Stevanovic Z S, Salter M W, Whiteside C I
Department of Medicine, Hospital for Sick Children, University of Toronto, Ontario, Canada.
Am J Physiol. 1996 Jul;271(1 Pt 2):F21-9. doi: 10.1152/ajprenal.1996.271.1.F21.
The role of extracellular chloride in the regulation of mesangial cell calcium responsiveness to vasopressor peptides was explored. First, the components of vasopressor-stimulated calcium signaling were defined in rat mesangial cells cultured on coverslips and preloaded with fura 2. By spectrofluorometry, manganese uptake (reflecting divalent cation channel activation) was observed by quenching of fura 2, or intracellular cytosolic calcium concentration was calculated by dual-excitation ratiometric measurement. In cells depolarized with KCl (45 mM), enhanced manganese uptake or increased cytosolic calcium were inhibited with verapamil (10 microM). Pretreatment of mesangial cells with verapamil reduced the sustained calcium level in response to endothelin-1 (0.1 microM) by 65 +/- 6% (means +/- SE, n = 12) and to vasopressin (1 microM) by 62 +/- 12% (n = 8). Perforated cell patch-clamp measurement confirmed that endothelin-1 stimulated a sustained increase in cytosolic calcium or divalent cation entry only in the presence of simultaneous depolarization. In chloride-free buffer (chloride replaced with impermeant anions), sustained calcium response to endothelin-1 was reduced by 72 +/- 8 (n = 8) and by 65 +/- 4% (n = 8) in the presence of the chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (55 microM). In chloride-free buffer, cytosolic calcium (unstimulated) increased to > 200 nM by 30 min. These data indicate that reduced extracellular chloride increases mesangial cell basal cytosolic calcium and decreases the transient and sustained cytosolic calcium response to vasopressor peptides.
探讨了细胞外氯离子在调节系膜细胞对血管加压肽的钙反应性中的作用。首先，在盖玻片上培养并用fura 2预加载的大鼠系膜细胞中确定了血管加压素刺激的钙信号成分。通过荧光分光光度法，通过fura 2的淬灭观察锰摄取（反映二价阳离子通道激活），或通过双激发比率测量计算细胞内胞质钙浓度。在用氯化钾（45 mM）去极化的细胞中，维拉帕米（10 μM）可抑制增强的锰摄取或增加的胞质钙。用维拉帕米预处理系膜细胞可使内皮素-1（0.1 μM）引起的持续钙水平降低65±6%（平均值±标准误，n = 12），使血管加压素（1 μM）引起的降低62±12%（n = 8）。穿孔膜片钳测量证实，内皮素-1仅在同时去极化的情况下刺激胞质钙或二价阳离子持续增加。在无氯缓冲液（用非渗透性阴离子替代氯离子）中，内皮素-1引起的持续钙反应在存在氯离子通道抑制剂5-硝基-2-（3-苯丙基氨基）苯甲酸（55 μM）时降低了72±8（n = 8）和65±4%（n = 8）。在无氯缓冲液中，30分钟内未受刺激的胞质钙增加至>200 nM。这些数据表明，细胞外氯离子减少会增加系膜细胞基础胞质钙，并降低对血管加压肽的瞬时和持续胞质钙反应。
