A Review on the Sources, Structures, and Pharmacological Activities of Lucidenic Acids.

Ganoderma lucidum has long been used as a multi-purpose plant and functional food. The pharmacological properties of G. lucidum are primarily attributed to its polysaccharides and triterpenoids. Ganoderic and lucidenic acids are the two major triterpenoids groups in G. lucidum. Despite the discovery of 22 types of lucidenic acids, research on lucidenic acids is significantly less extensive compared to that on ganoderic acid. To the best of our knowledge, for the first time, in this review, we aimed to summarize the sources, contents, chemical structures, and pharmacological effects, including anti-cancer, anti-inflammatory, antioxidant, anti-viral, neuroprotective, anti-hyperlipidemic, anti-hypercholesterolemic, and anti-diabetic properties, of lucidenic acids. Studies on lucidenic acids are still preliminary and have several limitations. Therefore, more in-depth studies with optimal designs are essential for the development of lucidenic acids as medicines, functional foods, and nutraceuticals.

A defective flexible loop contributes to the processing and gating defects of the predominant cystic fibrosis-causing mutation.

People with the genetic disease cystic fibrosis (CF) often carry a deletion mutation ΔF508 on the gene encoding the CF transmembrane conductance regulator (CFTR) Cl- channel. This mutation greatly reduces the CFTR maturation process and slows the channel opening rate. Here, we investigate whether residues near F508 contribute to these defects in ΔF508-CFTR. Most deletion mutations, but not alanine substitutions, of individual residues from positions 503 to 513 impaired CFTR maturation. Interestingly, only protein processing of ΔY512-CFTR, like that of ΔF508-CFTR, was greatly improved by low-temperature culture at 27°C or small-molecule corrector C18. The 2 mutant Cl- channels were equally slow to open, suggesting that they may share common structural flaws. Studies on the H3-H4 loop that links residues F508 and Y512 demonstrate that G509A/V510G mutations, moving G509 1 position backward in the loop, markedly enhanced ΔF508-CFTR maturation and opening rate while promoting protein stability and persistence of the H3 helix in ΔF508 nucleotide-binding domain 1. Moreover, V510A/S511A mutations noticeably increased ΔY512-CFTR maturation at 27°C and its opening rate. Thus, loop abnormalities may contribute to ΔF508- and ΔY512-CFTR defects. Importantly, correcting defects from G509 displacement in ΔF508-CFTR may offer a new avenue for drug discovery and CF treatments.-Chen, X., Zhu, S., Zhenin, M., Xu, W., Bose, S. J., Wong, M. P.-F., Leung, G. P. H., Senderowitz, H., Chen, J.-H. A defective flexible loop contributes to the processing and gating defects of the predominant cystic fibrosis-causing mutation.

Chemical and pharmacological evaluations on the extract of Scutellaria baicalensis Georgi (Huang-Qin) prepared by various extraction methods.

BACKGROUND: This study reported a comprehensive approach (comparing the extraction yields, chemical profiles, antioxidant properties and CYP450-inhibitory effects) to evaluated the effectiveness of various extraction methods [microwave-assisted extraction using water (MAE-W), heat reflux extraction using water (HRE-W), ultrasonic extraction using 70 % ethanol and ultrasonic extraction using ethanol (UE-E)] for Huang-Qin (HQ), the dried root of Scutellaria baicalensis Georgi. RESULTS: The HQ extraction efficiency by MAE-W was the best. The chemical profiles of extracts obtained using HRE-W and MAE-W were similar; whereas more flavones but less flavone glycosides were detected in the UE-E extract. There was no difference in the antioxidant properties among different extracts. In vitro human liver microsome assays illustrated that all extracts possessed herb-drug interaction potentials but the UE-E extract are shown with a potent interaction with CYP3A4-metabolized drugs. CONCLUSION: MAE-W is a favorable method for the preparation of HQ extracts based on extraction yield, pharmacological properties and safety.

In vitro vitamin K(2) and 1α,25-dihydroxyvitamin D(3) combination enhances osteoblasts anabolism of diabetic mice.

In this study, we evaluated the anabolic effect and the underlying cellular mechanisms involved of vitamin K2 (10 nM) and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) (10 nM), alone and in combination, on primary osteoblasts harvested from the iliac crests of C57BL/KsJ lean (+/+) and obese/diabetic (db/db) mice. A lower alkaline phosphatase (ALP) activity plus a reduced expression of bone anabolic markers and bone formation transcription factors (osteocalcin, Runx2, Dlx5, ATF4 and OSX) were consistently detected in osteoblasts of db/db mice compared to lean mice. A significantly higher calcium deposits formation in osteoblasts was observed in lean mice when compared to db/db mice. Co-administration of vitamin K2 (10 nM) and 1,25(OH)2D3 (10 nM) caused an enhancement of calcium deposits in osteoblasts in both strains of mice. Vitamins K2 and 1,25(OH)2D3 co-administration time-dependently (7, 14 and 21 days) increased the levels of bone anabolic markers and bone formation transcription factors, with a greater magnitude of increase observed in osteoblasts of db/db mice. Combined vitamins K2 plus 1,25(OH)2D3 treatment significantly enhanced migration and the re-appearance of surface microvilli and ruffles of osteoblasts of db/db mice. Thus, our results illustrate that vitamins K2 plus D3 combination could be a novel therapeutic strategy in treating diabetes-associated osteoporosis.

Equilibrative Nucleoside Transporters 1 and 4: Which One Is a Better Target for Cardioprotection Against Ischemia-Reperfusion Injury?

The cardioprotective effects of adenosine and adenosine receptor agonists have been studied extensively. However, their therapeutic outcomes in ischemic heart disease are limited by systemic side effects such as hypotension, bradycardia, and sedation. Equilibrative nucleoside transporter (ENT) inhibitors may be an alternative. By reducing the uptake of extracellular adenosine, ENT1 inhibitors potentiate the cardioprotective effect of endogenous adenosine. They have fewer systemic side effects because they selectively increase the extracellular adenosine levels in ischemic tissues undergoing accelerated adenosine formation. Nonetheless, long-term inhibition of ENT1 may adversely affect tissues that have low capacity for de novo nucleotide biosynthesis. ENT1 inhibitors may also affect the cellular transport, and hence the efficacy, of anticancer and antiviral nucleoside analogs used in chemotherapy. It has been proposed that ENT4 may also contribute to the regulation of extracellular adenosine in the heart, especially under the acidotic conditions associated with ischemia. Like ENT1 inhibitors, ENT4 inhibitors should work specifically on ischemic tissues. Theoretically, ENT4 inhibitors do not affect tissues that rely on ENT1 for de novo nucleotide synthesis. They also have no interaction with anticancer and antiviral nucleosides. Development of specific ENT4 inhibitors may open a new avenue in research on ischemic heart disease therapy.

Relaxation effect of a novel Danshensu/tetramethylpyrazine derivative on rat mesenteric arteries.

Danshen (Radix Salviae miltiorrhizae) and ChuanXiong (Ligusticum wallichii) are two traditional herbal medicines commonly used in China for the treatment of cardiovascular diseases. The active components in Danshen and ChuanXiong are Danshensu (DSS, (R)-3, 4-dihydroxyphenyllactic acid) and tetramethylpyrazine (TMP), respectively. In the present study, a new compound named ADTM, which is a conjugation of DSS and TMP, was synthesized and its effect on the contractility of rat mesenteric arteries was examined. The relaxation effect of ADTM on rat mesenteric arteries was studied using myography. The effects of ADTM on Ca(2+) channels were measured by Ca(2+) imaging and patch-clamp techniques. The results showed that ADTM caused a concentration-dependent relaxation of rat mesenteric arteries. This relaxation effect was not affected by the removal of endothelium or inhibitors of nitric oxide synthase, cyclooxygenase, guanylyl cyclase and adenylyl cyclase. Potassium channel blockers including tetraethylammonium, iberiotoxin, apamin, 4-aminopyridine, BaCl2 and glibenclamide also failed to inhibit the relaxation response to ADTM. ADTM inhibited CaCl2-induced contractions and reduced the Ca(2+) influx in isolated mesenteric arterial muscle cells. Our results suggest that ADTM may be a novel relaxing agent. Its mechanism of action involves the direct blockade of voltage-gated Ca(2+) channels in vascular smooth muscle cells, resulting in a decrease in Ca(2+) influx into the cells.

Oleanolic Acid Loaded PEGylated PLA and PLGA Nanoparticles with Enhanced Cytotoxic Activity against Cancer Cells.

Oleanolic acid (OA) is a natural triterpenoid with anticancer properties, but its hydrophobic nature and poor aqueous solubility pose challenges in pharmaceutical formulation development. The present study aimed at developing OA-loaded mPEG-PLGA or mPEG-PLA nanoparticles (NPs) to improve the delivery of OA. The NPs were prepared by nanoprecipitation, and their physicochemical properties were characterized. The OA encapsulation efficiency of the NPs was between 40 and 75%. The size of the OA-loaded NPs was around 200-250 nm, which fell within the range required for tumor targeting by means of the enhanced permeability and retention (EPR) effect, and the negatively charged NPs remained physically stable for over 20 weeks with no aggregation observed. The OA-loaded NPs produced significant cytotoxic effects through apoptosis in cancer cell lines. Overall, the OA-loaded mPEG-PLGA NPs and mPEG-PLA NPs shared similar physicochemical properties. The former, especially the OA-loaded mPEG-P(D,L)LGA NPs, were more cytotoxic to cancer cells and therefore were more efficient for OA delivery.

Inhalable dry powder formulations of siRNA and pH-responsive peptides with antiviral activity against H1N1 influenza virus.

Pulmonary delivery of siRNA has considerable therapeutic potential for treating viral respiratory infectious diseases including influenza. By introducing siRNA that targets the conserved region of viral genes encoding nucleocapsid protein (NP), viral mRNAs can be degraded and viral replication can be inhibited in mammalian cells. To enable siRNA to be used as an antiviral agent, the nucleic acid delivery barrier must be overcome. Effective local delivery of siRNA to lung tissues is required to reduce the therapeutic dose and minimize systemic adverse effects. To develop a formulation suited for clinical application, complexes of pH-responsive peptides, containing either histidine or 2,3-diaminopropionic acid (Dap), and siRNA were prepared into dry powders by spray drying with mannitol, which was used as a bulking agent. The spray-dried (SD) powders were characterized and found to be suitable for inhalation with good stability, preserving the integrity of the siRNA as well as the biological and antiviral activities. The formulations mediated highly effective in vitro delivery of antiviral siRNA into mammalian lung epithelial cells, leading to significant inhibition of viral replication when the transfected cells were subsequently challenged with H1N1 influenza virus. SD siRNA powders containing pH-responsive peptides are a promising inhalable formulation to deliver antiviral siRNA against influenza and are readily adapted for the treatment of other respiratory diseases.

Thyroid hormone affects both endothelial and vascular smooth muscle cells in rat arteries.

Hypothyroidism impairs endothelium-dependent dilatations, while hyperthyroidism augments the production of endothelial nitric oxide. Thus, experiments were designed to determine if thyroid hormone causes endothelium-dependent responses, or alleviates diabetic endothelial dysfunction. Isometric tension was measured in rings with or without endothelium of arteries from normal and diabetic Sprague-Dawley rats. Release of 6-keto prostaglandin F1α and thromboxane B2 were measured by enzyme linked immunosorbent assay and protein levels [endothelial nitric oxide synthase (eNOS), cyclooxygenases (COX)] by immunoblotting. Triiodothyronine (T3) caused concentration-dependent (3×10(-6)-3×10(-5)M) relaxations in mesenteric (pEC50, 4.96±0.19) and femoral (pEC50, 4.57±0.35) arteries without endothelium. In femoral arteries of rats with diabetes, 5-methylamino-2-[[(2S,3R,5R,8S,9S)-3,5,9-trimethyl-2-(1-oxo-(1H-pyrrol-2- -yl)propan-2-yl)-1,7-dioxaspiro-(5,5)undecan-8-yl]methyl]benzooxazole-4-carboxylic acid (A23187, 3×10(-7) to 10(-6)M) caused partly endothelium-dependent contractions. After chronic T3-treatment with (10µg/kg/day; four weeks), the contractions to A23187 of preparations with and without endothelium were comparable, the thromboxane B2-release was reduced (by 38.1±9.2%). The pEC50 of 9, 11-dideoxy-11α, 9α-epoxymethanoprostaglandin F2α (U46619, TP-receptor agonist) was increased in T3-treated diabetic rats compared with controls (8.53±0.06 vs 7.94±0.09). The protein expression of eNOS increased (by 228%) but that of COX-1 decreased (by 35%) after chronic T3 treatment. In human umbilical vein endothelial cells incubated for one week with T3 (10(-10)-10(-7)M) in the presence but not in the absence of interleukin-1ß (1ng/ml), the expression of eNOS was increased compared to control. In conclusion, thyroid hormone acutely relaxes mesenteric and femoral vascular smooth muscle, but given chronically reduces the release of endothelium-derived vasoconstrictor prostanoids while enhancing the responsiveness of TP receptors of vascular smooth muscle.

Uptake and protective effects of ergothioneine in human endothelial cells.

Ergothioneine is a thiourea derivative of histidine found in food, especially mushrooms. Experiments in cell-free systems and chemical assays identified this compound as a powerful antioxidant. Experiments were designed to test the ability of endothelial cells to take up ergothioneine and hence benefit from protection against oxidative stress. Reverse-transcription polymerase chain reaction and Western blotting demonstrated transcription and translation of an ergothioneine transporter in human brain microvascular endothelial cells (HBMECs). Uptake of [(3)H]ergothioneine occurred by the organic cation transporter novel type-1 (OCTN-1), was sodium-dependent, and was reduced when expression of OCTN-1 was silenced by small interfering RNA (siRNA). The effect of ergothioneine on the production of reactive oxygen species (ROS) in HBMECs was measured using dichlorodihydrofluorescein and lucigenin, and the effect on cell viability was studied using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. ROS production and cell death induced by pyrogallol, xanthine oxidase plus xanthine, and high glucose were suppressed by ergothioneine. The antioxidant and cytoprotective effects of ergothioneine were abolished when OCTN-1 was silenced using siRNA. The expression of NADPH oxidase 1 was decreased, and those of glutathione reductase, catalase, and superoxide dismutase enhanced by the compound. In isolated rat basilar arteries, ergothioneine attenuated the reduction in acetylcholine-induced relaxation caused by pyrogallol, xanthine oxidase plus xanthine, or incubation in high glucose. Chronic treatment with the compound improved the response to acetylcholine in arteries of rats with streptozotocin-induced diabetes. In summary, ergothioneine is taken up by endothelial cells via OCTN-1, where the compound then protects against oxidative stress, curtailing endothelial dysfunction.

Differential ligand binding affinities of human estrogen receptor-α isoforms.

Rapid non-genomic effects of 17ß-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66) and the truncated isoforms, estrogen receptor-α46 (ER46) and estrogen receptor-α36 (ER36). However, the binding affinities of the membrane estrogen receptors (mERs) remain unknown due to the difficulty of developing of stable mER-transfected cell lines with sufficient mER density, which has largely hampered biochemical binding studies. The present study utilized cell-free expression systems to determine the binding affinities of 17ß-estradiol to mERs, and the relationship among palmitoylation, membrane insertion and binding affinities. Saturation binding assays of human mERs revealed that [³H]-17ß-estradiol bound ER66 and ER46 with Kd values of 68.81 and 60.72 pM, respectively, whereas ER36 displayed no specific binding within the tested concentration range. Inhibition of palmitoylation or removal of the nanolipoprotein particles, used as membrane substitute, reduced the binding affinities of ER66 and ER46 to 17ß-estradiol. Moreover, ER66 and ER46 bound differentially with some estrogen receptor agonists and antagonists, and phytoestrogens. In particular, the classical estrogen receptor antagonist, ICI 182,780, had a higher affinity for ER66 than ER46. In summary, the present study defines the binding affinities for human estrogen receptor-α isoforms, and demonstrates that ER66 and ER46 show characteristics of mERs. The present data also indicates that palmitoylation and membrane insertion of mERs are important for proper receptor conformation allowing 17ß-estradiol binding. The differential binding of ER66 and ER46 with certain compounds substantiates the prospect of developing mER-selective drugs.

DNA-loaded chitosan oligosaccharide nanoparticles with enhanced permeability across Calu-3 cells.

Chitosan oligosaccharide (oligoCS) is a low molecular weight chitosan and its potential for DNA delivery is described here. DNA-loaded oligoCS nanoparticles were prepared by ionic gelation using thiamine pyrophosphate (TPP) as cross-linker. The nanoparticles with oligoCS:DNA: TPP weight ratio of 50:1:25 were approximately 170 nm in diameter with a zeta potential of +40 mV, and were used in the permeability study. The cytotoxicity of oligoCS solutions and nanoparticles was evaluated by MTT assay. The concentrations that exhibited minimal cytotoxicity were employed to investigate their effect on trans-epithelial electrical resistance (TEER) and cellular uptake across the Calu-3 cell layer which was used as a nasal epithelial model. OligoCS nanoparticles were able to cause a significant and reversible decrease in TEER and promote efficient cellular uptake. In addition, the oligoCS nanoparticles were able to enhance paracellular permeability to a greater extent than oligoCS solutions at an equivalent concentration. However, the oligoCS nanoparticles were too large to cross the cell layers through the paracellular route. The transcellular pathway appeared to be the major mechanism of the transportation of oligoCS nanoparticles across the cell layers. OligoCS nanoparticles also allowed efficient DNA incorporation, thereby providing the possibility of controlled nucleic acids release and absorption across epithelial surface.

Involvement of organic cation transporter-3 and plasma membrane monoamine transporter in serotonin uptake in human brain vascular smooth muscle cells.

The serotonin (5-HT) uptake system is supposed to play a crucial part in vascular functions by "fine-tuning" the local concentration of 5-HT in the vicinity of 5-HT(2) receptors in vascular smooth muscle cells. In this study, the mechanism of 5-HT uptake in human brain vascular smooth muscle cells (HBVSMCs) was investigated. [(3)H]5-HT uptake in HBVSMCs was Na(+)-independent. Kinetic analyses of [(3)H]5-HT uptake in HBVSMCs revealed a K(m) of 50.36 ± 10.2 mM and a V(max) of 1033.61 ± 98.86 pmol/mg protein/min. The specific serotonin re-uptake transporter (SERT) inhibitor citalopram, the specific norepinephrine transporter (NET) inhibitor desipramine, and the dopamine transporter (DAT) inhibitor GBR12935 inhibited 5-HT uptake in HBVSMCs with IC(50) values of 97.03 ± 40.10, 10.49 ± 5.98, and 2.80 ± 1.04 µM, respectively. These IC(50) values were 100-fold higher than data reported by other authors, suggesting that those inhibitors were not blocking their corresponding transporters. Reverse transcription-polymerase chain reaction results demonstrated the presence of mRNA for organic cation transporter (OCT)-3 and plasma membrane monoamine transporter (PMAT), but the absence of OCT-1, OCT-2, SERT, NET, and DAT. siRNA knockdown of OCT-3 and PMAT specifically attenuated 5-HT uptake in HBVSMCs. It is concluded that 5-HT uptake in HBVSMCs was mediated predominantly by a low-affinity and Na(+)-independent mechanism. The most probable candidates are OCT-3 and PMAT, but not the SERT.

Iatrogenic mitochondriopathies: a recent lesson from nucleoside/nucleotide reverse transcriptase inhibitors.

The use of nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) has revolutionized the treatment of infection by human immunodeficiency virus (HIV) and hepatitis-B virus. NRTIs can suppress viral replication in the long-term, but possess significant toxicity that can seriously compromise treatment effectiveness. The major toxicity of NRTIs is mitochondrial toxicity. This manifests as serious side effects such as myopathy, peripheral neuropathy and lactic acidosis. In general, it is believed that the mitochondrial pathogenesis is closely related to the effect of NRTIs on mitochondrial DNA polymerase-γ. Depletion and mutation of mitochondrial DNA during chronic NRTI therapy may lead to cellular respiratory dysfunction and release of reactive oxidative species, resulting in cellular damage. It is now apparent that the etiology is far more complex than originally thought. It appears to involve multiple mechanisms as well as host factors such as HIV per se, inborn mitochondrial mutation, and sex. Management of mitochondrial toxicity during NRTI therapy remains a challenge. Interruption of NRTI therapy and substitution of the causative agents with alternative better-tolerated NRTIs represents the mainstay of management for mitochondrial toxicity and its clinical manifestations. A range of pharmacological approaches has been proposed as treatments and prophylaxes.

Anti-Parkinsonian drug discovery from herbal medicines: what have we got from neurotoxic models?

ETHNOPHARMACOLOGICAL RELEVANCE: Herbal medicines are used to treat Parkinson's disease (PD) in ancient medical systems in Asian countries such as India, China, Japan and Korea based on their own anecdotal or experience-based theories. AIM OF THE REVIEW: To systematically summarize and analyze the anti-Parkinsonian activities of herbal preparations (including active compounds, herbal extracts and formulations) investigated in the neurotoxic models of PD and provide future references for basic and clinical investigations. MATERIALS AND METHODS: All the herbal materials tested on in vitro and in vivo neurotoxic models of PD were retrieved from PubMed database by using pre-set searching strings. The relevant compounds and herbal extracts with anti-Parkinsonian activities were included and analyzed according to their chemical classifications or biological activities. RESULTS: A total of 51 herbal medicines were analyzed. A diversity of compounds isolated from herbal materials were reported to be effective on neurotoxic models of PD by modulating multiple key events or signaling pathways implicated in the pathogenesis of PD. The main structure types of these compounds belong to catechols, stilbenoids, flavonoids, phenylpropanoids and lignans, phenylethanoid glycosides and terpenes. Although some herbal extracts and formulations have shown positive results on PD animal models, the relative compounds accounting for the effects and the underlying mechanisms remain to be further investigated. CONCLUSIONS: Herbal medicines can be an alternative and valuable source for anti-Parkinsonian drug discovery. Compounds classified into stilbenoids, flavonoids, catechols and terpenes may be the most promising candidates for further investigation. Some well-studies compounds such as baicalein, puerarin, resveratrol, curcumin and ginsenosides deserve further consideration in clinical trials. In-depth experimental studies are still needed to evaluate the efficacy of herbal extracts and formulations in PD models.

Physiological and pharmacological roles of vascular nucleoside transporters.

Adenosine modulates various vascular functions such as vasodilatation and anti-inflammation. The local concentration of adenosine in the vicinity of adenosine receptors is fine tuned by 2 classes of nucleoside transporters: equilibrative nucleoside transporters (ENTs) and concentrative nucleoside transporters (CNTs). In vascular smooth muscle cells, 95% of adenosine transport is mediated by ENT-1 and the rest by ENT-2. In endothelial cells, 60%, 10%, and 30% of adenosine transport are mediated by ENT-1, ENT-2, and CNT-2, respectively. In vitro studies show that glucose per se increases the expression level of ENT-1 via mitogen-activating protein kinase-dependent pathways. Similar results have been demonstrated in diabetic animal models. Hypertension is associated with the increased expression of CNT-2. It has been speculated that the increase in the activities of ENT-1 and CNT-2 may reduce the availability of adenosine to adenosine receptors, thereby weakening the vascular functions of adenosine. This may explain why patients with diabetes and hypertension suffer greater morbidity from ischemia and atherosclerosis. No oral hypoglycemic agents can inhibit ENTs, but an exception is troglitazone (a thiazolidinedione that has been withdrawn from the market). ENTs are also sensitive to dihydropyridine-type calcium-channel blockers, particularly nimodipine, which can inhibit ENT-1 in the nanomolar range. Those calcium-channel blockers are noncompetitive inhibitors of ENTs, probably working through the reversible interactions with allosteric sites. The nonsteroidal anti-inflammatory drug sulindac sulfide is a competitive inhibitor of ENT-1. In addition to their original pharmacological actions, it is believed that the drugs mentioned above may regulate vascular functions through potentiation of the effects of adenosine.

Inhibition of TNF-α-mediated endothelial cell-monocyte cell adhesion and adhesion molecules expression by the resveratrol derivative, trans-3,5,4'-trimethoxystilbene.

Resveratrol (RSV) has been shown to have anti-inflammatory activity and to have a protective role against atherosclerosis. Here it is shown, for the first time, that its derivative trans-3,5,4'-trimethoxystilbene (TMS) may be a more potent anti-inflammatory agent than resveratrol. A comparative analysis of the inhibitory activities of related stilbenes, resveratrol, TMS and polydatin (PD), on monocyte adhesion to TNF-α-activated endothelial cells showed TMS to be the most effective, with PD being the least effective. RSV and its analogues inhibited, albeit differentially, the protein and mRNA expression levels of inducible cell adhesion molecules, ICAM-1 and VCAM-1, in cultured endothelial cells. The mechanism of the inhibitory effects of these stilbenes on endothelial cell-monocyte cell adhesion can be attributed mainly to inhibition of NF-κB pathway activation. The results demonstrate that all three investigated stilbene compounds, especially TMS, exhibit a potent inhibitory effect on inflammation-induced cell-cell adhesion, expression of adhesion molecules and activation of the NF-κB pathway.

Genistein enhances relaxation of the spontaneously hypertensive rat aorta by transactivation of epidermal growth factor receptor following binding to membrane estrogen receptors-α and activation of a G protein-coupled, endothelial nitric oxide synthase-dependent pathway.

Genistein, a phytoestrogen present in soybeans, has well established vasodilator properties. The present study examined the mechanisms involved in the rapid vascular effects of genistein. Endothelium-dependent relaxations and contractions, induced by acetylcholine and the calcium ionophore A23187, were obtained in isolated aortic rings from male spontaneously hypertensive rats (SHR). Acute exposure to genistein potentiated relaxations and reduced contractions induced by the two agonists. Both effects of genistein were not affected by transcription- and translation-inhibitors or by tyrosine kinase inhibition. The potentiation of acetylcholine and A23187-induced relaxation by genistein was inhibited by NF023 and GP antagonist-2A, selective G(i) and G(q) α-subunit antagonists, respectively, but not by NF449, a selective G(s) α-subunit antagonist. These G protein antagonists did not alter the inhibitory effect of genistein on acetylcholine and A23187-induced contractions. The potentiation of A23187-induced relaxations by genistein was not inhibited by the conventional estrogen receptor (ER) antagonist, ICI 182,780, but inhibited by the specific ER-α antagonist, MPP, and by the epidermal growth factor receptor (EGFR) inhibitor, AG1478. It was mimicked by heparin-binding epidermal growth factor (HB-EGF). Activation of EGFR and endothelial nitric oxide synthase (eNOS) was detected in genistein-treated rings using Western blotting. These data suggest that the rapid vascular actions of genistein are mediated by non-genomic pathways and are unrelated to its tyrosine kinase inhibitory properties. Furthermore, genistein transactivates EGFR through membrane ERα via G protein-coupled pathways. This in turn enhances eNOS phosphorylation and hence endothelial function in the aorta of the SHR.

Formononetin, an isoflavone, relaxes rat isolated aorta through endothelium-dependent and endothelium-independent pathways.

We evaluated the vasorelaxation effects of formononetin, an isoflavone/phytoestrogen found abundantly in Astragalus mongholicus Bunge, on rat isolated aorta and the underlying mechanisms involved. Cumulative administration of formononetin, genistein, daidzein and biochanin A relaxed phenylephrine-preconstricted aorta. Formononetin and biochanin A caused a similar magnitude of relaxation whereas daidzein was least potent. Mechanical removal of endothelium, L-NAME (100 microM) and methylene blue (10 microM) suppressed formononetin-induced relaxation. Formononetin increased endothelial nitric oxide (NO) synthase (eNOS), but not inducible NO synthase, activity with an up-regulation of eNOS mRNA and p-eNOS(Ser1177) protein expression. In endothelium-denuded preparations, formononetin-induced vasorelaxation was significantly reduced by glibenclamide (3 microM) and iberiotoxin (100 nM), and a combination of glibenclamide (3 microM) plus iberiotoxin (100 nM) abolished the relaxation. In contrast, formononetin-elicited endothelium-independent relaxation was not altered by ICI 182,780 (10 microM, an estrogen receptor (ER alpha/ER beta) antagonist) or mifepristone (10 microM, a progesterone receptor antagonist). In single aortic smooth muscle cells, formononetin caused opening of iberiotoxin-sensitive Ca(2+)-activated K(+) (BK(Ca)) channels and glibenclamide-sensitive adenosine triphosphate (ATP)-dependent K(+) (K(ATP)) channels. Thus, our results suggest that formononetin caused vascular relaxation via endothelium/NO-dependent mechanism and endothelium-independent mechanism which involves the activation of BK(Ca) and K(ATP) channels.

Epac1 mediates protein kinase A-independent mechanism of forskolin-activated intestinal chloride secretion.

Intestinal Cl- secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca2+]i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl- secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (I(sc)) measurement in response to agonist-stimulated Cl- secretion. FSK-stimulated Cl- secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 microM), and the [Ca2+]i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 microM). Both FSK and the Epac activator 8-pCPT-2'-O-Me-cAMP (50 microM) elevated [Ca2+]i, activated Ras-related protein 2, and induced Cl- secretion in intact or basolateral membrane-permeabilized T84 cells and mouse ileal sheets. The effects of 8-pCPT-2'-O-Me-cAMP were completely abolished by BAPTA-AM, but not by H89. In contrast, T84 cells with silenced Epac1 had a reduced I(sc) response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2'-O-Me-cAMP on Cl- secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2'-O-Me-cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl- conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl->Br->I- permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca2+]i signaling pathway is involved in cAMP-stimulated Cl- secretion, which is carried by a novel, previously undescribed Cl- channel.