Hepatocyte growth factor protects endothelial cells against gamma ray irradiation-induced damage

Aim： To investigate the effect of HGF on proliferation, apoptosis and migratory ability of human vascular endothelial cells against gamma ray irradiation. Methods： ECV304 cells derived from adult human umbilical vein endothelial cells （HUVEC） were irradiated with a single gamma ray dose of 20 Gy. Immunocytochemistry and Western blot analysis were used to detect c-Met protein expression and HGF/c-Met signal pathway. In the HGF-treated groups, ECV304 cells were incubated with HGF （20 or 40 ng/mL） 3 h prior to irradiation. At 48 h post- irradiation, the proliferation of ECV304 cells was measured by MTT assay, the apoptosis was assessed by flow cytometry, and the migratory ability of ECV304 cells was measured by transwell chamber assay, Results： c-Met protein is expressed in ECV304 cells and can be activated by HGF. Gamma ray irradiation inhibits proliferation and migration of ECV304 cells in a dose-dependent manner. HGF significantly promoted the proliferation of ECV304 cells, and flow cytom- etry revealed that HGF can inhibit apoptosis of ECV304 cells. Transwell chamber assay also showed that HGF increases migration activity of endothelial cells. Conclusion： HGF may afford protection to vascular endothelial cells against gamma ray irradiation-induced damage.     

Dauricine inhibits insulin-like growth factor-Ⅰ-induced hypoxia inducible factor la protein accumulation and vascular endothelial growth factor expression in human breast cancer cells

Aim： To investigate the effects of dauricine （Dau） on insulin-like growth factor-Ⅰ （IGF-Ⅰ）-induced hypoxia inducible factor 1α （HIF-1α） and vascular endothelial growth factor （VEGF） expression in human breast cancer cells （MCF-7）. Methods： Serum-starved MCF-7 cells were pretreated for 1 h with different concentrations of Dau, followed by incubation with IGF-Ⅰ for 6 h. HIF-1α and VEGF protein expression levels were analyzed by Western blotting and ELISA, respectively. HIF-1α and VEGF mRNA levels were determined by real-time PCR. In vitro angiogenesis was observed via the human umbilical vein endothelial cell （HUVEC） tube formation assay. An in vitro invasion assay on HUVECs was performed. Results： Dau significantly inhibited IGF-Ⅰ-induced HIF-1α protein expression but had no effect on HIF-1α mRNA expression. However, Dau remarkably suppressed VEGF expression at both protein and mRNA levels in response to IGF-Ⅰ. Mechanistically, Dau suppressed IGF-Ⅰ-induced HIF-1α and VEGF protein expression mainly by blocking the activation of PI-3K/AKT/mTOR signaling pathway. In addition, Dau reduced IGF-Ⅰ-induced HIF-1α protein accumulation by inhibiting its synthesis as well as by promoting its degradation. Functionally, Dau inhibited angiogenesis in vitro. Moreover, Dau had a direct effect on IGF-Ⅰ-induced invasion of HUVECs. Conclusion： Dau inhibits human breast cancer angiogenesis expression, which may provide a novel potential mechanism for by suppressing HIF-1α protein accumulation and VEGF the anticancer activities of Dau in human breast cancer.     

Response of mesenchymal stem cells to shear stress in tissueengineered vascular grafts

Aim： Recent studies have demonstrated that mesenchymal stem cells （MSCs） can differentiate into endothelial cells. The effect of shear stress on MSC differentiation is incompletely understood, and most studies have been based on two-dimensional systems. We used a model of tissue-engineered vascular MSC differentiation. grafts （TEVGs） to investigate the effects of shear stress on Methods： MSCs were isolated from canine bone marrow. The TEVG was constructed by seeding MSCs onto poly-ε- caprolactone and lactic acid （PCLA） scaffolds and subjecting them to shear stress provided by a pulsatile bioreactor for four days （two days at 1 dyne/cm^2 to 15 dyne/cm^2 and two days at 15 dyne/cm^2）. Results： Shear stress significantly increased the expression of endothelial cell markers, such as platelet-endothelial cell adhesion molecule-1 （PECAM-1）, VE-cadherin, and CD34, at both the mRNA and protein levels as compared with static control cells. Protein levels of alpha-smooth muscle actin （α-SMA） and calponin were substantially reduced in shear stresscultured cells. There was no significant change in the expression of α-SMA, smooth muscle myosin heavy chain （SMMHC） or calponin at the mRNA level. Conclusion： Shear stress upregulated the expression of endothelial cell-related markers and downregulated smooth muscle-related markers in canine MSCs. This study may serve as a basis for further investigation of the effects of shear stress on MSC differentiation in TEVGs.     

Genipin inhibits endothelial exocytosis via nitric oxide in cultured human umbilical vein endothelial cells

Aim： Exocytosis of endothelial Weibel-Palade bodies, which contain von Willebrand factor （VWF）, P-selectin and other modulators, plays an important role in both inflammation and thrombosis. The present study investigates whether genipin, an aglycon of geniposide, inhibits endothelial exocytosis. Methods： Human umbilical vein endothelial cells （HUVECs） were isolated from umbilical cords and cultured. The concentration of VWF in cell supernatants was measured using an ELISA Kit. P-selectin translocation on the cell surface was analyzed by cell surface ELISA. Cell viability was measured using a Cell Counting Kit-8. Mouse bleeding times were measured by amputating the tail tip. Western blot analysis was used to determine the amount of endothelial nitric oxide synthase （eNOS） and phospho-eNOS present. Nitric oxide （NO） was measured in the cell supernatants as nitrite using an NO Colorimetric Assay. Results： Genipin inhibited thrombin-induced VWF release and P-selectin translocation in HUVECs in a dose- and time- dependent manner. The drug had no cytotoxic effect on the cells at the same doses that were able to inhibit exocytosis. The functional study that demonstrated that genipin inhibited exocytosis in vivo also showed that genipin prolonged the mouse bleeding time. Furthermore, genipin activated eNOS phosphorylation, promoted enzyme activation and increased NO production. L-NAME, an inhibitor of NOS, reversed the inhibitory effects of genipin on endothelial exocytosis. Conclusion： Genipin inhibits endothelial exocytosis in HUVECs. The mechanism by which this compound inhibits exocytosis may be related to its ability to stimulate eNOS activation and NO production. Our findings suggest a novel antiinflammatory mechanism for genipin. This compound may represent a new treatment for inflammation and/or thrombosis in which excess endothelial exocytosis plays a pathophysiological role.     

Ginsenoside Rgl promotes endothelial progenitor cell migration and proliferation

Aim： To investigate the effect of ginsenoside Rgl on the migration, adhesion, proliferation, and VEGF expression of endothelial progenitor cells （EPCs）.
Methods： EPCs were isolated from human peripheral blood and incubated with different concentrations of ginsenoside Rgl （0.1, 0.5, 1.0, and 5.0 μmol/L） and vehicle controls. EPC migration was detected with a modified Boyden chamber assay. EPC adhesion was determined by counting adherent cells on fibronectin-coated culture dishes. EPC proliferation was analyzed with the 3.（4,5.dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay. In vitro vasculogenesis was assayed using an in vitro vasculogenesis detection kit. A VEGF-ELISA kit was used to measure the amount of VEGF protein in the cell culture medium.
Results： Ginsenoside Rgl promoted EPC adhesion, proliferation, migration and in vitro vasculogenesis in a dose- and timedependent manner. Cell cycle analysis showed that 5.0 μmol/L ofginsenoside Rgl significantly increased the EPC proliferative phase （S phase） and decreased the resting phase （G0/G1 phase）. Ginsenoside Rgl increased vascular endothelial growth factor production. Conclusion： The results indicate that ginsenoside Rgl promotes proliferation, migration, adhesion and in vitro vasculogenesis.     

Adenosine A1 receptor-mediated transactivation of the EGF receptor produces a neuroprotective effect on cortical neurons in vitro

Aim： To understand the mechanism of the transactivation of the epidermal growth factor receptor （EGFR） mediated by the adenosine A1 receptor （A1R）.
Methods： Primary cultured rat cortical neurons subjected to oxygen-glucose deprivation （OGD） and HEK293/A1R cells were treated with the A1R-specific agonist N^6-cyclopentyladenosine （CPA）. Phospho-EGFR, Akt, and ERK1/2 were observed by Western blot. An interaction between EGFR and A1R was detected using immunoprecipitation and immunocytochemistry.
Results： The A1R agonist CPA causes protein kinase B （Akt） activation and protects primary cortical neurons from oxygen-glucose deprivation （OGD） insult. A1R and EGFR co-localize in the membranes of neurons and form an immunocomplex. A1R stimulation induces significant EGFR phosphorylation via a PI3K and Src kinase signaling pathway; this stimulation provides a neuroprotective effect in cortical neurons. CPA leads to sustained phosphorylation of extracellularly regulated kinases 1 and 2 （ERK1/2） in cortical neurons, but only to transient phosphorylation in HEK 293/A1R cells. The response to the A1R agonist is mediated primarily through EGFR transactivation that is dependent on pertussis toxin （PTX）-sensitive G1 protein and metalloproteases in HEK 293/A1R.
Conclusion： A1R-mediated EGFR transactivation confers a neuroprotective effect in primary cortical neurons. PI3 kinase and Src kinase play pivotal roles in this response.     

PPAR-γ agonists inhibit TGF-β1-induced chemokine expression in human tubular epithelial cells

Aim： Peroxisome proliferator-activated receptor-γ （PPAR-γ） has a wide range of biological functions, including anti-inflammation. In this study, we investigated the inhibitory effects of PPAR-γ on transforming growth factor β1 （TGF-β1）-induced interleukin-8 （IL-8） and monocyte chemoattractant protein-1 （MCP-1） expression in renal tubular epithelial cells （HK-2）.
Methods： HK-2 cells were pretreated with 15d-PGJ2 or troglitazone （TGL） and then treated with TGF-β1. Expression of MCP-1 and IL-8 was measured using real-time PCR and ELISA.
Results： Treatment with 5 ng/mL TGF-β1 for 24 h increased both MCP-1 and IL-8 mRNA and protein levels in HK-2 cells. Both 15d-PGJ2 at 2.5 and 5 12mol/L and TGL at 2.5 12mol/L exhibited inhibitory effects on TGF-β1-induced MCP-1 expression. Additionally, 15d-PGJ2 at 2.5 and 5 μmol/L and TGL at 2.5 μmol/L inhibited TGF-β1-induced expression of IL-8.
Conclusion： PPAR-γ agonists （15d-PGJ2 and TGL） could inhibit the TGF-β1-induced expression of chemokines in HK-2 cells. Our results suggest that PPAR-γ agonists have the potential to be used as a treatment regimen to reduce inflammation in renal tubulointerstitial disease.     

The protective effect of the RAS inhibitor on diabetic patients with nephropathy in the context of VEGF suppression

Aim： The aim of the present study was to explore whether renin angiotensin system （RAS） inhibitor can reduce the production of vascular endothelium growth factor （VEGF）. Further, we sought to elucidate the correlation between VEGF level and certain clinical parameters, such as albumin excretion rate （AER）, before and after treatment with angiotensin type 1 receptor blocker. Methods： We recruited 166 type 2 diabetic patients at various stages of diabetic nephropathy （DN） and 46 healthy control subjects for a cross-sectional study. We recruited another 42 hypertensive type 2 diabetic patients with microalbuminuria for a longitudinal study involving a 6-month irbesartan treatment protocol. Urinary VEGF （uVEGF） levels were determined using ELISA. Results： In the cross-sectional study, hypertensive type 2 diabetic patients who received RAS inhibitor presented lower uVEGF levels than those who did not receive the RAS inhibitor. Statistical analysis indicated that uVEGF level was independently correlated with the AER. In the longitudinal study involving the 6-month irbesartan treatment, we demonstrated that uVEGF levels decreased significantly in patients who achieved a 50% AER reduction （remission group, n=32）. In contrast, uVEGF levels remained unchanged in patients who did not exhibit a 50% AER reduction （nonremission group, n=10）. Furthermore, the change in uVEGF was significantly correlated with the change in AER （r=0.65, P〈0.01） before and after 6 months of irbesartan treatment. This result held true even after we had adjusted for the decrease in average blood pressure. Conclusion： The protective effect of the RAS inhibitor in DN patients is associated with the suppression of VEGF. Accordingly, it may be possible to use uVEGF as a marker of DN progression. We suggest that uVEGF may be an important target for therapeutic intervention in the context of DN.     

Composite glycidyl methacrylated dextran （Dex-GMA）/gelatin nanoparticles for localized protein delivery

Aim： Localized delivery of growth factors has significant potential as a future therapeutic strategy in tissue engineering and regenerative medicine. A nanoparticle vehicle was created and evaluated in this study with the intent to deliver growth factors for periodontal regeneration.
Methods： Novel composite nanoparticles based on glycidyl methacrylate derivatized dextrans （Dex-GMA） and gelatin were fabricated by a facile method without using any organic solvents. The configurations of the resultant nanoparticles were evaluated by transmission electron microscopy, scanning electron microscopy, and atomic force microscope. Their surfaces were characterized by zeta-potential measurements, after which their properties including swelling, degradation, drug release, and cytotoxicity were also investigated using in vitro models. Results： The particle size of Dex-GMA/gelatin nanoparticles （DG-NPs） ranged from 20 to 100 nm and showed a mono-dis- perse size distribution （mean diameter 53.7 nm） and a strongly negative surface zeta potential （-20 mV）. The DG-NPs were characterized by good swelling and degradation properties in media including dextranase. The in vitro drug release studies showed that the efficient bone morphogenetic protein （BMP） release from DG-NPs was maintained for more than 12 d under degradation conditions, where more than 90% of the loaded BMP was released. No any relevant cell damage caused by DG-NPs was found in the cytotoxicity tests for a period of 24 h.
Conclusion： These combined results demonstrate that DG-NPs fulfill the basic prerequisites for growth factor delivery. With further in vivo studies, those nanoparticles may offer a promising vehicle for the delivery of active drugs to the perio- dontium.     

The antiproliferative effects of somatostatin receptor subtype 2 in breast cancer cells

Aim： Somatostatin receptor subtype 2 （SSTR2） is the principal mediator of somatostatin＇s （SST） antiproliferative effects on normal and cancer cells. Therefore, we investigated whether the enhanced expression of SSTR2 could inhibit the proliferation of tumor cells, and, if so, the mechanisms that might be involved.
Methods： SSTR2 expression levels were determined by qRToPCR in several tumor cell lines. Then, a plasmid plRES2-EGFP-SSTR2 （pSlG） was constructed and stably transfected into MCF-7 cells （MCF-7/pSIG）. After SSTR2 overexpression was identified by qRT-PCR, immunofluorescence staining and a receptor binding assay, the MCF-7/pSIG cells were analyzed by PI staining for apoptosis and cell cycle arrest was tested by flow cytometry for epidermal growth factor receptor （EGFR） expression. The EGF-stimulated proliferation of MCF-7 cells was assayed by MTT.
Results： The human breast cancer cell line MCF-7 expresses a lower level of SSTR2, thereby partly accounting for the decreased response to SST. The overexpression of SSTR2 in MCF-7 cells resulted in apoptosis, cytostasis and G1/S cell cycle arrest. Furthermore the expression of EGFR, together with EGF-stimulated proliferation, was markedly decreased in the MCF-7/pSlG cells.
Conclusion： Enhanced SSTR2 expression played an antiproliferative role in MCF-7 cells through inducing apoptosis and G1/S cell cycle arrest, and also by decreasing EGFR expression, thereby counteracting the growth-stimulating effect of EGF. Our data seem to indicate that developing a new therapeutic agent capable of upregulating SSTR expression could potentially be a way to block tumor progression.     

Allosteric activation mechanism of the cys-loop receptors

Binding of a neurotransmitter to its ionotropic receptor opens a distantly located ion channel, a process termed allosteric activation. Here we review recent advances in the molecular mechanism by which the cys-loop receptors are activated with emphasis on the best studied nicotinic acetylcholine receptors （nAChRs）. With a combination of affinity labeling, mutagenesis, electrophysiology, kinetic modeling, electron microscopy （EM）, and crystal structure analysis, the allosteric activation mechanism is emerging. Specifically, the binding domain and gating domain are interconnected by an allosteric activation network. Agonist binding induces conformational changes, resulting in the rotation of a β sheet of aminoterminal domain and outward movement of loop 2, loop F, and cys-loop, which are coupled to the M2-M3 linker to pull the channel to open. However, there are still some controversies about the movement of the channel-lining domain M2. Nine angstrom resolution EM structure of a nAChR imaged in the open state suggests that channel opening is the result of rotation of the M2 domain. In contrast, recent crystal structures of bacterial homologues of the cys-loop receptor family in apparently open state have implied an M2 tilting model with pore dilation and quaternary twist of the whole pentameric receptor. An elegant study of the nAChR using protonation scanning of M2 domain supports a similar pore dilation activation mechanism with minimal rotation of M2. This remains to be validated with other approaches including high resolution structure determination of the mammalian cys-loop receptors in the open state.     

Addictive evaluation of cholic acid-verticinone ester,a potential cough therapeutic agent with agonist action of opioid receptor

Aim： The purpose of this work was to search for potential drugs with potent antitussive and expectorant activities as well as a low toxicity, but without addictive properties. Cholic acid-verticinone ester （CA-Ver） was synthesized based on the clearly elucidated antitussive and expectorant activities of verticinone in bulbs of Fritillaria and different bile acids in Snake Bile. In our previous study, CA-Ver showed a much more potent activity than codeine phosphate. This study was carried out to investigate the central antitussive mechanism and the addictive evaluation of CA-Ver. Methods： Testing on a capsaicin-induced cough model of mice pretreated with naloxone, a non-selective opioid receptor antagonist, was performed for the observation of CA-Ver＇s central antitussive mechanism. We then took naloxone-induced withdrawal tests of mice for the judgment of CA-Ver＇s addiction. Lastly, we determined the opioid dependence of CA-Ver in the guinea pig ileum. Results： The test on the capsaicin-induced cough model showed that naloxone could block the antitussive effect of CA-Ver, suggesting the antitussive mechanism of CA-Ver was related to the central opioid receptors. The naloxone-urged withdrawal tests of the mice showed that CA-Ver was not addictive, and the test of the opioid dependence in the guinea pig ileum showed that CA-Ver had no withdrawal response. Conclusion： These findings suggested that CA-Ver deserved attention for its potent antitussive effects related to the central opioid receptors, but without addiction, and had a good development perspective.     

BMP6 reverses TGF-β1-induced changes in HK-2 cells： implications for the treatment of renal fibrosis

Aim： The aim of the study was to investigate the potential role of BMP6 in TGF-β1-mediated changes in HK-2 cells.
Methods： BMP6 was purified via heparin affinity and reverse phase liquid chromatography. The purity, specificity, and bioactivity of BMP6 were determined by SDS-PAGE, Western blot assays, and the induction of alkaline phosphatase （ALP） activity, respectively. Cell proliferation, morphology, and expression levels of α-SMA and E-cadherin were assessed by cell viability, microscopy, and Western blot assays, respectively. In addition, cell adhesion abilities were determined by counting the number of attached cells, The expression of fibroneetin, collagen Ⅳ, matrix metalloproteinases 2 （MMP-2）, and tissue inhibitors of matrix metalloproteinases 2 （TIMP-2） were analyzed using RT-PCR. MMP-2 activity was analyzed by zymography, whereas the activation of the MAPKs and Smad signaling were analyzed using Western blot assays and a reporter gene assay, respectively.
Results： Our results indicated that recombinant BMP6 induced ALP activity Jn a dose-dependent and time-course-dependent manner. Treatment with TGF-β1 reduced both the cell proliferation and the expression of E-cadherin, induced a morphological transformation, decreased the expression and activity of MMP-2, and increased the expression levelsof α-SMA, flbronectin, and TIMP-2 in HK-2 cells. All of these effects were inhibited when cells were treated with TGF-β1 in combination with rhBMP6, whereas rhBMP6 alone demonstrated no such effect. Treatment with TGF-β1, rhBMP6, or a combination of both had no effect on the expression of collagen IV. In addition, the administration of rhBMP6 prevented the enhanced adhesion behavior triggered by TGF-β1. Furthermore, the addition of rhBMP6 abrogated the JNK and Smad2/3 signaling that was activated by TGF-β1.
Conclusion： BMP6 ameliorated the TGF-131-induced changes in HK-2 cells. The suppression of TGF-β1-mediated JNK and Smad2/3 signaling activation were implicated in these effect     

Pivotal effects of phosphodiesterase inhibitors on myocyte contractility and viability in normal and ischemic hearts

Phosphodiesterases （PDEs） are enzymes that degrade cellular cAMP and cGMP and are thus essential for regulating the cyclic nucleotides. At least 11 families of PDEs have been identified, each with a distinctive structure, activity, expression, and tissue distribution. The PDE type-3, -4, and -5 （PDE3, PDE4, PDE5） are localized to specific regions of the cardiomyocyte, such as the sarcoplasmic reticulum and Z-disc, where they are likely to influence cAMP/cGMP signaling to the end effectors of contractility. Several PDE inhibitors exhibit remarkable hemodynamic and inotropic properties that may be valuable to clinical practice. In particular, PDE3 inhibitors have potent cardiotonic effects that can be used for short-term inotropic support, especially in situations where adrenergic stimulation is insufficient. Most relevant to this review, PDE inhibitors have also been found to have cytoprotective effects in the heart. For example, PDE3 inhibitors have been shown to be cardioprotective when given before ischemic attack, whereas PDE5 inhibitors, which include three widely used erectile dysfunction drugs （sildenafil, vardenafil and tadalafil）, can induce remarkable cardioprotection when administered either prior to ischemia or upon reperfusion. This article provides an overview of the current laboratory and clinical evidence, as well as the cellular mechanisms by which the inhibitors of PDE3, PDE4 and PDE5 exert their beneficial effects on normal and ischemic hearts. It seems that PDE inhibitors hold great promise as clinically applicable agents that can improve cardiac performance and cell survival under critical situations, such as ischemic heart attack, cardiopulmonary bypass surgery, and heart failure.     

Notch1 signaling inhibits growth of EC109 esophageal carcinoma cells through downmodulation of HPV18 E6/E7 gene expression

Aim： To investigate the role of the Notch1 signaling pathway in growth arrest of an esophageal carcinoma cell line （EC109） in vitro and the mechanism involved. Methods： An intracellular domain of Notch1 （ICN） was transfected into cultured EC109 cells by lipofectamine transfection. Subsequently, the proliferation of the transfected cells was measured by an MTT assay. Cell cycle distribution was analyzed by flow cytometry. Human papillomavirus type 18 （HPV18） E6/E7 mRNA expression was detected by RT-PCR, and p53 protein expression was detected by Western blot. Results： Activation of Notch1 signaling resulted in inhibition of EC109 cell proliferation with the induction of G2/M arrest, downmodulation of HPV18 E6/E7 gene expression, and upregulation of p53 expression. Conclusion：Repression of HPV18 E6/E7 expression by Notch1 signaling results in the activation of p53-mediated pathways with concomitant growth suppression of HPV18-positive EC109 cells.     

Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

Aim： Several nicotinic acetylcholine receptor （nAChR） subunits have been engineered as fluorescent protein （FP） fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR α7 subunit without compromising formation of functional receptors, Methods： A gene construct was generated to introduce yellow fluorescent protein （YFP）, in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR α7 subunit （α7Y）. SH-EP1 cells were transfected with mouse nAChR wild type α7 subunits （α7） or with α7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125I-labeled α-bungarotoxin （I-Bgt） binding, laser scanning confocal microscopy, and total internal reflectance fluorescence （TIRF） microscopy. Results： Whole-cell currents revealed that α7Y nAChRs and α7 nAChRs were functional with comparable EC50 values for the α7 nAChR-selective agonist, choline, and IC50 values for the α7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that α7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little α7Y localized to the plasma membrane, typical of α7 nAChRs. Conclusion： nAChRs composed as homooligomers of α7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of α7 nAChRs, α7Y nAChRs may be used to elucidate properties of α7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.     

The impact of extracellular and intracellular Ca2＋ on ethanol-induced smooth muscle contraction

Aim： To evaluate the impact of extracellular and intracellular Ca2＋ on contractions induced by ethanol in smooth muscle. Methods： Longitudinal smooth muscle strips were prepared from the gastric fundi of mice. The contractions of smooth muscle strips were recorded with an isometric force displacement transducer. Results： Ethanol （164 mmol/L） produced reproducible contractions in isolated gastric fundal strips of mice. Although lidocaine （50 and 100 μmol/L）, a local anesthetic agent, and hexamethonium （100 and 500 μmol/L）, a ganglionic blocking agent, failed to affect these contractions, verapamil （1-50 μmol/L） and nifedipine （1-50 pmol/L）, selective blockers of L-type Ca2＋ channels, significantly inhibited the contractile responses of ethanol. Using a Ca2＋-free medium nearly eliminated these contractions in the same tissue. Ryanodine （1-50 μmol/L） and ruthenium red （10-100 pmol/L）, selective blockers of intracellular Ca2＋ channels/ryanodine receptors; cyclopiazonic acid （CPA; 1-10 μmol/L）, a selective inhibitor of sarcoplasmic reticulum （SR） Ca2＋-ATPase; and caffeine （0.5-5 mmol/L）, a depleting agent of intracellular Ca2＋ stores, significantly inhibited the contractile responses induced by ethanol. In addition, the com- bination of caffeine （5 mmol/L） plus CPA （10 μmol/L）, and ryanodine （10 μmol/L） plus CPA （10 μmol/L）, caused further inhibition of contractions in response to ethanol. This inhibition was significantly different from those associated with caffeine, ryanodine or CPA. Furthermore the combination of caffeine （5 mmol/L）, ryanodine （10 μmol/L） and CPA（IO pmol/L） eliminated the contractions induced by ethanol in isolated gastric fundal strips of mice. Conclusion： Both extracellular and intracellular Ca2＋ may have important roles in regulating contractions induced by ethanol in the mouse gastric fundus.     

The peroxisome proliferator-activated receptor-α （PPAR-α） agonist,AVE8134, attenuates the progression of heart failure and increases survival in rats

Aim： To investigate the efficacy of the peroxisome proliferator-activated receptor-α （PPARa） agonist, AVE8134, in cellular and experimental models of cardiac dysfunction and heart failure.
Methods： In Sprague Dawley rats with permanent ligation of the left coronary artery （post-MI）, AVE8134 was compared to the PPARy agonist rosiglitazone and in a second study to the ACE inhibitor ramipril. In DOCA-salt sensitive rats, efficacy of AVE8134 on cardiac hypertrophy and fibrosis was investigated. Finally, AVE8134 was administered to old spontaneously hypertensive rats （SHR） at a nonblood pressure lowering dose with survival as endpoint. In cellular models, we studied AVE8134 on hypertrophy in rat cardiomyocytes nitric oxide signaling in human endothelial cells （HUVEC） and LDL-uptake in human MonoMac-6 cells.
Results： In post-MI rats, AVE8134 dose-dependently improved cardiac output, myocardial contractility and relaxation and reduced lung and left ventricular weight and fibrosis. In contrast, rosiglitazone exacerbated cardiac dysfunction. Treatment at AVE8134 decreased plasma proBNP and arginine and increased plasma citrulline and urinary NOx/creatinine ratio. In DOCA rats, AVE8134 prevented development of high blood pressure, myocardial hypertrophy and cardiac fibrosis, and ameliorated endothelial dysfunction. Compound treatment increased cardiac protein expression and phosphorylation of eNOS. In old SHR, treatment with a low dose of AVE8134 improved cardiac and vascular function and increased life expectancy without lowering blood pressure. AVE8134 reduced phenylephrine-induced hypertrophy in adult rat cardiomyocytes. In HUVEC, Ser-1177-eNOS phosphorylation but not eNOS expression was increased. In monocytes, AVE8134 increased the expression of CD36 and the macrophage scavenger receptor 1, resulting in enhanced uptake of oxidized LDL.
Conclusion： The PPARa agonist AVE8134 prevents post-MI myocardial hypertrophy, fibrosis and cardiac dysfunction. AVE8134 has beneficial effects against hy