Hughes A R, Bird G S, Obie J F, Thastrup O, Putney J W
Laboratory of Cellular and Molecular Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Mol Pharmacol. 1991 Aug;40(2):254-62.
The effects of epidermal growth factor on Ca2+ signaling in A431 cells were investigated. Epidermal growth factor induced a transient Ca2+ signal in the absence of external Ca2+ and a sustained response in the presence of extracellular Ca2+, indicating an ability to mobilize intracellular Ca2+ as well as the ability to increase Ca2+ entry from the extracellular space. The Ca(2+)-ATPase inhibitor thapsigargin also activated Ca2+ entry, and neither epidermal growth factor nor the guanine nucleotide-dependent protein-linked receptor agonist bradykinin activated additional Ca2+ entry over that due to thapsigargin. In nominally Ca(2+)-free medium, the addition of bradykinin to A431 cells rapidly but transiently increased inositol 1,4,5-trisphosphate and, in parallel fashion, transiently increased cytosolic Ca2+. Unexpectedly, under these experimental conditions, epidermal growth factor elicited a small but significant Ca2+ signal after the addition of bradykinin. Experiments were designed to determine whether the Ca2+ response to epidermal growth factor after bradykinin results from mobilization of Ca2+ by an inositol 1,4,5-trisphosphate-independent mechanism. Epidermal growth factor stimulated additional inositol 1,4,5-trisphosphate formation in bradykinin-treated cells. Furthermore, the Ca2+ signals elicited by both bradykinin and epidermal growth factor were blocked in cells microinjected with the inositol 1,4,5-trisphosphate receptor antagonist heparin, whereas the intracellular Ca(2+)-ATPase inhibitor thapsigargin still mobilized Ca2+. Finally, histamine, a less efficacious guanine nucleotide-dependent protein-linked receptor agonist, as well as photolyzed, microinjected, caged inositol 1,4,5-trisphosphate, also mobilized Ca2+ after bradykinin. The results of this study show (i) that epidermal growth factor activates intracellular Ca2+ release as well as Ca2+ entry, the latter most likely resulting from an indirect effect due to the depletion of intracellular Ca2+ pools, (ii) that the actions of epidermal growth factor on Ca2+ homeostasis can be fully accounted for by inositol 1,4,5-trisphosphate formation, and (iii) that the ability of A431 cells to produce Ca2+ signals when epidermal growth factor is applied after bradykinin can be explained by the rapid and complete desensitization of the bradykinin stimulated phospholipase C activity.
研究了表皮生长因子对A431细胞中Ca2+信号的影响。表皮生长因子在无细胞外Ca2+时诱导短暂的Ca2+信号，在有细胞外Ca2+时诱导持续性反应，表明其具有动员细胞内Ca2+的能力以及增加Ca2+从细胞外空间内流的能力。Ca(2+)-ATP酶抑制剂毒胡萝卜素也能激活Ca2+内流，表皮生长因子和鸟嘌呤核苷酸依赖性蛋白偶联受体激动剂缓激肽均未激活超过毒胡萝卜素诱导的额外Ca2+内流。在名义上无Ca(2+)的培养基中，向A431细胞添加缓激肽可迅速但短暂地增加肌醇1,4,5-三磷酸，并以平行方式短暂增加胞质Ca2+。出乎意料的是，在这些实验条件下，添加缓激肽后表皮生长因子引发了一个小但显著的Ca2+信号。设计实验以确定缓激肽后对表皮生长因子的Ca2+反应是否源于通过不依赖肌醇1,4,5-三磷酸的机制动员Ca2+。表皮生长因子刺激缓激肽处理的细胞中额外的肌醇1,4,5-三磷酸形成。此外，缓激肽和表皮生长因子引发的Ca2+信号在显微注射肌醇1,4,5-三磷酸受体拮抗剂肝素的细胞中被阻断，而细胞内Ca(2+)-ATP酶抑制剂毒胡萝卜素仍能动员Ca2+。最后,组胺，一种效力较弱的鸟嘌呤核苷酸依赖性蛋白偶联受体激动剂，以及光解、显微注射、笼化的肌醇1,4,5-三磷酸，在缓激肽后也能动员Ca2+。本研究结果表明：(i)表皮生长因子激活细胞内Ca2+释放以及Ca2+内流，后者很可能是由于细胞内Ca2+池耗竭的间接作用所致；(ii)表皮生长因子对Ca2+稳态的作用可完全由肌醇1,4,5-三磷酸的形成来解释；(iii)缓激肽后应用表皮生长因子时A431细胞产生Ca2+信号的能力可通过缓激肽刺激的磷脂酶C活性的快速和完全脱敏来解释。
