Alleviating depressive-like behavior in DSS-induced colitis mice: Exploring naringin and poncirin from Poncirus trifoliata extracts.

Patients with inflammatory bowel diseases (IBDs), including ulcerative colitis (UC) and Crohn's disease (CD), often have concomitant mental disorders such as depression and anxiety. Therefore, a bidirectional approach involving the gut and brain axes is necessary for the prevention and treatment thereof. In this study, we explored the potential of Poncirus trifoliata extract (PT), traditionally known for its neuroprotective effects against gastrointestinal diseases, as a natural treatment agent for IBD in a dextran sulfate sodium (DSS)-induced colitis model. Oral administration of PT ameliorated weight loss and inflammatory responses in mice with DSS-induced colitis. Furthermore, PT treatment effectively restored the colon length and ameliorated enterocyte death by inhibiting DSS-induced reactive oxygen species (ROS)-mediated necroptosis. The main bioactive components of PT, poncirin and naringin, confirmed using ultra-performance liquid chromatography-quadrupole time-of-flight (UPLC-qTOF), can be utilized to regulate necroptosis. The antidepressant-like effects of PT were confirmed using open field test (OFT) and tail suspension test (TST). PT treatment also restored vascular endothelial cell integrity in the hippocampus. In the Cornu Ammonis 1 (CA1) and dentate gyrus (DG) regions of the hippocampus, PT controlled the neuroinflammatory responses of proliferated microglia. In conclusion, PT, which contains high levels of poncirin and naringin, has potential as a bidirectional therapeutic agent that can simultaneously improve IBD-associated intestinal and mental disorders.

Poncirus trifoliata (L.) Raf. Seed Extract Induces Cell Cycle Arrest and Apoptosis in the Androgen Receptor Positive LNCaP Prostate Cancer Cells.

Prostate cancer (PCa) is the second most common male cancer. Its incidence derives from the interaction between modifiable and non-modifiable factors. The progression of prostate cancer into a more aggressive phenotype is associated with chronic inflammation and increased ROS production. For their biological properties, some phytochemicals from fruits and vegetable emerge as a promise strategy for cancer progression delay. These bioactive compounds are found in the highest amounts in peels and seeds. Poncirus trifoliata (L.) Raf. (PT) has been widely used in traditional medicine and retains anti-inflammatory, anti-bacterial, and anticancer effects. The seeds of P. trifoliata were exhaustively extracted by maceration with methanol as the solvent. The cell proliferation rate was performed by MTT and flow cytometry, while the apoptosis signals were analyzed by Western blotting and TUNEL assay. P. trifoliata seed extract reduced LNCaP and PC3 cell viability and induced cell cycle arrest at the G0/G1phase and apoptosis. In addition, a reduction in the AKT/mTOR pathway has been observed together with the up-regulation of stress-activated MAPK (p38 and c-Jun N-terminal kinase). Based on the study, the anti-growth effects of PT seed extract on prostate tumor cells give indications on the potential of the phytochemical drug for the treatment of this type of cancer. However, future in-depth studies are necessary to identify which components are mainly responsible for the anti-neoplastic response.

Larvicidal constituents from Poncirus trifoliata root extracts.

In the search for effective and environmentally friendly mosquito control agents, we have examined natural sources, such as microbes and plants, and the synthetic analogs of natural products. These plants and microbes have evolved in their ecological niches to produce defensive compounds against other competing organisms in their surroundings such as microbes, plants, and insects as a means to enhance their survival. Thus, some of these plants and microbes have bioactive compounds with insecticidal, fungicidal, and phytotoxic activities. In our previous research, we successfully isolated bioactive constituents from natural sources. We have carried out synthetic modifications and total synthesis of marginally active isolated compounds to achieve significantly higher active compounds. We have focused on plants in the Rutaceae family as the members of this family are known to possess bioactive compounds with algicidal, antifungal, insecticidal, and fungicidal activities. In this article, we report the isolation and structure elucidation of mosquito larvicidal constituents from Poncirus trifoliata (Rutaceae) root extract.

Anticancer effects of Poncirus fructus on hepatocellular carcinoma through regulation of apoptosis, migration, and invasion.

Poncirus fructus (PF) is a phytochemical compound extracted from the dry, immature fruits of Poncirus trifoliate. PF is traditionally used to treat gastrointestinal disorders, allergies, and inflammatory disease. In East Asia, PF is also known for its anticancer properties. There are numerous reports on the anticancer and anti­inflammatory effects of PF in a wide range of cancers and gastrointestinal diseases, respectively. However, the role of PF in inducing apoptosis and suppressing the invasiveness of hepatocellular carcinoma (HCC) remains unclear. This study investigated the ability of PF to induce apoptosis and inhibit the invasiveness and migratory ability of HCC cell lines (Hep3B and Huh7). Wound healing, Transwell migration and invasion, and colony­formation assays, as well as flow cytometry, were used to analyze cell proliferation, migration, invasion, and apoptosis. Epithelial­mesenchymal transition (EMT)­related and apoptotic proteins were assessed by western blotting. The mitochondrial membrane potential of the Hep3B and Huh7 cells was observed with tetramethylrhodamine ethyl ester. The reactive oxygen species (ROS) level was determined by dihydroethidium (DHE) staining. PF treatment significantly decreased the proliferation of Hep3B and Huh7 cells in a dose­dependent manner, reduced the mitochondrial membrane potential, increased ROS levels, decreased the protein levels of Bcl­2, and increased the protein levels of Bax and cleaved caspase­3 and 9, suggesting that PF mediated HCC apoptosis via a mitochondrial pathway. Our findings showed that PF prevented HCC cell migration and invasion by inhibiting the EMT process and downregulating MMP­2 and MMP­9 activities. The results suggest the potential anticancer effects of PF by inhibiting proliferation, inducing apoptosis, and reducing the invasion and migration of HCC cells.

How the cells were injured and the secondary metabolites in the shikimate pathway were changed by boron deficiency in trifoliate orange root.

Boron (B) deficiency is frequently observed in citrus orchards as a major cause for loss of productivity and quality. The structural and morphological responses of roots to B deficiency have been reported in some plants. The study was conducted to get novel information about the B-deficient-induced cellular injuries and target secondary metabolites in the shikimate pathway. Fluorescent vital staining, paraffin section, transmission electron microscopy (TEM) and target metabolomics were to investigate the responses of the cell viability and structure, and target aromatic metabolites in the shikimate pathway in B-deficient trifoliate orange roots. Boron deprivation-induced ROS accumulation accelerated the membrane peroxidation, resulting in weakened cell vitality and cell rupture in roots. In addition, B deficiency increased phenylalanine (Phe), tyrosine (Try) in roots, thereby promoting the biosynthesis of salicylic acid, caffeic acid and ferulic acid. B-starvation up-regulated salicylic acid and lignin while reduced 3-indoleacetic acid (IAA) content. These adverse effects might be involved in the structural and morphological changes in B-deficient roots. What is more, the results provide a new insight into the mechanism of B deficiency-induced structural damage and elongation inhibition on roots.

Poncirus Trifoliata (L.) Raf. Extract Inhibits the Development of Atopic Dermatitis-like Lesions in Human Keratinocytes and NC/Nga mice.

This study investigated the anti-allergic effect of Poncirus trifoliata (L.) Raf. (PT) on human keratinocytic HaCaT cells in vitro and on 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis-like lesions in vivo. The release of TARC, MCP-1, IL-6 and IL-8 is increased by IFN-γ and TNF-α in HaCaT cells, and PT extract suppressed the increased production of TARC, MCP-1, IL-6, and IL-8. PT extract recovered the expression of filaggrin decreased by IFN-γ and TNF-α. in vivo experiment, PT administration decreased the skin severity score, thickening of the epidermis, movement of inflammatory cells into the dermis, and serum IgE level as compared to DNCB treatment. Moreover, the decrease of filaggrin and loricrin induced by DNCB treatment was recovered by PT administration. The levels of IL-4, IL-5, IL-13 and eotaxin in mouse splenocytes increased after treatment with concanavalin A, and the secretions of IL-4, IL-5, IL-13 and eotaxin were lower in the PT-treated group than in the DNCB group. These findings may indicate that PT is useful in drug development for the treatment of AD.

The Effects of Poncirus fructus on Insulin Resistance and the Macrophage-Mediated Inflammatory Response in High Fat Diet-Induced Obese Mice.

Obesity is a chronic low-grade inflammatory condition in which hypertrophied adipocytes and adipose tissue immune cells, mainly macrophages, contribute to increased circulating levels of proinflammatory cytokines. Obesity-associated chronic low-grade systemic inflammation is considered a focal point and a therapeutic target in insulin resistance and metabolic diseases. We evaluate the effect of Poncirus fructus (PF) on insulin resistance and its mechanism based on inflammatory responses in high-fat diet (HFD)-induced obese mice. Mice were fed an HFD to induce obesity and then administered PF. Body weight, epididymal fat and liver weight, glucose, lipid, insulin, and histologic characteristics were evaluated to determine the effect of PF on insulin resistance by analyzing the proportion of macrophages in epididymal fat and liver and measured inflammatory gene expression. PF administration significantly decreased the fasting and postprandial glucose, fasting insulin, HOMA-IR, total-cholesterol, triglycerides, and low-density lipoprotein cholesterol levels. The epididymal fat tissue and liver showed a significant decrease of fat accumulation in histological analysis. PF significantly reduced the number of adipose tissue macrophages (ATMs), F4/80+ Kupffer cells, and CD68+ Kupffer cells, increased the proportion of M2 phenotype macrophages, and decreased the gene expression of inflammatory cytokines. These results suggest that PF could be used to improve insulin resistance through modulation of macrophage-mediated inflammation and enhance glucose and lipid metabolism.

Antinociceptive properties of 25-methoxy hispidol A, a triterpinoid isolated from Poncirus trifoliata (Rutaceae) through inhibition of NF-κB signalling in mice.

The 25-methoxy hispidol A (25-MHA) is a triterpenoid, isolated from the immature fruit of Poncirus trifoliata (Rutaceae). The pretreatment with 25-MHA markedly (p < 0.001) attenuated the formalin-induced biphasic responses as well as acetic acid-induced writhing responses. The intraperitoneal administration of 25-MHA significantly attenuated the mechanical hyperalgesia (p < 0.001) and allodynia (p < 0.05). Similarly, 25-MHA also significantly attenuated (p < 0.001) complete Freund's adjuvant (CFA)-induced paw edema in mice. The 25-MHA treatment significantly attenuated the production of nuclear kappa B (NF-κB) (p65 nuclear subunit). The cytokines are the important mediators of inflammation and pain; however, treatment with 25-MHA exhibited significant inhibition (p < 0.001) on the mRNA expression levels of various inflammatory mediators. The 25-MHA administration also significantly enhanced antioxidant enzymes (p < 0.001) and inhibited the oxidative stress markers. The current study indicates that 25-MHA significantly (p < 0.001) inhibited the nitric oxide (NO) in mice plasma. Similarly, the haematoxylin and eosin (H&E) staining shows that 25-MHA administration significantly inhibited the inflammatory process in the mice paw tissue compared with the CFA-treated group. The 25-MHA treatment did not exhibited any toxicity on the liver, kidney, muscles strength, and motor co-ordination in mice. The 25-MHA was coadministered with the various drugs such as tramadol, piroxicam, and gabapentin to observe the synergistic effect.

Antiviral activity of Poncirus trifoliata seed extract against oseltamivir-resistant influenza virus.

The emergence of oseltamivir-resistant variants of influenza virus has highlighted the necessity for the development of more effective novel antiviral drugs. To date, numerous researchers have focused on developing antiviral drugs using natural resources, such as traditional herbal medicines. Poncirus trifoliata is widely used in oriental medicine as a remedy for gastritis, dysentery, inflammation and digestive ulcers. In this study, we investigated the potential antiviral effect of the Poncirus trifoliata orange seed extract against influenza virus. An ethanol extract of Poncirus trifoliata seeds (PTex) inhibited the activity of influenza viruses, in particular, oseltamivir- resistant strains, in Madin-Darby canine kidney cells. In contrast to oseltamivir, PTex exerted a significant inhibitory effect on the cellular penetration pathway of the virus rather than HA receptor binding. The potent antiviral effect and novel working mechanism of PTex support its further development as an effective natural antiviral drug with a wide spectrum of activity against influenza and oseltamivir-resistant viruses.

Phytotherapeutic effects of the fruits of Poncirus trifoliata (L.) Raf. on cancer, inflammation, and digestive dysfunction.

Poncirus trifoliata (L.) Raf. belongs to the family Rutaceae in the genus Poncirus. Its fruits are widely used to alleviate symptoms of various disorders. The mature fruit (MF) possesses anticancer and antiinflammatory activities. Extracts of the dried, immature fruit, Poncirus fructus (PF) are widely used as a traditional medicine for ameliorating symptoms of digestive dysfunction in East Asia. Molecular and cellular mechanisms underlying the effects of MF and PF extracts on cancer, inflammation, and gastrointestinal disorders have been extensively studied in the past decade. This review summarizes recent findings on the anticancer and antiinflammatory effects of MF and the prokinetic effects of PF. Although the therapeutic effects of MF and PF have been clearly elucidated, in-depth further clinical studies are still required to completely verify the clinical efficacy and safety of the fruits of P. trifoliata (L.) Raf.

Anti-Obesity and Hypoglycemic Effects of Poncirus trifoliata L. Extracts in High-Fat Diet C57BL/6 Mice.

The present study investigated the possible anti-obesity and hypoglycemic effects of Poncirus trifoliata L. extracts. Mature fruit were divided into flavedo (PF) and juice sacs (PJ), and extracts from them were tested on C57BL/6 mice fed a high-fat diet (HFD) for thirteen weeks. Both fruit extracts (40 mg/kg body weight, respectively) showed anti-obesity and hypoglycemic effects. Consumption of PF and PJ extracts reduced body weight by 9.21% and 20.27%, respectively. Liver and adipose weights, fasting glucose, serum triglyceride (TG), and low density lipoprotein cholesterol (LDL-c) levels decreased significantly, while serum high density lipoprotein cholesterol (HDL-c) and oral glucose tolerance levels increased significantly in response to two fruit extracts. These effects were due in part to the modulation of serum insulin, leptin, and adiponectin. Furthermore, transcript levels of fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1) were reduced while those of carnitine palmitoyltransferase 1α (CPT1α) and insulin receptor substrate 2 (IRS2) were increased in the liver of C57BL/6 mice, which might be an important mechanism affecting lipid and glucose metabolism. Taken together, P. trifoliata fruit can be potentially used to prevent or treat obesity and associated metabolic disorders.

Studies on antimicrobial activity of Poncirus trifoliata ethyl extract fraction against methicillin-resistant Staphylococcus aureus and to elucidate its antibacterial mechanism.

Traditional medicinal plants contain a wide variety of chemicals that have potent antibacterial activity. To find an alternative agent of overcoming the problems of methicillin-resistant Staphylococcus aureus (MRSA), the antibacterial mechanism of Ponciruss trifoliata against MRSA was investigated. Ethyl acetate (EtOAc)-soluble extract of P. trifoliata methanolic extract was evaluated for antibacterial activity using minimum inhibitory concentration (MIC). An EtOAc sub-fraction 08 (EA08) from silica-gel open column chromatography exhibited strong anti-MRSA activity. Apart from the study to isolate single compound from EA08, a synergistic antibacterial effect between the sub-fraction and ß-lactam antibiotics against MRSA was determined. In order to elucidate the antibacterial restoring mechanism of EA08 on MRSA, mRNA expression of mecA gene and production penicillin-binding protein 2a (PBP2a) encoded by mecA gene were monitored. EA 08 showed the strongest antibacterial activity with MIC value of 256 µg ml(-1). MIC of oxacillin against MRSA was dramatically reduced from 512 to 16 µg ml(-1) in combination with 256 µg ml(-1) of EA08. The fractional inhibitory concentration index of oxacillin was measured at 0.53 in combination with EA08 against MRSA, suggesting that EA08-oxacillin combinations exert synergetic effect against MRSA. The analysis of RT-PCR and Western blotting profiles revealed that EA08 inhibited mRNA expression of mecA gene and production PBP2a, which is a key determinant for ß-lactam antibiotic resistance, in a dose-dependent manner. These results indicated that EA08 eventually led to the reduction or inhibition of PBP2a production through translational inhibition in MRSA.

Characteristic Volatile Components of Trifoliate Orange Peel (Poncirus trifoliata).

The volatile components of the peel of trifoliate orange {Poncirus trifoliata (L.) Raf.}, family Rutaceae, were investigated using SAFE technique after solvent extraction. Limonene was the most abundant component in the peel aroma extract, followed by myrcene, trans-D-ocimcne, -indole, P-caryophyllenc, (ME,6E)-ra-famescne, germacrene D, and j-phcllandrcne. In this study, the single sulfur- and nitrogen-containing compound, 4-methyl-5-vinylthiazole, and two macrocyclic lactones, cyclododecanolide and (7Z,IOZ,13Z)-hexadecatrien-16-olide, were identified as citrus aroma components for thefirst time. As aresultof AEDA for the polar fraction of the aroma extract, indole, ethyl octanoate and those macrocyclic lactones with musky notes were found to be responsible f or the characteristic aroma profile of the peel of trifoliate orange. The enantiomeric distributions of the four odor-active components, linalool, P-citronellol, ethyl 2-methylbutanoate, and 2-methylbutanoic acid, were also determined by means of multidimensional chiral GC/MS.

New Glabretal Triterpenes from the Immature Fruits of Poncirus trifoliata and Their Selective Cytotoxicity.

Two new glabretal triterpenes, pancastatins A (1) and B (2), were isolated from the immature fruits of Poncirus trifoliata. Their chemical structures were elucidated by spectroscopic analyses including one- and two-dimensional NMR and high-resolution electrospray ionization mass spectrometry. Compounds 1 and 2 exhibited selective cytotoxicity against PANC-1 pancreatic cancer cells under low-glucose stress conditions.

Growth inhibition of various human cancer cell lines by imperatorin and limonin from poncirus trifoliata rafin. Seeds.

We examined the anticancer effects of limonin and imperatorin on various human cancer cells by MTT assay, and the results showed that imperatorin inhibited the cell growth of SNU 449 (liver cancer) and HCT-15 (colon cancer) cells in a dose-dependent manner, while limonin had less effect. Exposure of different concentrations of limonin and imperatorin caused morphological changes in cancer cells, but not in normal dermal fibroblast cells. Limonin and imperatorin induced apoptotic cell death concurrent to cell cycle arrest in SNU 449 and HCT-15 cells. Limonin and imperatorin up regulated proapoptotic protein Bax expression and down regulated anti-apoptotic protein Bcl-2 expression in a dose-dependent manner in HCT-15 and SNU 449 cells. In conclusion, our data demonstrate that limonin and imperatorin have anticancer potential which is associated with promoting cell apoptosis through expression of apoptosis-related proteins.

[Effects of arbuscular mycorrhizal fungi on root system morphology and sucrose and glucose contents of Poncirus trifoliata].

The effects of inoculation with Glomus mosseae, G. versiforme, and their mixture on plant growth, root system morphology, and sucrose and glucose contents of trifoliate orange (Poncirus trifoliata L.) were studied by pot culture. The results showed that all the inoculated treatments significantly increased the plant height, stem diameter, leaf number, and shoot and root biomass. In addition, the mycorrhizal treatments significantly increased the number of 1st, 2nd, and 3rd lateral roots. Inoculation with arbuscular mycorrhizal fungi significantly increased the root projected area, surface area, volume, and total root length (mainly 0-1 cm root length), but decreased the root average diameter. Meanwhile, G. versiforme showed the best effects. Mycorrhizal inoculation significantly increased the leaf sucrose and root glucose contents, but decreased the leaf glucose and root sucrose contents. Owing to the 'mycorrhizal carbon pool' in roots, inoculation with arbuscular mycorrhizal fungi resulted in high glucose content and low sucrose content of roots, which would facilitate the root growth and development, thereby the establishment of better root system morphology of host plants.

Volatile profiles of flavedo, pulp and seeds in Poncirus trifoliata fruits.

BACKGROUND: Poncirus trifoliata, also known as trifoliate orange, is a tree native to China and Korea and widely used all over the world as a rootstock breeding material. In this study the differences among the volatile profiles of flavedo, pulp and seeds from two cultivars (var. trifoliata and var. monstrosa) grown in Italy (Marche, Abruzzo and Sicily) were determined. Headspace solid phase microextraction and hydrodistillation techniques were used in combination with GC/FID and GC/MS to obtain the volatile profiles of the samples. RESULTS: Both techniques permitted the differentiation of fruit parts based on the main volatile components: the flavedo was characterized by monoterpene hydrocarbons such as limonene and myrcene, the seeds were characterized by sesquiterpene hydrocarbons such as (E)-caryophyllene and germacrene D, while the pulp showed an intermediate composition. The main differences in volatile profiles obtained by the two techniques were analyzed by chemometric techniques such as principal component analysis. CONCLUSION: The study did not highlight significant differences in volatiles between the two cultivars of trifoliate orange, while few differences in the number of volatiles in the fruit parts were revealed by the two techniques.

Content changes of bitter compounds in 'Guoqing No.1' Satsuma mandarin (Citrus unshiu Marc.) during fruit development of consecutive 3 seasons.

The main bitter compounds (nomilin, limonin and naringin) in the fruit tissues of 'Guoqing No.1' Satsuma mandarin (Citrus unshiu Marc.) were determined throughout the fruit development of 3 consecutive growing seasons. Although fluctuating largely at the corresponding developing stages of the 3 years, the contents of these compounds in fruit tissues mostly displayed a declining trend, which implied that the rhythm of the metabolism of these bitter compounds was not consistent among years and was largely growing season dependent. Regarding their distribution, fruit flavedo might be a weak sink that contained the lowest level of naringin, while the segment membrane accumulated large amount of limonin and nomilin, which indicated a possible tissue bias pattern for biosynthesis or accumulation of those compounds. Partial correlation coefficient analysis revealed a synergistic accumulation of naringin and the two limonoid aglycones in fruit tissues during fruit development, indicating an integrated metabolism of flavonoids and limonoids.

Smooth muscle relaxation activity of an aqueous extract of dried immature fruit of Poncirus trifoliata (PF-W) on an isolated strip of rat ileum.

We demonstrated that an aqueous extract of dried immature fruit of Poncirus trifoliate (PF-W) produces relaxation of intestinal smooth muscle using the ileac strips of a rat. Furthermore, the underlying mechanism of its relaxant activity was investigated. PF-W was prepared using the standard extraction protocol. A 1.5 - 2 cm long rat ileac strip was placed in an organ bath with Tyrode's solution and smooth muscle contractility was recorded by connecting it to a force transducer. Various compounds were added to the organ baths, and changes in muscular contractility were measured. PF-W concentration-dependently induced relaxation of rat ileac strips that were contracted both spontaneously and via acetylcholine treatment. Various potassium channel blockers did not inhibit the relaxation by PF-W. No difference in the effect of PF-W was observed between ileac strips treated with low (20 mM) and high concentrations (60 mM) of KCl. PF-W inhibited the contraction of rat ileac strips induced by extracellular calcium. PF-W acts as a potent smooth muscle relaxant, implicating its possible action as a rapid acting reliever for abdominal pains and a cure for intestinal convulsion. Considering that PF-W also exhibits prokinetic activity, its use in various gastrointestinal disorders seems promising.

Poncirus trifoliate fruit modulates pacemaker activity in interstitial cells of Cajal from the murine small intestine.

ETHNOPHARMACOLOGICAL RELEVANCE: Poncirus fructus (PF) has been widely used as a traditional medicine in Eastern Asia, especially to ameliorate the symptoms of gastrointestinal (GI) disorders related to abnormal GI motility. AIM OF THE STUDY: Poncirus fructus (PF), also known as Poncirus trifoliata (L.) Raf. (Rutaceae), is widely used as a traditional medicine in Eastern Asia mainly to ameliorate the symptoms of gastrointestinal (GI) disorders related to abnormal GI motility. In a previous study, a methanol extract of PF was found to have particularly potent gastroprokinetic effects. Interstitial cells of Cajal (ICCs) are pacemaker cells in the gastrointestinal tract, but the action mechanisms of PF extract in mouse small intestinal ICCs have not been investigated. Therefore, in the present study, we investigated the effects of a methanol extract of PF (MPF) in mouse small intestinal ICCs. In addition, we sought to identify the receptors involved. MATERIALS AND METHODS: Enzymatic digestions were used to dissociate ICCs from small intestines. The whole-cell patch-clamp configuration was used to record potentials (current clamp) from cultured ICCs. In addition, we analyzed intracellular Ca(2+) concentrations ([Ca(2+)]i). RESULTS: MPF decreased the amplitudes of pacemaker potentials in ICCs, and depolarized resting membrane potentials in a concentration dependent manner. Y25130 (a 5-HT3 receptor antagonist) and RS39604 (a 5-HT4 receptor antagonist) blocked MPF-induced membrane depolarizations, whereas SB269970 (a 5-HT7 receptor antagonist) did not. Pretreatment with Na(+) or Ca(2+)-free solution or thapsigargin (a Ca(2+)-ATPase inhibitor in endoplasmic reticulum) abolished the generation of pacemaker potentials and suppressed MPF-induced activity. [Ca(2+)]i analysis showed that MPF increased [Ca(2+)]i. Furthermore, treatments with PD 98059, SB203580, or JNK II inhibitor blocked MPF-induced membrane depolarizations in ICCs. CONCLUSION: These results suggest that MPF modulates pacemaker potentials through 5-HT3 and 5-HT4 receptor-mediated pathways via external Na(+) and Ca(2+) influx, and via Ca(2+) release from internal stores in a mitogen-activated protein kinase dependent manner. The study shows MPF is a good candidate for the development of a gastroprokinetic agent. In view of the effects of MPF on ICCs, further research is required, particularly to identify the active compound(s) involved and to determine their action mechanisms.