We studied the Ca2+ movement induced by activation of α1A-, α1B- and α1D-adrenoceptor subtypes in transfected HEK-293 cells with the fura- probe. All these α1-AR subtypes induced both Ca2+ release and Ca2+ entry. The effect on Ca2+ release in α1b transfected HEK-293 cells was bigger than that in α1a and α1b transfected HEK-293 cells, and the effects on Ca2+ entry were the same in ala, alb and aid transfected HEK-293 cells. The Ca2+ entry was inhibited by 1mM NiSO4, but not by nifedipine. Cyclopiazonic acid (CPA) produced a biphasic Ca2+ signal response in Ca2+ medium, and only induced a transient response in Ca2+-free medium. After depletion of CPA-sensitive Ca2+ pool by 10uM CPA in Ca2+-free medium, 10uM adrenaline (Adr) still transiently increased [Ca2+]i in three different α1-adrenoceptor subtype transfected HEK-293 cells. However, after depletion of drenaline-sensitive Ca2+ pool by 10uM Adr, CPA transiently elevated [Ca2+], only in ala and aid transfected HEK-293 cells, not in a,, transfected HEK-293 cells. U73122, a phospholipase C (PLC) inhibitor, inhibited both Ca2+ release and Ca2+ entry induced by activation of α1B-AR,α1B and α1D subtypes in transfected HEK-293 cells. These results suggest that HEK-293 cell line contains two functionally separate intracellular Ca2+ pools, CPA-sensitive and Adr-sensitive pools. Activation of a,,-AR stimulates Ca2+ release from both CPA-sensitive and Adr-sensitive Ca2+ pools. α1A and α1D subtypes induce Ca2+release only from Adr-sensitive Ca2+ pool.

Patch-clamp whole-cell recording techniques were used to study ATP-induced Cl- current and the effects of Cl- channel blockers on NO release in bovine aortic endothelial cells. ATP evoked an outward rectified Cl- current with a reversal potential of 2973mV. This outward rectified Cl- current was dependent on Ca2+ influx, but not Ca2+ release. In Ca2+-free medium, ATP did not produce the Cl- current; however, subsequent addition of Ca2+ to the medium evoked an ATP-induced outward rectified Cl- current. Furosemide, glibenclamide, and DIDS inhibited ATP-activated Cl- current in a concentrationdependent manner with maximal inhibition of 8874%, 9371%, and 7973%, respectively. The IC50 values of furosemide, glibenclamide, and DIDS were 6.272.6, 29.6712.3, and 21.0713.4mM, respectively. These effects of Cl- channel blockers matched those on NO release from endothelial cells. Our data suggest that ATP-induced Ca2+ entry followed by increased [Ca2+]i activates Ca2+-activated Cl- channels which mediate NO release from endothelial cells. Drug Dev. Res. 57: 000-000, 2003

Recent growing evidence suggests that chloride (Cl-) channels are critical to the cell cycle. In cultured rat aortic vascular smooth muscle cells (VSMCs), we have previously found that Cl- channel blockers inhibit endothelin-1 (ET-1)-induced cell proliferation. The present study was designed to further identify the specific Cl- channels responsible for VSMC proliferation. Due to the lack of a specific blocker or opener of any known Cl- channels, we used the antisense strategy to investigate the potential role of ClC-3, a member of the voltage-gated Cl- channel gene family, in cell proliferation of cultured rat aortic VSMCs. With [3H]-thymidine incorporation and immunoblots, we found that ET-1-induced cell proliferation was parallel to a significant increase in the endogenous expression of ClC-3 protein. Transient transfection of rat aortic VSMCs with antisense oligonucleotide specific to ClC-3 caused an inhibition in ET-1-induced expression of ClC-3 protein and cell proliferation of VSMCs in the same concentration- and time-dependent pattern, whereas sense and missense oligonucleotides resulted in no effects on ClC-3 protein xpression and cell proliferation. These results strongly suggest that ClC-3 may be the Cl-channel involved in VSMC proliferation and thus provide compelling molecular evidence linking a specific Cl- channel to cell proliferation. The full text of this article is available at http://www.circresaha.org. (Circ Res. 2002; 91: e28-e32.)

AIM: To investigate the effects of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) on cell proliferation and apoptosis in ECV304 endothelial cells and related molecular mechanism. METHODS: MTT, Hoechst33258, TUNEL, Flow cytometry, DNA ladder, RT-PCR, Western blot, and electrophoretic mobility shift assay (EMSA) analysis were employed. RESULTS: The 15d-PGJ2 induced apoptosis in ECV304 endothelial cells in a dose-dependent manner (the percentage of apoptosis was enhanced from 10.0%±1.3% to 32.8%±1.6%), which was accompanied by inhibition of NF-κB and AP-1 DNA binding activity, down-regulation of c-myc, upregulation of Gadd45 and p53, and activation of p38 kinase. However, the expression of p21 was found no significant change. CONCLUSION: peroxisome proliferator-activated receptor gamma ligand, 15d-PGJ2, can inhibit proliferation and induce apoptosis in ECV304 endothelial cells through different mechanisms.

目的：研究随着糖尿病的发生发展，大鼠主动脉平滑肌对苯肾上腺素等激动剂收缩反应的变化及其可能机制。方法：用链尿菌素诱导糖尿病后，在第2、6、12周，取主动脉环进行实验观察。结果：苯肾上腺素的浓度依赖性收缩反应曲线，与对照相比：在第2周，低浓度时（0.01-0.03μmol·L-1）明显增加（P<0.01），最大反应无明显变化；在第6周，各浓度点均显著增加，且最大收缩反应增加约40%；然而，在第12周1）苯肾上腺素10μmol·L-1引起的最大收缩反应趋向降低（P<0.05），2）在无Ca2+液，也较对照明显减小（P<0.05），3）在无Ca2+液，在尼非地平1μmol·-L-1和苯肾上腺素10μmol·L-1存在下，复Ca2+引起的收缩在两组问的差异未见显著性，4）在正常Krebs‘液，环匹阿尼酸10μmol·L-1引起的收缩反应较对照也显著减小（P<0.001)。结论：（1）在糖尿病的第2周，平滑肌α-1肾上腺素能受体的敏感性增加。（2）糖尿病大鼠主动脉平滑肌对苯肾上腺素收缩反应的异常变化，与通过电压依赖性钙通道的Ca+内流大小、胞内功能性Ca2+池大小及其胞内Ca2+池耗竭后所引起的充电性内流变化密切相关。