ANTI-HUMAN PLATELET TETRASPANIN(CD9)MONOCLONAL ANTIBODIES INDUCE PLATELET INTEGRIN αbβ3 ACTIVATION IN A Fc RECEPTOR INDEPENDENT FASHION

This study characterized the activation of platelet integrin α bβ3 induced by two anti human platelet tetraspanin monoclonal antibodies(mAbs),HI117 and SJ9A4. Methods.Using 125 I labeled human fibrinogen(Fg),specific Fg binding to human platelets induced by HI117 and SJ9A4 was measured as indication of activation of platelet integrin αbβ3 by the two mAbs. Results.HI117 and SJ9A4(10μg/ml and 20μg/ml) induced evident specific Fg binding to human platelets,suggesting that the two mAbs evoked activation of platelet integrin αbβ3.Further study indicated that HI117 and SJ9A4 induced integrin αⅡbβ3 activation independent of platelet Fc receptors, and that HI117 and SJ9A4 induced integrin αbβ3 activation was inhibited by sphingosing, aspirin, apyrase, and/or PGI2. Conclusion.The anti platelet tetraspanin(CD9)mAbs,HI117 and SJ9A4, can induce platelet integrin αⅡbβ3 activation independent of Fc receptors.Three signaling pathways,i.e.thromboxane,secreted ADP, and cAMP pathways may be involved in the process,with protein kinase C activation presumably being the common step of the three pathways.

Angiotensin Ⅱ type 1 receptor modulation of L-type calcium currents in guinea-pig ventricular cells

Objective To study the cellular mechanism of the effect of Ang Ⅱ on ICa,L in single guinea-pig ventricular cells by using losartan and 1-(5-Isoquinolinylsulfonyl)-2-Methyl-Piperazine (H-7) as the Ang Ⅱ type 1 receptor (AT1) inhibitor and protein kinase C inhibitor, respectively.Methods Patch clamp techniques were used to study the cellular mechanism of the effect of Ang Ⅱ on ICa,L in single guinea-pig ventricular cells.Results In the whole cell patch clamp recording model, Ang Ⅱ stimulated ICa,L in a concentration dependent manner; the maximal effect was obtained at 100?nmol/L (n=9). At 30?nmol/L, Ang Ⅱ stimulated peak ICa,L from 11.3±0.6?pA/pF to 15.3±0.6?pA/pF (at +10?mV, n=9, P<0.05). 100?nmol/L Losartan, a specific AT1 receptor inhibitor, had no effect on ICa,L (n=9), but the effect of Ang Ⅱ on ICa,L was inhibited by 100?nmol/L Losartan. Ang Ⅱ on ICa,L was also inhibited by 20?μmol/L H-7, a specific protein kinase C inhibitor, whereas H-7 alone has no effect on ICa,L (n=9).Conclusion Ang Ⅱ stimulates ICa,L in guinea-pig ventricular cells by binding to AT1 through a transduction pathway involving protein kinase C.

Isoflurane induces expression of vascular endothelial growth factor through activating protein kinase C in myocardial cells

Objective： Vascular endothelial growth factor （VEGF） plays important roles in establishing collateral circulation of ischemic myocardium. This study aimed to investigate the effect of isoflurane on VEGF expression and the potential intracellular signal transduction pathway in cultured rat myocardial cells in order to further reveal the molecular mechanism of myocardial preservation of isoflurane. Methods： Primary myocardial cells of Sprague-Dawley rats were isolated and cultured. They were divided randomly into control group, isoflurane group, protein kinase C （PKC） inhibitor group and PKC inhibitor＋isoflurane group where cells were respectively incubated without any treatment, treated by 0.5, 1.0 and 1.5 minimum alveolar concentration （MAC） of isoflurane for 6 hours, by PKC inhibitor calphostin C at a final concentration of 50 nmol/L and by 50 nmol/L calphosfin C＋ 1.0 MAC isoflurane for 6 hours. VEGF expression was detected by enzyme-linked immunosorbent assay （ELISA） and the expression levels of PKC isoforms were determined by Western immunoblotting method. Results： Isoflurane increased the VEGF expression in myocardial cells in a dose-dependent way. VEGF levels were significantly higher in 1.0 and 1.5 MAC isoflurane groups than in the control group （both P〈0.01）. The effect of isoflurane on upregulating VEGF expression was blocked by PKC inhibitor calphostin C （P〈0.01）, but calphostin C did not alter VEGF expression （P〉0.05）. Isoflurane induced the activation and translocation of PKC Immunoblotting analysis revealed that the immunoreactivity of PKC ε increased significantly in the membrane fractions and deceased significantly in the kytoplasm fractions for cells treated with 1.0 MAC isoflurane as compared with the untreated cells, but not of PKC a, PKCα and PKCζ （P〈0.01）. Conclusion： Isoflurane induces myocardial cells to release VEGF through activating PKCε from the endochylema to the cytomembrane, suggesting a possible novel mechanism of isoflurane protecting myocardial cells.

Protein kinase C-δ and -β coordinate flow-induced directionality and deformation of migratory human blood T-lymphocytes

T-tym phocyte migration under flow is critical for immune responses, but the mechanisms by which flow modulates the migratory beha- viors of T-lymphocytes remain unclear. Human peripheral blood T-lymphocytes （PBTLs）, when stimulated with phorboL 12-myristate 13-acetate （PMA）, stretched their ceU bodies dramatically and moved alongthe flow direction. In contrast, stromal ceil-derived factor- lα-stimulated PBTI.s deformed and migrated in a random manner. Here we elucidated the molecular mechanisms underlying flow- induced directionality and deformation of PMA-stimulated PBTLs. PMA primed PBTLs for polarization under flow, with protein kinase C （PKC）-δ enriched in the leading edge, PKC-β1 in the microtubuie organizing center, and PKC-1311 in the uropod and peripheral region. PKC-δ regulated cell protrusions in the leading edge through Tiaml/Racl/caLmoduUn, whereas PKC-β regulated RhoA/Rho- associated kinase activity and microtubule stability to modulate uropod contractility and detachment. Our findings indicate that PKC-δ and -β coordinate in the cell Leading edge and uropod, respectively, to modu｜ate the directionality and deformability of migratory T-Lymphocytes under flow.

针刺对肝郁化火型不寐患者不同神经递质表达的影响

目的:比较针刺与药物曲唑酮治疗肝郁化火型不寐患者的疗效差异及其对不同神经递质表达的影响。方法:将70例确诊为肝郁化火型不寐患者随机分为观察组和对照组,每组35例。观察组采用针刺疗法,穴取神门、百会、印堂、合谷、太冲等,每次留针20min,每日1次,2周为一疗程,治疗2个疗程;对照组口服曲唑酮治疗,100mg,每日1次,2周为一疗程,治疗2个疗程。比较两组患者治疗前后匹兹堡睡眠质量指数(PSQI)、Asberg副作用量表评分(SERS)、5-羟色胺(5-HT)、去甲肾上腺素(NE)等神经递质含量以及外周血蛋白激酶C(PKC)、脑源性神经营养因子(BDNF)基因表达水平。结果:两组治疗后PSQI、SERS评分均较治疗前明显降低(均P<0.05),治疗后,观察组患者PSQI评分、SERS评分明显低于对照组(均P<0.05);两组治疗后血清NE含量、PKC水平明显降低,血清5-HT含量、BDNF mRNA表达明显增高(均P<0.05);观察组NE含量、PKC水平明显低于对照组(P<0.05);观察组血清5-HT含量及BDNF mRNA表达明显高于对照组(均P<0.05)。结论:针刺疗法能够改善肝郁化火型不寐患者的睡眠质量,同时降低患者血清NE,提高5-HT,增加BDNF表达,其作用均优于西药曲唑酮,是肝郁化火型不寐的有效治疗手段之一。