects are absent in inflamed animals and might be one pathophysiological aspect behind BPS/IC.

Airway Relaxation Effects of Water-Soluble Sclerotial Extract From Lignosus rhinocerotis.

Lignosus rhinocerotis has a long history of use by the indigenous community within East Asia to treat a range of health conditions including asthma and chronic cough. To date, there is limited scientific evidence to support its therapeutic effects in relieving these airways conditions. In this study, we examined the effects of the different molecular weight fractions [high-molecular-weight (HMW), medium-molecular-weight (MMW), and low-molecular-weight (LMW)] obtained from the cold water sclerotial extract (CWE) of L. rhinocerotis on airways patency using airway segments isolated from Sprague Dawley rat in an organ bath set-up. It is demonstrated that the HMW and MMW fractions exhibited higher efficacy in relaxing the pre-contracted airways when compared to the CWE and LMW fraction. In addition, the HMW fraction markedly supressed carbachol-, 5-hydroxytrptamine-, and calcium-induced airway contractions. CWE demonstrated a lower efficacy than the HMW fraction but it also significantly attenuated carbachol- and calcium-induced airway contractions. Results showed that the bronchorelaxation effect of CWE and fractions is mediated via blockade of extracellular Ca2+ influx. The composition analysis revealed the following parts of carbohydrate and proteins, respectively: HMW fraction: 71 and 4%; MMW fraction: 35 and 1%; and LMW fraction: 22 and 0.3%. Our results strongly suggest that the polysaccharide–protein complex or proteins found in the HMW and MMW fractions is likely to contribute to the bronchorelaxation effect of CWE.

Changes of Bladder M1,3 Muscarinic Receptor Expression in Rats Fed with Short-Term/Long-Term High-Fat Diets.

To investigate the effect of high-fat diet (HFD) on bladder M1,3 muscarinic receptor expression and contractile function in the rat. Eight-week-old male rats were divided into two groups including one with HFD for 8 weeks (short-term) and the other for 24 weeks (long-term). Each group was compared to age-matched rats fed with normal chow as controls. The body weight, food intake amount and blood biochemistry were monitored. Bladder muscle contractile responses to acetylcholine (0.1-10 μM), bethanechol (10 μM) and KCl (50 mM) were studied in an organ bath set-up. Bladder M1 and M3 muscarinic receptor protein expressions were measured by Western blotting analysis. Increase in body weight as well as blood triglyceride, cholesterol and sugar levels compared to controls were noted in both 8- and 24-week HFD rats. Eating appetite change with increased food and water intakes was noted in the HFD rats. Significantly decreased bladder contractile responses to acetylcholine and bethanechol were shown in both HFD groups. On the other hand, decreased bladder contractile response to KCl was demonstrated in the 24-week group but not the 8-week group. The expressions of bladder M1 and M3 muscarinic receptor proteins were significantly and progressively decreased by HFD feeding from 8 to 24 weeks. High-fat diet induces obesity and polyphagia in rats. Short-term and long-term HFD feeding decrease rat bladder M1 and M3 receptor expressions as well as contractile responses to the agonistic stimulation. In addition, bladder muscle dysfunction develops after long-term HFD feeding.

A novel set-up for the ex vivo analysis of mechanical properties of mouse aortic segments stretched at physiological pressure and frequency.

Cyclic stretch is known to alter intracellular pathways involved in vessel tone regulation. We developed a novel set-up that allows straightforward characterization of the biomechanical properties of the mouse aorta while stretched at a physiological heart rate (600beatsmin-1 ). Active vessel tone was shown to have surprisingly large effects on isobaric stiffness. The effect of structural vessel wall alterations was confirmed using a genetic mouse model. This set-up will contribute to a better understanding of how active vessel wall components and mechanical stimuli such as stretch frequency and amplitude regulate aortic mechanics. Cyclic stretch is a major contributor to vascular function. However, isolated mouse aortas are frequently studied at low stretch frequency or even in isometric conditions. Pacing experiments in rodents and humans show that arterial compliance is stretch frequency dependent. The Rodent Oscillatory Tension Set-up to study Arterial Compliance is an in-house developed organ bath set-up that clamps aortic segments to imposed preloads at physiological rates up to 600beatsmin-1 . The technique enables us to derive pressure-diameter loops and assess biomechanical properties of the segment. To validate the applicability of this set-up we aimed to confirm the effects of distension pressure and vascular smooth muscle tone on arterial stiffness. At physiological stretch frequency (10Hz), the Peterson modulus (EP ; 293 (10) mmHg) for wild-type mouse aorta increased 22% upon a rise in pressure from 80-120mmHg to 100-140mmHg, while, at normal pressure, EP increased 80% upon maximal contraction of the vascular smooth muscle cells. We further validated the method using a mouse model with a mutation in the fibrillin-1 gene and an endothelial nitric oxide synthase knock-out model. Both models are known to have increased arterial stiffness, and this was confirmed using the set-up. To our knowledge, this is the first set-up that facilitates the study of biomechanical properties of mouse aortic segments at physiological stretch frequency and pressure. We believe that this set-up can contribute to a better understanding of how cyclic stretch frequency, amplitude and active vessel wall components influence arterial stiffening.

A new dynamic organ bath setup to assess isobaric stiffness parameters of periodically stretched isolated mouse aortic segments

A new dynamic organ bath setup to assess isobaric stiffness parameters of periodically stretched isolated mouse aortic segments

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice.

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Joint action of herbal components influence the effects of STW 5 on inflammatory processes and disturbed motility

STW 5 (Iberogast®) contains nine herbal extracts affecting inflammatory processes and disturbed motility in the gut. The aim of the study was to investigate the effect of individual ethanolic extracts (in concentration used in STW 5) and selected combinations on targets related to inflammation. Cytotoxicity was examined in CaCo-2 cells using the lactate dehydrogenase (LDH) assay. THP-1 cells were used to determine TNFα release using an ELISA. ACh-induced contractions of rat ileum preparations were measured in an organ bath setup. STW 5 (500 µg/ml), STW 5-II (5.1 µg/ml, containing only 6 of the 9 extracts) and lemon balm (59.9 µg/ml) reduced LPS (10 ng/ml)-induced LDH leakage from CaCo-2 cells by 74 – 75%. Iberis amara (29.1 µg/ml), peppermint (38.9 µg/ml), chamomile (116.3 µg/ml), angelica (84.7 µg/ml) and milk thistle (15.2 µg/ml) inhibited LDH leakage by 25 – 37%. Liquorice (84.9 µg/ml), caraway (30.9 µg/ml) and celandine (61.9 µg/ml) had no effect. The combinations of Iberis amara and peppermint as well as peppermint and milk thistle revealed synergistic or additive effects, whereas the combination of chamomile and angelica evoked additive or antagonistic effects depending on their compositions. STW 5, STW 5-II, Iberis amara, peppermint, liquorice, caraway, milk thistle and lemon balm in concentration used in the LDH assay inhibited LPS (100 ng/ml)-induced TNFα release to 51 – 67%. The combinations of Iberis amara and peppermint as well as chamomile and liquorice had additive or antagonistic effects depending on their compositions. STW 5 and STW 5-II reduced the acetylcholine (ACh)-induced contractions in rat ileum preparations to 81 – 83%. The individual extracts inhibited the contractions to 83 – 91% except lemon balm. The combination of Iberis amara and peppermint as well as liquorice and caraway had additive or antagonistic effects depending on the ratios. Our results highlighted the interactions between the extracts of STW 5 and confirm the concept of multi-target actions. Keywords: Iberogast, Cytotoxixity, TNF-alpha, CaCo-2 cells, THP-1 cells, synergy, drug interaction

Effect of Muscarinic and Nicotinic Receptor Antagonism on Rat Gastric Motor Activity

Background/Aims: Our aim was to investigate whether muscarinic and nicotinic receptors mediate nitric oxide release during motor events in the rat stomach. Methods: Isolated rat stomach volume changes were monitored in an organ bath setup with an intragastric balloon coupled to a barostat and studied in basal conditions and during electrical vagal stimulation (EVS). In conscious rats, the intragastric pressure (IGP) was measured during test meal infusion. Results: In the presence of N^G-nitro-L-arginine methyl ester (L-NAME; 0.1 mmol/l), EVS induced significant gastric contractions (mean ± SEM = 0.27 ± 0.04 ml; n = 6) that could be blocked by atropine (3 µmol/l) and hexamethonium (0.1 mmol/l). In the presence of atropine and/or hexamethonium, EVS-induced relaxations could not be blocked by L-NAME, while exogenous nitric oxide could still relax the stomach. In conscious rats, atropine (1 mg kg^–1) initially decreased IGP, while during further distension it increased IGP. In the presence of L-NAME (30 mg kg^–1) atropine consistently decreased IGP. L-NAME alone significantly increased IGP during the test meal infusion, but this effect was reduced in the presence of atropine. Conclusion: These findings indicate a role for nicotinic and muscarinic receptors in the vagal-stimulation-induced activation of nitrergic nerves in the rat stomach.

Selective desensitization of the 5‐HT4 receptor‐mediated response in pig atrium but not in stomach

The time dependency of the effect of 5-HT(4) receptor agonists depends on many specific regulatory mechanisms, which vary between tissues. This has important implications with regard to the effects of endogenous 5-HT, as well as to the clinical use of 5-HT(4) receptor agonists, and might contribute to tissue selectivity of agonists. The progression and desensitization of 5-HT(4) receptor-mediated responses were evaluated in an organ bath set-up using two, clinically relevant, porcine in vitro models: gastric cholinergic neurotransmission and atrial contractility. Exposure of gastric tissue to 5-HT or to the selective 5-HT(4) receptor agonists prucalopride and M0003 results in a sustained non-transient effect during exposure; after washout, the response to a subsequent challenge with 5-HT shows no clear desensitization. Incubation of left atrial tissue with 5-HT resulted in a transient response, leading after washout to a marked desensitization of the subsequent response to 5-HT. The selective 5-HT(4) receptor agonists prucalopride and M0003 induce only very weak atrial responses whereas they are very effective in desensitizing the atrial response to 5-HT. The observations also suggest that the properties of prucalopride and M0003 to bind to and/or activate the 5-HT(4) receptor differ from those of 5-HT. This difference might have contributed to the observed desensitization. The high potency of prucalopride and M0003 in desensitizing the response to 5-HT together with their low efficacy in the atrium emphasizes the cardiac safety of this class of 5-HT(4) receptor agonists.