Submaximal stimulation of porcine endothelial cells causes focal Ca2+ elevation beneath the cell membrane
Submaximal stimulation of porcine endothelial cells causes focal Ca2+ elevation beneath the cell membrane
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DOI：
10.1111/j.1469-7793.1998.109bx.x
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发表时间：
1998-01-01
影响因子：
5.5
通讯作者：
Sturek, M
中科院分区：
文献类型：
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作者：
Graier, WF;Paltauf-Doburzynska, J;Sturek, M
1. 内皮细胞的活化与胞浆内Ca2+浓度的增加有关，通常用胞浆内Ca2+染料（如fura-2和Calcium Green-1）监测。我们验证了猪冠状动脉内皮细胞在弱刺激过程中发生局灶性质下Ca2+升高的假设，这是由线粒体膜和浅内质网（SER）附近的细胞膜Na+ - Ca2+交换控制的。用fura-2或Green-1钙监测总体Ca2+浓度（[Ca2+](b)），用FFP-18测定浆下Ca2+浓度（[Ca2+](sp)）。利用激光扫描和反褶积显微镜估计了SER网络的分布。氟化钠（10 mmol l(-1)）和缓激肽（Bk; 1 nmol l(-1)）的亚最大浓度刺激Ca2+进入，但没有增加[Ca2+](b)。尽管肌醇1,4,5-三磷酸Ca2+释放对这两种刺激的响应是相似的，Ca2+进入形成和细胞内va对NaF的响应超过了对1 nmol l(-1) Bk的响应的四倍，这表明除了通过细胞内Ca2+释放刺激外，还有另外的2+进入途径。NaF对Ca2+4的影响。通过降低细胞外Na+来预防Na+ - Ca2+交换活性，揭示细胞内Ca2+释放响应于NaF和1 nmol l(-1) Bk，表明[Ca2+]增加(b)。因此;NaF减少了bk释放的Ca2+池，而线粒体Ca2+含量（用FCCP或寡霉素释放）和细胞内储存的Ca2+量（用离子霉素释放）与正常Na+条件下NaF处理的细胞相比增加。25 μ mol l(-1) ryanodine减少了低n +条件下naf诱导的[Ca2+]增加(b)和bk释放Ca2+池的消耗(s)，表明Ca2+诱导的Ca2+释放（CICR）参与。在同时记录[Ca2+](sp)（用FFP-18）和[Ca2+](b)（用钙绿-1）时，1 nmol l(-1) Bk或10 mmol l(-1) NaF在浆下区产生局灶性[Ca2+]升高，而在核周区没有增加。10 mmol mol l(-1) nocodazole使SER在1 nmol l(-1) Bk和10 mmol l(-1) Bk的作用下塌陷并释放Ca2+，而thapsigarin的作用没有改变。f条件,7。这些数据表明，在内皮细胞中，局灶性，质膜下Ca2+升高响应于小或慢的IF，形成是由于Ca2+从SER向质膜释放矢量，然后通过Na+-Ca2+交换挤压Ca2+。虽然这些局部Ca2+升高不能用Ca2+染料检测[Ca2+](b)，但预防Ca2+挤压或SER破坏产量增加[Ca2+](b)部分是由于CICR.8。所有的数据都支持我们的假设，即在弱刺激的内皮细胞中，细胞内Ca2+释放和[Ca2+]升高仅限于质下区。我们提出，SER与质膜的相关部分合作，控制Ca2+稳态，Ca2+分布和Ca2+进入。在讨论Ca2+信号时，需要考虑这种质下Ca2+控制单元（SCCU）的存在，特别是当细胞质Ca2+染料，如fura-2或钙绿-1被使用时。
1. Endothelial cell activation is correlated with increased cytosolic Ca2+ concentration, often monitored with cytoplasmic Ca2+ dyes, such as fura-2 and Calcium Green-1. We tested the hypothesis that during weak stimulation of porcine coronary artery endothelial cells, focal, subplasmalemmal Ca2+ elevations occur which are controlled by cell membrane Na+ - Ca2+ exchange near mitochondrial membrane and superficial endoplasmic reticulum (SER).2. Bulk Ca2+ concentration ([Ca2+](b)) was monitored using fura-2 or Calcium Green-1 and subplasmalemmal Ca2+ concentration ([Ca2+](sp)) was determined with FFP-18. The distribution of the SER network was estimated using laser scanning and deconvolution microscopy.3. Sodium fluoride (10 mmol l(-1)) and submaximal concentrations of bradykinin (Bk; 1 nmol l(-1)) stimulated Ca2+ entry with no increase in [Ca2+](b). Although inositol 1,4,5-trisphosphate Ca2+ release in response to both stimuli were similar, Ca2+ entry in formation ana intracellular va response to NaF exceeded that in response to 1 nmol l(-1) Bk by fourfold, suggesting additional 2+ entry pathways but stimulation via intracellular Ca2+ release. effects of NaF on Ca2+4. Prevention of Na+ - Ca2+ exchange activity by decreasing extracellular Na+ unmasked intracellular Ca2+ release in response to NaF and 1 nmol l(-1) Bk, indicated by an increase in [Ca2+](b). Thereby; NaF depleted Bk-releasable Ca2+ pools, while mitochondrial Ca2+ content (released with FCCP or oligomycin) and the amount of Ca2+ stored within the cells (released with ionomycin) was increased compared with cells treated with NaF under normal Na+ conditions. The NaF-initiated increase in [Ca2+](b) and depletion of Bk-releasable Ca2+ pool(s) in the low-N+ condition was diminished by 25 mu mol l(-1) ryanodine, indicating the involvement of Ca2+ induced Ca2+ release (CICR).5. In simultaneous recordings of [Ca2+](sp) (with FFP-18) and [Ca2+](b) (with Calcium Green-1), 1 nmol l(-1) Bk or 10 mmol l(-1) NaF yielded focal [Ca2+] elevation in the subplasmalemmal region with no increase in the perinuclear area.6. Treatment with 10 mu mol l(-1) nocodazole caused the SER to collapse and unmasked Ca2+ release in response to 1 nmol l(-1) Bk and 10 mmol l(-1) while the effect of thapsigargin was not changed. f conditions,7. These data show that in endothelial cells, focal, subplasmalemmal Ca2+ elevations in response to small or slow IF, formation occur due to vectorial Ca2+ release from the SER towards the plasmalemma followed by Ca2+ extrusion by Na+-Ca2+ exchange. While these local Ca2+ elevations are not detectable with Ca2+ dyes for the determination of [Ca2+](b), prevention of Ca2+ extrusion or SER disruption yields increases in [Ca2+](b) partially due to CICR.8. All of the data support our hypothesis that in weakly stimulated endothelial cells, intracellular Ca2+ release and [Ca2+] elevation are limited to the subplasmalemmal region. We propose that the SER co-operates with associated parts of the plasma membrane to control Ca2+ homeostasis, Ca2+ distribution and Ca2+ entry The existence of such a subplasmalemmal Ca2+ control unit (SCCU) needs to be considered in discussions of Ca2+ signalling, especially when cytoplasmic Ca2+ dyes, such as fura-2 or Calcium Green-1, are used.