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.

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.

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.

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.

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.

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.

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.

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.

The role of prostaglandin E and thromboxane-prostanoid receptors in the response to prostaglandin E2 in the aorta of Wistar Kyoto rats and spontaneously hypertensive rats.

AIMS: The present study examined the hypothesis that prostaglandin E2 (PGE2) through activation of prostaglandin E (EP) receptor contributes to endothelium-dependent contractions. METHODS AND RESULTS: Western blotting revealed that the protein expression of EP1 receptor was significantly down-regulated in the aorta of the spontaneously hypertensive rat (SHR), but there was no significant difference in the expression of EP2, EP4, and total EP3 receptors between preparations of Wistar Kyoto rats (WKY) and SHR. Isometric tension studies showed that low concentrations of PGE2 caused endothelium-dependent relaxations in WKY but not in aortas of the SHR. High concentrations of PGE2 evoked contractions predominately through the activation of thromboxane-prostanoid (TP) receptors in the WKY, but involves the dual activation EP and TP receptors in the SHR. SQ29,548, BAYu3405 and Terutroban (TP receptor antagonists), and AH6809 (non-selective EP receptor antagonist) abolished, while SC19220 (preferential EP1 receptor antagonist) did not inhibit endothelium-dependent contractions. Both SC19220 and AH6809 significantly inhibited contractions to U46619 (TP receptor agonist). CONCLUSION: The present study demonstrates that the contraction caused by PGE2 in the SHR aorta is dependent on the activation of EP1 and TP receptors, but that endothelium-dependent contractions do not require the former. Thus, PGE2 is unlikely to be an endothelium-derived contracting factor in this artery. The ability of AH6809 to inhibit endothelium-dependent contractions can be attributed to its partial antagonism at TP receptors. Nevertheless, the impairment of PGE2-mediated relaxation may contribute to endothelial dysfunction in the aorta of the SHR.

Inhibitory effects of epoxyeicosatrienoic acids on volume-activated chloride channels in rat mesenteric arterial smooth muscle.

Epoxyeicosatrienoic acids (EETs) are synthesized from arachidonic acid by cytochrome P450 epoxygenases in endothelial cells. It has previously been shown that EETs activate K(+) channels, which are important for the hyperpolarization and dilation of blood vessels. However, the effects of EETs on other ion channels have been less well studied. We investigated the effects of EETs on volume-activated Cl(-) channels (VACCs) in rat mesenteric arterial smooth muscle cells. Whole-cell patch clamp recording demonstrated that hypotonic solution and guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) induced a 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)- and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS)-sensitive VACC current in the primary cultured rat mesenteric arterial smooth muscle cells. The VACC current was inhibited by EETs and the order of potency was 8,9-EET>5,6-EET>11,12-EET>14,15-EET. The inhibitory effects of EETs could be reversed by 14,15 epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, an EET analog), Rp-cGMP and KT-5823 (protein kinase G inhibitors). Interestingly, the inhibitory effects of EETs on VACCs were not influenced by Rp-cAMP (a protein kinase A antagonist) but it could be abolished by NF-449 (a Gs protein inhibitor), indicating the involvement of cAMP but not protein kinase A. In conclusion, our results demonstrate that EETs inhibit VACCs in rat mesenteric arterial smooth muscle cells through a cGMP-dependent pathway, which is probably due to the cross-activation by cAMP. This mechanism may be involved in the regulation of cell volume and membrane potential.

Inhibition of human equilibrative nucleoside transporters by dihydropyridine-type calcium channel antagonists.

Dihydropyridine-type calcium channel antagonists, in addition to having a vasodilatory effect, are known to inhibit cellular uptake of nucleosides such as adenosine. However, the nucleoside transporter subtypes involved and the mechanism by which this occurs are not known. Therefore, we have studied the inhibitory effects of dihydropyridines on both human equilibrative nucleoside transporters, hENT-1 and hENT-2, which are the major transporters mediating nucleoside transport in most tissues. Among the dihydropyridines tested, nimodipine proved to be the most potent inhibitor of hENT-1, with an IC(50) value of 60+/-31 muM, whereas nifedipine, nicardipine, nitrendipine, and felodipine exhibited 100-fold less effective inhibitory activity. Nifedipine, nitrendipine, and nimodipine inhibited hENT-2 with IC(50) values in the micromolar range; however, nicardipine and felodipine had no significant effect on hENT-2. Removal of the 4-aryl ring or changing the nitro group at the 4-aryl ring proved not to be detrimental to the inhibitory effects of dihydropyridines on hENT-1, but resulted in a drastic decrease in their inhibitory effects on hENT-2. Kinetic studies revealed that nimodipine and nifedipine reduced V(max) of [(3)H]uridine transport without affecting K(m). The inhibitory effects of nimodipine and nifedipine could be washed out. In addition, nimodipine and nifedipine inhibited the rate of NBTGR-induced dissociation of [(3)H]NBMPR from hENT-1 cell membrane. We conclude that dihydropyridines are non-competitive inhibitors of hENT-1 and hENT-2, probably working through reversible interactions with the allosteric sites. The inhibitory potencies of dihydropyridines may be associated with the structure of the 4-aryl ring, as well as the ester groups at the C-3 and C-5 positions.

Comparison of vascular relaxation, lipolysis and glucose uptake by peroxisome proliferator-activated receptor-gamma activation in +db/+m and +db/+db mice.

In this study, we determined the in vitro effect of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activation on the aortic relaxation, lipolysis and insulin-induced [(3)H]-glucose uptake of the abdominal (omental) adipocytes of the non-diabetic (+db/+m) and obese/diabetic (+db/+db) mice. The expression of PPAR-gamma (mRNA and protein) in aorta and adipose tissues was evaluated and compared. Cumulative application of ciglitazone, pioglitazone and troglitazone (PPAR-gamma agonists) caused a concentration-dependent aortic relaxation (sensitive to 2-chloro-5-nitro-N-phenylbenzamide (GW9662) (1 microM, a selective PPAR-gamma antagonist) and N(omega)-nitro-l-arginine methyl ester (l-NAME) (20 microM, a nitric oxide synthase inhibitor)) with a maximum relaxation of approximately 30% (3 microM) in +db/+m mice, whereas no relaxation was observed in +db/+db mice. All PPAR-gamma agonists examined did not alter the basal lipolysis of both species, but forskolin caused a concentration-dependent lipolysis, with a greater magnitude observed in +db/+m mice. Insulin (0.1 and 1 microM) caused an enhancement of [(3)H]-glucose uptake into adipocytes with a greater magnitude in +db/+m mice. In contrast, none of the PPAR-gamma agonists tested (0.1, 1 and 10 microM) altered the basal and the insulin (0.1 microM)-induced [(3)H]-glucose uptake into adipocytes of both species. In addition, there was no difference in PPAR-gamma expression (mRNA and protein) in the aorta and adipose tissues between the species. In conclusion, our results demonstrate that PPAR-gamma is present in the abdominal (omental) adipose tissue and thoracic aorta. An acute activation of PPAR-gamma produced a small ( approximately 30%) aortic relaxation (nitric oxide/endothelium-dependent) of +db/+m mice. However, all PPAR-gamma agonists examined have no acute effect on lipolysis and the insulin-induced glucose uptake into adipocytes of both +db/+m and +db/+db mice.

The role of mitochondrial and plasma membrane nucleoside transporters in drug toxicity.

Many anticancer and antiviral drugs are nucleoside analogues, which interfere with nucleotide metabolism and DNA replication to produce pharmacological effects. Clinical efficacy and toxicity of nucleoside drugs are closely associated with nucleoside transporters because they mediate the transport of nucleoside drugs across biological membranes. Two families of human nucleoside transporters (equilibrative nucleoside transporters and concentrative nucleoside transporters) have been extensively studied for several decades. They are widely distributed, from the plasma membrane to membranes of organelles such as mitochondria, and the distribution differs in different tissues. In addition, they have different specificities to nucleoside drugs. The characteristics of equilibrative and concentrative nucleoside transporters affect the therapeutic outcomes achieved with anticancer and antiviral nucleoside drugs. In this review, an overview of the role of mitochondrial and plasma membrane nucleoside transporters in nucleoside drug toxicity is provided. Rational design and therapeutic application of nucleoside analogues are also discussed.

Cell-cell interaction underlies formation of fluid in the male reproductive tract of the rat.

The epithelia lining the epididymides of many species consists of several cell types. We have provided evidence that the basal cells are essential to the integrated functions of the epithelium. Basal cells, but not principal cells, and other cells in the epididymis express TRPC3 and COX-1. We have isolated basal cells from intact rat epididymis using antibody-coated Dynabeads and subjected them to whole-cell patch-clamp measurement of nonselective cation channel activity, a feature of TRPC3 protein, and Fluo-3 fluorescence measurement of intracellular Ca2+ concentration. The results show that a nonselective cation current blockable by La3+ (0.1 mM), Gd3+ (0.1 mM), or SKF96365 (20 microM) could be activated by lysylbradykinin (200 nM). In cells loaded with Fluo-3, addition of lysylbradykinin (100 nM) caused a sustained increase of intracellular Ca2+. This effect was blocked by Gd3+ (0.1 mM) or SKF96365 (20 microM) and was not observed in Fluo-3-loaded principal cells. Stimulation of basal cell/principal cell cocultures with lysylbradykinin (200 nM) evoked in principal cells a current with CFTR-Cl- channel characteristics. Isolated principal cells in the absence of basal cells did not respond to lysylbradykinin but responded to PGE2 (100 nM) with activation of a CFTR-like current. Basal cells, but not principal cells, released prostaglandin E2 when stimulated with lysylbradykinin (100 nM). The release was blocked by SKF96365 (20 microM) and BAPTA-AM (0.05 or 0.1 mM). Confluent cell monolayers harvested from a mixture of disaggregated principal cells and basal cells responded to lysylbradykinin (100 nM) and PGE2 (500 nM) with an increase in electrogenic anion secretion. The former response was dependent on prostaglandin synthesis as piroxicam blocked the response. However, cell cultures obtained from principal cells alone responded to PGE2 but not to bradykinin. These results support the notion that basal cells regulate principal cells through a Ca2+ and COX signaling pathway.

Chronic hypoxia-induced upregulation of store-operated and receptor-operated Ca2+ channels in pulmonary arterial smooth muscle cells: a novel mechanism of hypoxic pulmonary hypertension.

Chronic hypoxic pulmonary hypertension is associated with profound vascular remodeling and alterations in Ca(2+) homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Recent studies show that transient receptor potential (TRPC) genes, which encode store-operated and receptor-operated cation channels, play important roles in Ca(2+) regulation and cell proliferation. However, the influence of chronic hypoxia on TRPC channels has not been determined. Here we compared TRPC expression, and store- and receptor-operated Ca(2+) entries in PASMCs of normoxic and chronic hypoxic rats. Reverse-transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining showed consistently that TRPC1, TRPC3, and TRPC6 were expressed in intralobar pulmonary arteries (PAs) and PASMCs. Application of 1-oleoyl-2-acetyl-sn-glycerol (OAG) to directly activate receptor-operated channels, or thapsigargin to deplete Ca(2+) stores, caused dramatic increase in cation entry measured by Mn(2+) quenching of fura-2 and by Ca(2+) transients. OAG-induced responses were approximately 700-fold more resistant to La(3+) inhibition than thapsigargin-induced responses. siRNA knockdown of TRPC1 and TRPC6 specifically attenuated thapsigargin- and OAG-induced cation entries, respectively, indicating that TRPC1 mediates store-operated entry and TRPC6 mediates receptor-operated entry. In hypoxic PAs, there were 2- to 3-fold increases in TRPC1 and TRPC6 expression. They were accompanied by significant increases in basal, OAG-induced, and thapsigargin-induced cation entries in hypoxic PASMCs. Moreover, removal of Ca(2+) or inhibition of store-operated Ca(2+) entry with La(3+) and SK&F-96365 reversed the elevated basal [Ca(2+)](i) in PASMCs and vascular tone in PAs of chronic hypoxic animals, but nifedipine had minimal effects. Our results for the first time to our knowledge show that both store- and receptor-operated channels of PASMCs are upregulated by chronic hypoxia and contribute to the enhanced vascular tone in hypoxic pulmonary hypertension.

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.

Effect of thiazolidinediones on equilibrative nucleoside transporter-1 in human aortic smooth muscle cells.

Thiazolidinediones are a new class of anti-diabetic agents which increase insulin sensitivity by binding to the peroxisome proliferator-activated receptor gamma (PPAR(gamma)) and stimulating the expression of insulin-responsive genes involved in glucose and lipid metabolism. These drugs also have vasodilatory and anti-proliferative effects on vascular smooth muscle cells. However the mechanisms for these actions are not fully understood. Adenosine is a vasodilator and a substrate of equilibrative nucleoside transporters (ENT). The present study studied the effects of three thiazolidinediones, troglitazone, pioglitazone and ciglitazone, on ENT1 in the human aortic smooth muscle cells (HASMCs). Although incubating HASMCs for 48h with thiazolidinediones had no effect on ENT1 mRNA and protein levels, troglitazone acutely inhibited [3H]adenosine uptake and [3H]NBMPR binding of HASMCs with IC50 values of 2.35+/-0.35 and 3.99+/-0.57microM, respectively. The effect of troglitazone on ENT1 was PPAR(gamma)-independent and kinetic studies revealed that troglitazone was a competitive inhibitor of ENT1. In contrast, pioglitazone and ciglitazone had minimal effects on [3H]adenosine uptake by HASMCs. Troglitazone differs from pioglitazone and ciglitazone in that its side-chain contains a Vitamin E moiety. The difference in structure of troglitazone did not account for its inhibitory effect on ENT1 because Vitamin E did not inhibit [3H]adenosine uptake by HASMCs. Using the nucleoside transporter deficient PK15NTD cells stably expressing ENT1 and ENT2, it was found that troglitazone inhibited ENT1 but had no effect on ENT2. From these results, it is suggested that troglitazone may enhance the vasodilatory effect of adenosine by inhibiting ENT1. Pharmacologically, troglitazone is a novel inhibitor of ENT1.

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.