Department of Surgery, The Chinese University of Hong Kong, Hong Kong and TEDA International Cardiovascular Hospital, Medical College, Nankai University, Tianjin, China.
Cardiovasc Res. 2011 Aug 1;91(3):472-82. doi: 10.1093/cvr/cvr102. Epub 2011 Apr 14.
Intracellular Ca(2+) (Ca(2+)) regulation in endothelial cells depends on transient receptor potential channels (TRPs), and the role of canonical TRPs (TRPCs) during hypoxia-reoxygenation (H-R) is unclear. We hypothesized that TRPC3 contributes to endothelial nitric oxide (NO) release and that H-R may reduce TRPC3 activity and the associated endothelial function, including NO release.
Measurements of Ca(2+) and patch-clamp study in primary cultured porcine coronary endothelial cells, measurements of NO and endothelium-dependent relaxation in porcine coronary arteries, and RT-PCR and western blot were conducted. Pre-treatment with SKF96365 (an inhibitor of TRPCs) or the selective TRPC3 inhibitor Pyr3 significantly decreased bradykinin-induced vasorelaxation. One hour of hypoxia followed by reoxygenation significantly reduced the vasorelaxation (70.3 ± 6.4 vs. 88.9 ± 3.5%) and NO concentration (24.0 ± 1.3 vs. 45.2 ± 2.8 nmol/L), and they were restored by pre-incubation with the TRPC3/6/7 activator 1-oleoyl-2-acetyl-sn-glycerol (96.4 ± 1.8% and 41.1 ± 4.7 nmol/L, respectively). In porcine coronary endothelial cells, H-R inhibited bradykinin-activated membrane current (8.6 ± 0.4 vs. 14.0 ± 1.5 pA/pF) and Pyr3-sensitive TRPC3 current (3.8 ± 0.3 vs. 6.3 ± 0.6 pA/pF; P< 0.01). H-R also inhibited bradykinin-induced Ca(2+) influx and the Ca(2+) influx via TRPC3. Cell surface expression of TRPC3 was decreased after H-R.
We have, for the first time, demonstrated that Ca(2+) entry via endothelial TRPC3 contributes to NO release and have revealed that H-R is associated with inhibition of TRPC3 activity. Inhibition of channel trafficking to the cell surface is involved in the underlying mechanism of the decrease of TRPC3 current and the reduction in Ca(2+) entry through TRPC3 during H-R. This study suggests that TRPC3 may have the potential to be a new target for endothelial protection during H-R.
细胞内钙离子(Ca(2+))的调节依赖于瞬时受体电位通道(TRPs)，而在缺氧复氧(H-R)期间，经典 TRP (TRPCs)的作用尚不清楚。我们假设 TRPC3 有助于内皮一氧化氮(NO)的释放，并且 H-R 可能会降低 TRPC3 活性以及相关的内皮功能，包括 NO 的释放。
在原代培养的猪冠状动脉内皮细胞中进行Ca(2+)的测量和膜片钳研究，在猪冠状动脉中进行 NO 和内皮依赖性舒张的测量，以及 RT-PCR 和 Western blot 分析。用 SKF96365(TRPC 抑制剂)或选择性 TRPC3 抑制剂 Pyr3 预处理显著降低了缓激肽诱导的血管舒张。1 小时缺氧后再复氧显著降低了血管舒张(70.3±6.4% vs. 88.9±3.5%)和 NO 浓度(24.0±1.3 vs. 45.2±2.8 nmol/L)，并用 TRPC3/6/7 激活剂 1-油酰基-2-乙酰基-sn-甘油(96.4±1.8%和 41.1±4.7 nmol/L)预处理后可恢复。在猪冠状动脉内皮细胞中，H-R 抑制了缓激肽激活的膜电流(8.6±0.4 vs. 14.0±1.5 pA/pF)和 Pyr3 敏感的 TRPC3 电流(3.8±0.3 vs. 6.3±0.6 pA/pF；P<0.01)。H-R 还抑制了缓激肽诱导的 Ca(2+)内流和通过 TRPC3 的 Ca(2+)内流。H-R 后，内皮细胞表面的 TRPC3 表达减少。
我们首次证明了内皮细胞 TRPC3 介导的 Ca(2+)内流有助于 NO 的释放，并揭示了 H-R 与 TRPC3 活性的抑制有关。通道向细胞表面的转运抑制参与了 H-R 期间 TRPC3 电流减少和通过 TRPC3 的 Ca(2+)内流减少的潜在机制。本研究表明，TRPC3 可能成为 H-R 期间内皮保护的新靶点。
