Liquiritigenin enhances cyclic adenosine monophosphate production to mitigate inflammation in dendritic cells.

OBJECTIVE: This study aims to dissect the mechanism of traditional Chinese medicinal herbs against asthma; we chose to first focus on the main chemical components of licorice to investigate their contribution to asthmatic inflammation inhibition. METHODS: Production of cellular nucleotide molecules such as cAMP, cGMP, and cGAMP was examined by using enzyme-linked immunosorbent assay (ELISA). Enzyme-encoding genes were tested in vitro using quantitative real-time PCR and protein level was detected by Western blotting analysis. In addition, co-culturing of murine dendritic cells together with T cells was conducted to examine the expression of cytokine genes and host immune response. RESULTS: We found that one of the components within licorice, named liquiritigenin (LR), could efficiently enhance cAMP production in different cell lines. The augmentation of such molecules was linked to the high expression of cAMP synthesis genes and repressed expression of cAMP breaking down genes. In addition, the downstream immune response was also alleviated by the increase in cAMP levels by LR, suggesting the great potential of this molecule against inflammation. Subsequent immunological tests showed that LR could efficiently inhibit the expression of several cytokines and alter the NF-κB pathway and T cell polarization. CONCLUSION: Altogether, we have identified a promising antiasthmatic agent LR that could exhibit immunosuppressive function by elevating the cAMP level.

Electroacupuncture improves neuronal plasticity through the A2AR/cAMP/PKA signaling pathway in SNL rats.

Improvements in neuronal plasticity are considered to be conducive to recovery from neuropathic pain. Electroacupuncture (EA) is regarded as an effective rehabilitation method for neuropathic pain. However, the effects and potential mechanism associated with EA-induced repair of hyperesthesia are not fully understood. Evidence has suggested that the adenosine A2A receptor (A2AR) and the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway play an important role in improving neuropathic pain. Here, we examined the function of EA in promoting neuronal plasticity in spinal nerve ligation (SNL) rats. The A2AR antagonist SCH58261, A2AR agonist 2-p-(2-carboxyethyl)phenethylamino-50-N-ethylcarboxamido adenosine HCl (CGS21680) and A2AR siRNA were used to confirm the relationship between A2AR and the cAMP/PKA pathway as well as the effects of A2AR on EA-induced improvements in neurobehavioral state and neuronal plasticity. Mechanical withdrawal threshold (MWT), thermal withdrawal latency (TWL), HE staining, Western blotting, RT-PCR, immunofluorescence, enzyme-linked immunosorbent assay, Nissl staining, silver staining, Golgi-Cox staining and transmission electron microscopy were used to evaluate the changes in neurobehavioral performance, protein expression, neuronal structure and dendrites/synapses. The results showed that EA and CGS21680 improved the behavioral performance, neuronal structure and dendritic/synaptic morphology of SNL rats, consistent with higher expression levels of A2AR, cAMP and PKA. In contrast to the positive effects of EA, SCH58261 inhibited dendritic growth and promoted dendritic spine/synaptic remodeling. In addition, the EA-induced improvement in neuronal plasticity was inhibited by SCH58261 and A2AR siRNA, consistent with lower expression levels of A2AR, cAMP and PKA, and worse behavioral performance. These results indicate that EA suppresses SNL-induced neuropathic pain by improving neuronal plasticity via upregulating the A2AR/cAMP/PKA signaling pathway.

Lipoic Acid Stimulates cAMP Production in Healthy Control and Secondary Progressive MS Subjects.

Lipoic acid (LA) exhibits antioxidant and anti-inflammatory properties; supplementation reduces disease severity and T lymphocyte migration into the central nervous system in a murine model of multiple sclerosis (MS), and administration in secondary progressive MS (SPMS) subjects reduces brain atrophy compared to placebo. The mechanism of action (MOA) of LA's efficacy in suppression of MS pathology is incompletely understood. LA stimulates production of the immunomodulator cyclic AMP (cAMP) in vitro. To determine whether cAMP could be involved in the MOA of LA in vivo, we performed a clinical trial to examine whether LA stimulates cAMP production in healthy control and MS subjects, and whether there are differences in the bioavailability of LA between groups. We administered 1200 mg of oral LA to healthy control, relapsing remitting MS (RRMS) and SPMS subjects, and measured plasma LA and cAMP levels in peripheral blood mononuclear cells (PBMCs). There were no significant differences between the groups in pharmacokinetic (PK) parameters. Healthy and SPMS subjects had increased cAMP at 2 and 4 h post-LA treatment compared to baseline, while RRMS subjects showed decreases in cAMP. Additionally, plasma concentrations of prostaglandin E2 (PGE2, a known cAMP stimulator) were significantly lower in female RRMS subjects compared to female HC and SPMS subjects 4 h after LA ingestion. These data indicate that cAMP could be part of the MOA of LA in SPMS, and that there is a divergent response to LA in RRMS subjects that may have implications in the efficacy of immunomodulatory drugs. This clinical trial, "Defining the Anti-inflammatory Role of Lipoic Acid in Multiple Sclerosis," NCT00997438, is registered at https://clinicaltrials.gov/ct2/show/record/NCT00997438 .

Responsiveness of pituitary to galanin throughout the reproductive cycle of male European sea bass (Dicentrarchus labrax).

The neuropeptide galanin (Gal) is a putative factor regulating puberty onset and reproduction through its actions on the pituitary. The present study investigated the pituitary responsiveness to galanin and the patterns of galanin receptors (Galrs) expression throughout the reproductive cycle of two years old male European sea bass (Dicentrarchus labrax), an important aquaculture species. Quantitative analysis of pituitary and hypothalamus transcript expression of four galr subtypes revealed differential regulation according to the testicular developmental stage, with an overall decrease in expression from the immature stage to the mid-recrudescence stage. Incubation of pituitary cells with mammalian 1-29Gal peptide induced significant changes in cAMP concentration, with sensitivities that varied according to the testicular development stages. Furthermore 1-29Gal was able to stimulate both follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) release from pituitary cell suspensions. The magnitude of the effects and effective concentrations varied according to reproductive stage, with generalized induction of Fsh and Lh release in animals sampled in January (full spermiation). The differential expression of galrs in pituitary and hypothalamus across the reproductive season, together with the differential effects of Gal on gonadotropins release in vitro strongly suggests the involvement of the galaninergic system in the regulation the hypothalamus-pituitary-gonad axis of male sea bass. This is to our knowledge the first clear evidence for the involvement of galanin in the regulation of reproduction in non-mammalian vertebrates.

Inhibition of the cAMP/PKA/CREB Pathway Contributes to the Analgesic Effects of Electroacupuncture in the Anterior Cingulate Cortex in a Rat Pain Memory Model.

Pain memory is considered as endopathic factor underlying stubborn chronic pain. Our previous study demonstrated that electroacupuncture (EA) can alleviate retrieval of pain memory. This study was designed to observe the different effects between EA and indomethacin (a kind of nonsteroid anti-inflammatory drugs, NSAIDs) in a rat pain memory model. To explore the critical role of protein kinase A (PKA) in pain memory, a PKA inhibitor was microinjected into anterior cingulate cortex (ACC) in model rats. We further investigated the roles of the cyclic adenosine monophosphate (cAMP), PKA, cAMP response element-binding protein (CREB), and cAMP/PKA/CREB pathway in pain memory to explore the potential molecular mechanism. The results showed that EA alleviates the retrieval of pain memory while indomethacin failed. Intra-ACC microinjection of a PKA inhibitor blocked the occurrence of pain memory. EA reduced the activation of cAMP, PKA, and CREB and the coexpression levels of cAMP/PKA and PKA/CREB in the ACC of pain memory model rats, but indomethacin failed. The present findings identified a critical role of PKA in ACC in retrieval of pain memory. We propose that the proper mechanism of EA on pain memory is possibly due to the partial inhibition of cAMP/PKA/CREB signaling pathway by EA.

Pharmacologic analyses of four chicken melanocortin-4 receptor mutations.

The melanocortin-4 receptor (MC4R) is a critical regulator of mammalian food intake and energy expenditure, with receptor activation resulting in decreased food intake and increased energy expenditure. Recently, studies on role of MC4R in regulation of food intake have been extended to other species, such as chicken. Functional study of mutant MC4Rs is important in proving the causal link between MC4R mutation and production traits. Herein, we cloned chicken MC4R (cMC4R) complementary DNA and generated 4 mutant cMC4Rs (Q18H, G21R, S76L, and L299P) by site-directed mutagenesis and measured their expression by flow cytometry. Pharmacologic characteristics were analyzed with binding and signaling assays using 3 agonists. We showed that G21R had decreased cell surface and total expression (P < 0.05), whereas the other 3 mutants had similar total and cell surface expression levels as wild-type cMC4R. The 4 mutants had either decreased (Q18H, G21R, S76L; P < 0.05) or no (L299P) binding to radiolabeled [Nle(4), D-Phe(7)]-α-melanocyte-stimulating hormone (MSH). In signaling assays, Q18H was constitutively active. Q18H, G21R, and S76L had decreased responses to α-MSH stimulation (P < 0.05). L299P had decreased basal and ligand-stimulated signaling (P < 0.01). Nle(4), D-Phe(7)-MSH was the most potent agonist for cMC4R and therefore would be better suited for further in vivo studies. We conclude that the cloned cMC4R was a functional receptor and provided detailed functional data for these mutations, contributing to a better understanding of cMC4R variants associated with production traits.

A systematic study on the influence of the main ingredients of an ivy leaves dry extract on the ß2-adrenergic responsiveness of human airway smooth muscle cells.

The bronchospasmolytic and secretolytic effects of ivy leaves dry extracts can be explained by an increased ß2-adrenergic responsiveness of the bronchi. Recently, it was shown that α-hederin inhibits the internalization of ß2-adrenergic receptors (ß2AR) under stimulating conditions. α-Hederin pretreated alveolar type II cells and human airway smooth muscle cells revealed an increased ß2AR binding and an elevated intracellular cAMP level, respectively. In order to identify whether additional compounds also mediate an increased ß2-adrenergic responsiveness, we examined the ingredients of an ivy leaves dry extract (EA 575) protocatechuic acid, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, rutin, kaempferol-3-O-rutinoside, 3,4-, 3,5- and 4,5-dicaffeoylquinic acid, hederacoside B, and ß-hederin. Within all the tested substances, only ß-hederin inhibited the internalization of GFP-tagged ß2AR in stably transfected HEK293 cells. Using fluorescence correlation spectroscopy ß-hederin (1 µM, 24 h) pretreated HASM cells showed a statistically significant increase in the ß2AR binding from 33.0 ± 8.9% to 44.1 ± 11.5% which was distributed with 36.0 ± 9.5% for τbound1 and 8.1 ± 2.6% for τbound2, respectively (n = 8, p < 0.05). The increased binding was selectively found for the receptor-ligand complex with unrestricted lateral mobility (τbound1 of 0.9 ± 0.1 ms, D1 = 9.1 ± 0.2 µm(2)/s, n = 8), whereas the binding of ß2AR with hindered lateral mobility (τbound2 of 64.2 ± 47.6 ms, D2 = 0.15 ± 0.02 µm(2)/s, n = 8) was not affected. Compared to control cells, a statistically significant increase of 17.5 ± 6.4% (n = 4, p < 0.05) and 24.2 ± 5.8% (n = 4, p < 0.001) in the cAMP formation was found for ß-hederin pretreated HASM cells after stimulation with 10 µM of terbutaline and simultaneous stimulation with 10 µM terbutaline and 10 µM forskolin, respectively. Within this systematic study focusing on the influence of the ingredients of an ivy leaves dry extract on HASM cells it was possible to identify ß-hederin as further component presumably responsible for the ß2-mimetic effects.

The action of p-synephrine on hepatic carbohydrate metabolism and respiration occurs via both Ca(2+)-mobilization and cAMP production.

Citrus aurantium extracts, which contain large amounts of p-synephrine, are widely used for weight loss purposes and as appetite suppressants. In the liver, C. aurantium (bitter orange) extracts affect hemodynamics, carbohydrate metabolism, and oxygen uptake. The purpose of the present work was to quantify the action of p-synephrine and also to obtain indications about its mechanism of action, a task that would be difficult to accomplish with C. aurantium extracts due to their rather complex composition. The experimental system was the isolated perfused rat liver. p-Synephrine significantly stimulated glycogenolysis, glycolysis, gluconeogenesis, and oxygen uptake. The compound also increased the portal perfusion pressure and the redox state of the cytosolic NAD(+)/NADH couple. A Ca(2+)-dependency for both the hemodynamic and the metabolic effects of p-synephrine was found. p-Synephrine stimulated both cAMP overflow and the initial Ca(2+) release from the cellular stores previously labeled with (45)Ca(2+). The metabolic and hemodynamic actions of p-synephrine were strongly inhibited by α-adrenergic antagonists and moderately affected by ß-adrenergic antagonists. The results allow to conclude that p-synephrine presents important metabolic and hemodynamic effects in the liver. These effects can be considered as both catabolic (glycogenolysis) and anabolic (gluconeogenesis), they are mediated by both α- and ß-adrenergic signaling, require the simultaneous participation of both Ca(2+) and cAMP, and could be contributing to the overall stimulation of metabolism that usually occurs during weight loss periods.

Characterisation of structurally modified analogues of glucagon as potential glucagon receptor antagonists.

Acute in vitro and in vivo biological activities of four novel structural analogues of glucagon were tested. desHis(1)Pro(4)-glucagon, desHis(1)Pro(4)Glu(9)-glucagon, desHis(1)Pro(4)Glu(9)Lys(12)FA-glucagon and desHis(1)Pro(4)Glu(9)Lys(30)FA-glucagon were stable to DPP-4 degradation and dose-dependently inhibited glucagon-mediated cAMP production (p<0.05 to p<0.001). None stimulated insulin secretion in vitro above basal levels, but all inhibited glucagon-induced insulin secretion (p<0.01 to p<0.001). In normal mice all analogues antagonised acute glucagon-mediated elevations of blood glucose (p<0.05 to p<0.001) and blocked corresponding insulinotropic responses. In high-fat fed mice, glucagon-induced increases in plasma insulin (p<0.05 to p<0.001) and glucagon-induced hyperglycaemia were blocked (p<0.05 to p<0.01) by three analogues. In obese diabetic (ob/ob) mice only desHis(1)Pro(4)Glu(9)-glucagon effectively (p<0.05 to p<0.01) inhibited both glucagon-mediated glycaemic and insulinotropic responses. desHis(1)Pro(4)-glucagon and desHis(1)Pro(4)Glu(9)-glucagon were biologically ineffective when administered 8h prior to glucagon, whereas desHis(1)Pro(4)Glu(9)Lys(12)FA-glucagon retained efficacy (p<0.01) for up to 24h. Such peptide-derived glucagon receptor antagonists have potential for type 2 diabetes therapy.

The neuroprotective effects of an extract of Gastrodia elata.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastrodia elata (GE) Blume (family Orchidaceae) is a traditional Chinese herbal medicine for treating headaches, dizziness, tetanus, and epilepsy, indicating neuronal protective functions. AIM OF THE STUDY: To evaluate the neuroprotection of GE and its molecular mechanism in preventing serum deprivation-induced PC12 cell apoptosis. MATERIALS AND METHODS: An MTT assay and Hoechst staining were used to respectively validate serum deprivation-induced cell death and apoptosis. Cyclic (c)AMP formation and protein kinase (PK)A activity were also measured after GE treatment. Western blotting was used to detect the phosphorylation of the cAMP response element-binding (CREB) protein. Transient transfection of a dominant negative CREB was used to validate the importance of CREB. RESULTS: GE targeted the adenosine A(2A) receptor (A(2A)-R). GE increased cAMP formation, PKA activity, and phosphorylation of the CREB protein. GE-induced CREB protein phosphorylation and protection was blocked by a PKA inhibitor and overexpression of the dominant negative CREB, respectively. CONCLUSIONS: These results support the neuroprotective effects of GE. The protective mechanism might be mediated through an A(2A)-R/cAMP/PKA/CREB-dependent pathway.

Flavonol kaempferol improves chronic hyperglycemia-impaired pancreatic beta-cell viability and insulin secretory function.

Considerable evidence shows that chronic hyperglycemia can cause pancreatic beta-cell dysfunction, which contributes to progressive deterioration of glucose homeostasis and overt diabetes. In the present study, we found that kaempferol, a flavonol compound present in various Chinese medicinal herbs, has cytoprotective effects on cultured clonal beta-cells and pancreatic human islets. Kaempferol treatment dose-dependently promoted viability, inhibited cellular apoptosis, and reduced caspase-3 activity in beta-cells and human islets exposed to chronic high glucose, with 10 µM kaempferol exerting the maximum effect. In addition, kaempferol treatment improved the expression of anti-apoptotic proteins Akt and Bcl-2 that was significantly reduced in beta-cells and human islets chronically exposed to hyperglycemia. Furthermore, exposure of beta-cells and human islets to kaempferol restored high glucose-attenuated intracellular cAMP and ATP production. Inhibition of protein kinase A or Akt activation ablated the anti-apoptotic effect of kaempferol. These cytoprotective effects of kaempferol were associated with improved insulin secretory function and synthesis in beta-cells and human islets. These findings provide evidence that kaempferol may be a naturally occurring anti-diabetic compound by protecting pancreatic beta-cell survival and function in a hostile environment that would otherwise lead to type 2 diabetes.

A novel quercetin analogue from a medicinal plant promotes peak bone mass achievement and bone healing after injury and exerts an anabolic effect on osteoporotic bone: the role of aryl hydrocarbon receptor as a mediator of osteogenic action.

We recently reported that extracts made from the stem bark of Ulmus wallichiana promoted peak bone mass achievement in growing rats and preserved trabecular bone mass and cortical bone strength in ovariectomized (OVX) rats. Further, 6-C-ß-D-glucopyranosyl-(2S,3S)-(+)-3',4',5,7-tetrahydroxyflavanol (GTDF), a novel flavonol-C-glucoside isolated from the extracts, had a nonestrogenic bone-sparing effect on OVX rats. Here we studied the effects of GTDF on osteoblast function and its mode of action and in vivo osteogenic effect. GTDF stimulated osteoblast proliferation, survival, and differentiation but had no effect on osteoclastic or adipocytic differentiation. In cultured osteoblasts, GTDF transactivated the aryl hydrocarbon receptor (AhR). Activation of AhR mediated the stimulatory effect of GTDF on osteoblast proliferation and differentiation. Furthermore, GTDF stimulated cAMP production, which mediated osteogenic gene expression. GTDF treatments given to 1- to 2-day-old rats or adult rats increased the mRNA levels of AhR target genes in calvaria or bone marrow stromal cells. In growing female rats, GTDF promoted parameters of peak bone accrual in the appendicular skeleton, including increased longitudinal growth, bone mineral density, bone-formation rate (BFR), cortical deposition, and bone strength. GTDF promoted the process of providing newly generated bone to fill drill holes in the femurs of both estrogen-sufficient and -deficient rats. In osteopenic OVX rats, GTDF increased BFR and significantly restored trabecular bone compared with the ovaries-intact group. Together our data suggest that GTDF stimulates osteoblast growth and differentiation via the AhR and promotes modeling-directed bone accrual, accelerates bone healing after injury, and exerts anabolic effects on osteopenic rats likely by a direct stimulatory effect on osteoprogenitors. Based on these preclinical data, clinical evaluation of GTDF as a potential bone anabolic agent is warranted.

Exposure of human seminal vesicle tissue to phosphodiesterase (PDE) inhibitors antagonizes the contraction induced by norepinephrine and increases production of cyclic nucleotides.

OBJECTIVES: To investigate further the role of phosphodiesterase (PDE) isoenzymes in the control of human seminal vesicle (SV) smooth muscle contractility, we examined the functional responses of isolated SV tissue to various PDE inhibitors. It has been suggested that the application of inhibitors of the PDE type 5 may facilitate SV smooth muscle relaxation and, subsequently, retard ejaculatory response. METHODS: Using the organ bath technique, strip preparations of human SV were exposed for 5 minutes to 1 µM of the PDE inhibitors milrinone (PDE3 inhibitor), rolipram, Ro 20-1724 (PDE4 inhibitors), and sildenafil (PDE5 inhibitor). Norepinephrine (NE, alpha agonist) was then added (0,1 µM, 1 µM, and 10 µM) and isometric responses were recorded. A contraction-response curve to NE in the absence of PDE inhibitors was also generated. Drug effects on the production of cyclic adenosine monophosphate (AMP) and cyclic guanosine monophosphate (GMP) were measured by means of radioimmunometric assays. RESULTS: The contraction induced by NE was effectively antagonized by 1 µM of rolipram (83.3% inhibition), Ro 20-1724 (72.3% inhibition), sildenafil (41.6% inhibition), and milrinone (37.5% inhibition). The inhibition of force generation was paralleled by a 1.6-fold to 2.8-fold increase in tissue cyclic AMP (induced by milrinone, rolipram, Ro 20-1724), and a 12-fold rise in cyclic GMP (induced by sildenafil). CONCLUSION: The findings demonstrate that PDE inhibitors can counteract the contraction of human SV mediated by alpha-adrenergic receptors and enhance levels of cyclic nucleotides. This might be of importance with regard to the identification of new options for the pharmacological treatment of premature ejaculation.

Presence of a putative steroidal allosteric site on glycoprotein hormone receptors.

In a previous work we found that the insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), inhibits the accumulation of cAMP as induced by the bovine thyroid stimulating hormone (bTSH) in cells transfected with the TSH receptor. In this work, we demonstrate that the DDT molecular analogues, diethylstilbestrol and quercetine, are more potent inhibitors of the TSH receptor activity than DDT itself. The notion that all these compounds interfere with nuclear estrogen receptors, as either agonists (DDT and diethylstilbestrol) or antagonists (quercetin), prompted us to test the ability of the steroid hormone 17-beta-estradiol to inhibit the TSH receptor activity. We found that estrogen exposure causes a modest but significant inhibition of the bTSH induced cAMP accumulation both in transfected CHO-TSH receptor and Fischer Rat Thyroid Low Serum 5% (FRTL-5) cells. When applied to CHO cells transfected with the luteinizing hormone receptor, 17-beta-estradiol proved capable of inhibiting the hCG induced cAMP accumulation at a concentration as low as 10nM, though the effect was not greater than 35%. The effect of 17-beta-estradiol was not estrogen receptors mediated, as co-transfection of the estrogen receptor alpha and beta subunits with LH receptor caused cAMP to increase above the level attained by the sole hCG stimulation, and not to decrease it as expected. These data suggest the presence of a steroidal-like allosteric binding site on glycoprotein hormone receptors.

Sustained morphine treatment augments capsaicin-evoked calcitonin gene-related peptide release from primary sensory neurons in a protein kinase A- and Raf-1-dependent manner.

Studies have shown that long-term (5alpha,6alpha)-7,8-didehydro-4,5-epoxy-17-methylmorphinan-3,6-diol (morphine) treatment increases the sensitivity to painful heat stimuli (thermal hyperalgesia). The cellular adaptations contributing to sustained morphine-mediated pain sensitization are not fully understood. It was shown previously (J Neurosci 22:6747-6755, 2002) that sustained morphine exposure augments pain neurotransmitter [such as calcitonin gene-related peptide (CGRP)] release in the dorsal horn of the spinal cord in response to the heat-sensing transient receptor potential vanilloid 1 receptor agonist 8-methyl-N-vanillyl-6-nonenamide (capsaicin). In the present study, we demonstrate that sustained morphine-mediated augmentation of CGRP release from isolated primary sensory dorsal root ganglion neurons is dependent on protein kinase A and Raf-1 kinase. Our data indicate that, in addition to neural system adaptations, sustained opioid agonist treatment also produces intracellular compensatory adaptations in primary sensory neurons, leading to augmentation of evoked pain neurotransmitter release from these cells.

Investigation of beta(2)-adrenoceptor subtype selectivity and organ specificity for bedoradrine (KUR-1246), a novel tocolytic beta-adrenergic receptor stimulant.

OBJECTIVES: The aim of this study was to evaluate the beta-adrenergic receptor (beta-AR) selectivity, organ specificity and efficacy of delaying the onset of spontaneous delivery of bedoradrine (KUR-1246), a novel uterine relaxant. METHODS: beta-AR selectivity was evaluated in terms of the amount of cyclic adenosine monophosphate produced by bedoradrine, ritodrine and isoprenaline in Chinese hamster ovary cells expressing human beta(1)-, beta(2)-AR or beta(3)-AR. Inhibition of contractions of the atrium, trachea and proximal colon by bedoradrine were compared with those of the uterus in pregnant rats using an organ bath method. Finally, the delaying effect of bedoradrine on spontaneous labor was evaluated by an in vivo study using term pregnant rats. RESULTS: EC(50) values of bedoradrine for cyclic adenosine monophosphate production in Chinese hamster ovary cells via beta(1)-, beta(2)- and beta(3)-AR were 2400 +/- 30, 2.9 +/- 0.10 and 363 +/- 3 nmol/L, respectively, indicating that bedoradrine had 832- and 126-fold higher selectivity for beta(2)-AR than for beta(1)- and beta(3)-AR. EC(50) values of bedoradrine for the uterus, atrium, trachea and proximal colon were 1.01 +/- 0.27, 2300 +/- 356, 1610 +/- 299 and 219 +/- 23.5 nmol/L, respectively. Thus, bedoradrine was 2280-, 1590- and 217-fold more specific for the uterus than for the atrium, trachea and proximal colon, respectively. Bedoradrine delayed the spontaneous delivery of 21-day-pregnant rats in a dose-dependent manner. CONCLUSIONS: Bedoradrine is a promising drug for the treatment of preterm labor in obstetrical practice because it has better selectivity for beta(2)-AR and specificity for the uterus than currently used agents and may effectively delay spontaneous delivery.

Effects and mechanisms of total glucosides of paeony on synoviocytes activities in rat collagen-induced arthritis.

The aim of the study was to investigate the effects of TGP, an active compound extracted from the roots of Paeonia lactiflora Pall, on the activities of synoviocytes in rats with collagen-induced arthritis (CIA) and its possible mechanisms. CIA was induced in male Sprague-Dawley (SD) rats immunized with chicken type II collagen (CII) in Freund's complete adjuvant (FCA). Synoviocytes proliferation was determined by 3-(4, 5-2dimethylthiazal-2yl) 2, 5-diphenyltetrazoliumbromide (MTT) assay. Tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), prostaglandin E(2) (PGE(2)) and cyclic adenosine monophosphate (cAMP) levels in synoviocytes were measured by radioimmunoassay (RIA). E-prostanoid (EP)(2) and EP(4) receptors were analyzed by Western blot analysis. The results showed that TGP significantly inhibited the proliferation of synoviocytes, decreased the production of IL-1, TNF-alpha and PGE(2) and elevated the levels of cAMP. Further study showed that TGP could up-regulate the expression of EP(2) and EP(4). These results indicated that TGP might exert its anti-inflammatory effects through inhibiting the production of pro-inflammatory mediators in synoviocytes of CIA rats, which might be associated with its ability to regulate cAMP-dependent EP(2)/EP(4)-mediated pathway.

Wogonin inhibits microglial cell migration via suppression of nuclear factor-kappa B activity.

Previously, we and others have demonstrated that wogonin, an active component from the root of Scutellaria baicalensis Georgi, has a neuroprotective effect in cerebral ischemic insult. The neuroprotective effect of wogonin may at least in part be due to its anti-inflammatory properties. Microglial cells, well-known residential macrophages in the central nervous system, migrate to the ischemic lesion and play a pivotal role in the development of chronic inflammation. In the present study, we observed that wogonin potently inhibited microglial migration toward a chemokine, monocyte chemoattractant protein-1 (MCP-1). The anti-migratory effect of wogonin was provoked at nanomolar concentrations, at which wogonin did not significantly inhibit the production of cytokines and chemokines. NF-kappaB has previously shown to regulate microglial cell migration, and activation of cAMP-signaling pathway has also been associated with inhibition of microglial cell motility. In the present study, wogonin at low micromolar concentrations completely suppressed the activity of NF-kappaB in MCP-1-stimulated microglia, and NF-kappaB inhibitors such as N-acetyl cysteine and pyrrolidinedithiocarbamate inhibited the MCP-1-induced migration of microglial cells. However, wogonin did not stimulate the production of cAMP in microglial cells. Our results indicate that the anti-inflammatory activity of wogonin is exerted at least in part by suppressing microglial cell motility via inhibition of NF-kappaB activity.

Serotomide and safflomide modulate forskolin-stimulated cAMP formation via 5-HT1 receptor.

Serotomide (trans-N-caffeoylserotonin) and safflomide (trans-N-caffeoyltryptamine) belong to serotonin-derived phenylpropenoid amides found in plants. In this paper, serotomide and safflomide were investigated to determine their effects on serotonin receptor 5-HT1 in the renal epithelial (OK) cells, due to their structural similarity to 5-HT1 receptor ligands. At the concentration of 10 microM, serotomide was able to inhibit forskolin-stimulated cAMP formation in the OK cells by 31% (p<0.019). The inhibition was repressed by Nan-190 and spiperone (5-HT1 antagonists), suggesting that serotomide suppresses cAMP formation via binding to 5-HT1 receptors in the OK cells. Meanwhile, safflomide could not inhibit forskolin-stimulated cAMP formation at the same concentration (10 microM), but repress the inhibition of forskolin-stimulated cAMP by serotonin agonists (e.g., serotonin and 8-OH-DPAT) by 31% (p<0.018), suggesting that safflomide may block 5-HT1 receptors in a similar way to Nan-190 and spiperone. All together the data indicate that serotomide and safflomide may be potent compounds that respectively act to activate and to block 5-HT1 receptors on OK cells.

An alternative therapy for graves' disease: clinical effects and mechanisms of an herbal remedy.

Graves' disease, the most common cause of hyperthyroidism, is an autoimmune disorder. Antithyroid drugs have been selected as the first-line treatment of Graves' disease in Korea, Japan, and European countries. However, antithyroid drugs such as methimazole (MMI) and prophylthiouracil (PTU) have limitations in clinical applications because of their side effects. In this study, we performed a clinical trial and in vitro study to investigate the clinical effects and action mechanism of Ahnjeonbaekho-tang (AJBHT), an herbal remedy for Graves' disease. In a clinical study of Graves' disease patients who had side effects from antithyroid drugs, we found that treatment by AJBHT resulted in a reduction of serum triiodothyronine (T3) and free thyroxine (FT4) levels and an increase in thyroid stimulating hormone (TSH) levels (T3: p<0.0001, FT4: p=0.0012, TSH: p=0.0370, respectively). In vitro, AJBHT significantly inhibits FRTL-5 cell proliferation, DNA synthesis, cyclic AMP production, T4 synthesis, and the expression of thyroglobulin (Tg) mRNA in comparison with the control. These results suggest that AJBHT might suppress T(4) synthesis by modulating adenosine 3',5'-cyclic monophosphate (cAMP) and Tg expression, and therefore, AJBHT could be an alternative therapy for Graves' disease patients who have side effects from antithyroid drugs.