Endothelium-independent vasorelaxant effect of a Berberis orthobotrys root extract via inhibition of phosphodiesterases in the porcine coronary artery.

BACKGROUND: Berberis orthobotrys Bien ex Aitch. (Berberidaceae) is a plant indigenous of Pakistan that is locally used for the treatment of hypertension. HYPOTHESIS: This study evaluated the vasoactive properties of a Berberis orthobotrys root extract and its fractions, and investigated the role of the endothelium and the underlying mechanism. STUDY DESIGN: An aqueous methanolic extract of Berberis orthobotrys roots was prepared and submitted to a multi-step liquid-liquid fractionation with solvents of increasing polarity. Vascular reactivity of the different fractions was assessed using porcine coronary artery rings either with or without endothelium, and in the presence or absence of specific pharmacological tools. The ability of Berberis orthobotrys extracts to affect phosphodiesterase (PDE) activity was evaluated using a radioenzymatic method and purified phosphodiesterases. RESULTS: The aqueous methanol extract induced similar relaxations in coronary artery rings with and without endothelium, and, amongst the three derived preparations, the butanol fraction (BFBO) was slightly but significantly more effective than the ethyl acetate fraction and the aqueous residue in rings without endothelium. Analysis of the butanol fraction (BFBO) by LC-ELSD-MS indicated the presence of four major isoquinoline alkaloids including berberine. BFBO significantly potentiated the relaxations induced by cyclic GMP- and cyclic AMP-dependent relaxing agonists, and inhibited contractions to KCl, CaCl2, and U46619 in endothelium denuded rings. In contrast, BFBO did not affect relaxations to endothelium-dependent vasodilators. BFBO concentration-dependently inhibited the cyclic GMP-hydrolyzing activity of basal PDE1, calmodulin-activated PDE1 and PDE5, and of cyclic AMP-hydrolyzing activity of PDE3 and PDE4 with IC50 values ranging from 40 to 130µg/ml. CONCLUSION: The butanol fraction of the aqueous methanol extract of Berberis orthobotrys roots induced pronounced endothelium-independent relaxations and inhibited contractile responses by acting directly at the vascular smooth muscle in the coronary artery. Moreover, BFBO potentiated relaxations induced by both cyclic GMP- and cyclic AMP-dependent vasodilators most likely due to its ability to inhibit several vascular PDEs, and in particular PDE4 and PDE5.

The N-terminal region of the dopamine D2 receptor, a rhodopsin-like GPCR, regulates correct integration into the plasma membrane and endocytic routes.

BACKGROUND AND PURPOSE: Functional roles of the N-terminal region of rhodopsin-like GPCR family remain unclear. Using dopamine D(2) and D(3) receptors as a model system, we probed the roles of the N-terminal region in the signalling, intracellular trafficking of receptor proteins, and explored the critical factors that determine the functionality of the N-terminal region. EXPERIMENTAL APPROACH: The N-terminal region of the D(2) receptor was gradually shortened or switched with that of the D(3) receptor or a non-specific sequence (FLAG), or potential N-terminal glycosylation sites were mutated. Effects of these manipulations on surface expression, internalization, post-endocytic behaviours and signalling were determined. KEY RESULTS: Shortening the N-terminal region of the D(2) receptor enhanced receptor internalization and impaired surface expression and signalling; ligand binding, desensitization and down-regulation were not affected but their association with a particular microdomain, caveolae, was disrupted. Replacement of critical residues within the N-terminal region with the FLAG epitope failed to restore surface expression but partially restored the altered internalization and signalling. When the N-terminal regions were switched between D(2) and D(3) receptors, cell surface expression pattern of each receptor was switched. Mutations of potential N-terminal glycosylation sites inhibited surface expression but enhanced internalization of D(2) receptors. CONCLUSIONS AND IMPLICATIONS: Shortening of N-terminus or mutation of glycosylation sites located within the N-terminus enhanced receptor internalization but impaired the surface expression of D(2) receptors. The N-terminal region of the D(2) receptor, in a sequence-specific manner, controls the receptor's conformation and integration into the plasma membrane, which determine its subcellular localization, intracellular trafficking and signalling properties.

Delphinidin and cyanidin inhibit PDGF(AB)-induced VEGF release in vascular smooth muscle cells by preventing activation of p38 MAPK and JNK.

BACKGROUND AND PURPOSE: Red wine polyphenols (RWPs) inhibit the expression of vascular endothelial growth factor (VEGF), a major pro-angiogenic and pro-atherosclerotic factor, in vascular smooth muscle cells (VSMCs). The aim of this study was to identify which red wine polyphenols were inhibitory and to determine the mechanism underlying the inhibitory effects. EXPERIMENTAL APPROACH: Release of VEGF stimulated by platelet derived growth factor(AB) (PDGF(AB)), from human aortic VSMCs was measured by immunoassay and phosphorylation of kinases by Western blot analysis. The direct antioxidant properties of polyphenols were determined by electron paramagnetic resonance and the cellular formation of reactive oxygen species (ROS) by dichlorofluorescein. KEY RESULTS: The inhibitory effect of RWPs on PDGF(AB)-induced release of VEGF was mimicked by delphinidin but not by quercetin, catechins, resveratrol, gallic acid or caffeic acid. In the anthocyanin class, not only delphinidin but also cyanidin prevented VEGF release whereas malvidin and peonidin were without effect. RWPs, delphinidin and cyanidin directly scavenged ROS and prevented the PDGF(AB)-induced formation of ROS in VSMCs. Malvidin and peonidin did not scavenge ROS but prevented the cellular formation of ROS. Although the p38 MAPK, ERK1/2 and JNK pathways have been involved in the PDGF(AB)-induced expression of VEGF, in our experiments, only phosphorylation of p38 MAPK and JNK was inhibited by RWPs, delphinidin and cyanidin. CONCLUSIONS AND IMPLICATIONS: Anthocyanins presenting a hydroxyl residue at position 3' are able to inhibit PDGF(AB)-induced VEGF expression by preventing activation of p38 MAPK and JNK in VSMCs.

Anti-allergic and anti-inflammatory triterpenes from the herb of Prunella vulgaris.

The activity-guided fractionation of the extract of the herb of Prunella vulgaris (Labiatae) led to the isolation of four triterpenes, i.e., betulinic acid, ursolic acid, 2 alpha,3 alpha-dihydroxyurs-12-en-28-oic acid, and 2 alpha-hydroxyursolic acid. One of these compounds, 2 alpha,3 alpha-dihydroxyursolic acid, demonstrated significant inhibition on the release of beta-hexosaminidase from the cultured RBL-2H3 cells in a dose-dependent manner; the IC50 value was calculated to be 57 microM. When the isolated compounds were tested for their effects on the production of nitric oxide from cultured murine macrophages, RAW 264.7 cells, ursolic acid and 2 alpha-hydroxyursolic acid exhibited strong inhibitory activities (IC50 values, 17 and 27 microM, respectively).

Yomogin inhibits the degranulation of mast cells and the production of the nitric oxide in activated RAW 264.7 cells.

Yomogin (1), an eudesmane sesquiterpene isolated from Artemisia princeps, was tested for the effects on the degranulation process of cultured mast cells and on the nitric oxide production in LPS-activated murine macrophages. It demonstrated a significant inhibition on the release of beta-hexosaminidase from the cultured RBL-2H3 cells in a dose-dependent manner (IC50 value, 50 microM) and also exhibited a potent inhibition on the nitric oxide production from the activated RAW264.7 cells (IC50 value, 3 microM).

Inhibition of mast cell degranulation by tanshinones from the roots of Salvia miltiorrhiza.

The activity-guided fractionation of the extract of the root of Salvia miltiorrhiza B. (Labiatae, Tanshen), led to the isolation of four active components responsible for the anti-allergic activity in vitro. Among them, 15,16-dihydrotanshinone-I and cryptotanshinone demonstrated significant inhibition of the release of beta-hexosaminidase from cultured RBL-2H3 cells in a dose-dependent manner; the IC50 values were calculated as 16 and 36 microM, respectively.

Activation of Fc epsilon RI inhibits the pyruvate kinase through direct interaction with the gamma-chain.

The downstream signaling components of high-affinity IgE receptor (FcepsilonRI) were studied using yeast two-hybrid screening of the cDNA library constructed from RBL-2H3 cells. The cytoplasmic part of the gamma-chain but not that of the beta-chain was found to interact with pyruvate kinase in the yeast. The in-vitro-translated pyruvate kinase also specifically interacted with the bacterially expressed glutathione-S transferase fusion protein of the cytoplasmic part of the gamma-chain. When RBL-2H3 cells were challenged with antigen, the activity of pyruvate kinase gradually decreased, reaching the minimum activity around 5 min after the activation, and then slowly returned to the normal level. The dose-response curve (antigen vs. pyruvate kinase activity) plotted at 5 min after stimulation showed that the pyruvate kinase was dose-dependently inhibited and the maximum inhibition was reached at the concentration of 0.1 microgram/ml of antigen. Direct interaction between FcepsilonRI and pyruvate kinase was also demonstrated by co-immunoprecipitation in RBL-2H3 cells. These data suggest that pyruvate kinase is functionally linked with FcepsilonRI and might exert an important role in controlling cellular functions following the activation of FcepsilonRI.

Studies of structure activity relationship of flavonoids for the anti-allergic actions.

The structure activity relationship of flavonoids for anti-allergic actions was studied by determining the IC50 values for the degranulation. The hexosaminidase release from RBL-2H3 cells (degranulation marker) was employed as an estimate for the anti-allergic actions. Among 22 flavonoid compounds tested, luteolin, apigenin, diosmetin, fisetin, and quercetin were found to be most active with IC50 values less than 10 microM.