Changes of the free cytosolic Ca2+ concentration induced by shear stress were measured in Fura-2 acetoxymethyl ester-loaded endothelial cells from human umbilical cord veins. 2. We were able to induce Ca2+ transients in almost every cell by blowing a stream of physiological solution onto a single endothelial cell thereby inducing shear stress between 0 and 50 dyn cm-2. The Ca2+ response could be graded by varying the shear stress, and reached a half-maximal value at a shear stress of 30 dyn cm-2. 3. The shear stress responses critically depended on the extracellular Ca2+ concentration and were absent in a Ca(2+)-free solution. Repetitive application of short pulses of shear stress induced cumulative effects because of the slow decay of the shear stress Ca2+ responses (time constants 82.3 +/- 17.8 s from twenty-five cells). Application of a depolarizing high potassium solution to reduce the driving force for Ca2+ entry decreased the Ca2+ transients in some of the cells. 4. Application of shear stress in the presence of other divalent cations, such as nickel, cobalt or barium, always produced substantial changes in the ratio of the 390/360 nm fluorescence signal, indicating influx of these cations and subsequent quenching of the Fura-2 fluorescence. 5. Shear stress responses in the presence of 10 mM Ca2+ were completely blocked by application of 1 mM La3+. 6. Incubation of the cells with the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) did not alter the shear stress response, but completely blocked histamine-induced Ca2+ transients. 7. Small submaximal shear stress potentiated the Ca2+ transients induced by histamine. 8. We conclude that shear stress-dependent Ca2+ signals are induced by an influx of calcium that is not modulated via protein kinase C and not activated by membrane depolarization. The influx pathway is also permeable to divalent cations such as Ni2+, Co2+ and Ba2+, but is blocked by La3+.
摘要
在用人脐静脉Fura-2乙酰氧甲酯负载的内皮细胞中，测量了剪切应力诱导的游离胞质Ca2+浓度变化。2. 通过将生理溶液流吹到单个内皮细胞上，从而在0至50达因/平方厘米之间诱导剪切应力，我们能够在几乎每个细胞中诱导Ca2+瞬变。Ca2+反应可通过改变剪切应力进行分级，在30达因/平方厘米的剪切应力下达到半最大值。3. 剪切应力反应严重依赖于细胞外Ca2+浓度，在无Ca(2+)溶液中不存在。由于剪切应力Ca2+反应的缓慢衰减（来自25个细胞的时间常数为82.3±17.8秒），重复施加短脉冲剪切应力会产生累积效应。应用去极化高钾溶液以降低Ca2+进入的驱动力，会使一些细胞中的Ca2+瞬变减少。4. 在存在其他二价阳离子（如镍、钴或钡）的情况下施加剪切应力，总是会使390/360纳米荧光信号的比率产生实质性变化，表明这些阳离子的流入以及随后Fura-2荧光的淬灭。5. 在存在10 mM Ca2+的情况下，施加1 mM La3+可完全阻断剪切应力反应。6. 用佛波酯12-O-十四烷酰佛波醇-13-乙酸酯（TPA）孵育细胞不会改变剪切应力反应，但会完全阻断组胺诱导的Ca2+瞬变。7. 小的亚最大剪切应力会增强组胺诱导的Ca2+瞬变。8. 我们得出结论，剪切应力依赖性Ca2+信号是由钙的流入诱导的，该流入不通过蛋白激酶C调节，也不受膜去极化激活。该流入途径也可通透二价阳离子，如Ni2+、Co2+和Ba2+，但被La3+阻断。
