[Changes in the intracellular free calcium of cultured human epidermal keratinocytes].
[Changes in the intracellular free calcium of cultured human epidermal keratinocytes].
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培养的人表皮角质形成细胞胞内游离钙的变化。
DOI：
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发表时间：
1991
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影响因子：
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通讯作者：
C. Yasui
中科院分区：
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作者：
C. Yasui
本研究观察了培养的正常人表皮角质形成细胞（NHEK）胞内游离钙（[Ca ~（2+）]i）的变化，以确定腺苷酸环化酶（AC）系统和磷脂酶C激活系统是否参与了[Ca ~（2+）]i的增加。从新生儿包皮获得NHEK，并在补充有2%牛垂体提取物的无血清培养基（K-GM）中生长。[Ca2+]i通过荧光比率成像显微镜使用Fura-2作为指示剂测量。在AC系统的情况下，在肾上腺素、去甲肾上腺素、异丙肾上腺素和沙丁胺醇刺激下观察到[Ca 2 +]i的短暂增加。甲氧胺、可乐定和多巴酚丁胺均未引起[Ca 2 +]i升高。肾上腺素引起的[Ca ~（2+）]i升高可被普萘洛尔预处理抑制，但不被哌唑嗪或育亨宾抑制，表明肾上腺素通过β_2-肾上腺素能刺激引起[Ca ~（2+）]i升高。当分别用组胺、腺苷、GTP γ S、毛喉素和二丁酰cAMP刺激NHEK时，观察到类似的变化。细胞外Ca ~（2+）的缺乏对肾上腺素诱导的[Ca ~（2+）]i增加没有影响。看来，激活的蛋白激酶A，通过刺激性GTP结合蛋白的cAMP积累的基础上，引起释放的Ca 2+从细胞内存储。另一方面，当已知的药物激活磷脂酶C在各种各样的细胞类型进行了测试，在[Ca 2 +]i的瞬时增加被证明在响应于凝血酶，缓激肽和物质P的加入。这种反应不受EGTA的存在，这表明这些药物提高[Ca 2 +]i通过磷脂酰肌醇分解。加压素、血管紧张素II、5-羟色胺和乙酰胆碱均未引起[Ca 2 +]i增加。在此基础上，推测NHEK具有通过AC系统和磷脂酶C激活系统增加[Ca ~（2+）]i的作用机制。这似乎是可能的，这种上升[Ca 2 +]i启动钙依赖性细胞反应，如激活钙/钙调蛋白依赖性激酶，并随后调节人表皮角质形成细胞的增殖和分化。
Changes in the intracellular free calcium ([Ca2+]i) of cultured normal human epidermal keratinocytes (NHEK) were investigated in order to determine whether the adenylate cyclase cAMP (AC) system and phospholipase C activating system are involved in increasing [Ca2+]i. NHEK were obtained from neonatal foreskin and grown in serum-free medium (K-GM) supplemented with 2% bovine pituitary extract. [Ca2+]i was measured by fluorescence ratio imaging microscopy using Fura-2 as the indicator. In the case of the AC system, transient increases in [Ca2+]i were observed in response to stimulation with epinephrine, norepinephrine, isoproterenol and salbutamol. Methoxamine, clonidine and dobutamine did not induce any [Ca2+]i increase. The [Ca2+]i increase evoked by epinephrine was inhibited by pretreatment with propranolol, but not by prazosin or yohimbine, indicating that epinephrine-induced [Ca2+]i elevation via beta 2-adrenergic stimulation. Similar changes were observed when NHEK were stimulated with histamine, adenosine, GTP gamma S, forskolin and dibutyryl cAMP respectively. The absence of extracellular Ca2+ had no effect on the epinephrine-induced [Ca2+]i increase. It appears that activated protein kinase A, based on cAMP accumulation via stimulatory GTP binding protein, elicited the release of Ca2+ from intracellular stores. On the other hand, when drugs known to activate phospholipase C in a wide variety of cell types were tested, a transient increase in [Ca2+]i was demonstrated in response to the addition of thrombin, bradykinin and substance P. This reaction was not affected by the presence of EGTA, suggesting that these drugs raise [Ca2+]i via phosphatidylinositol breakdown. Vasopressin, angiotensin II, serotonin and acetylcholine did not induce any increase in [Ca2+]i. On the basis of these studies, it was concluded that NHEK possess the mechanism which increase [Ca2+]i via AC system and phospholipase C activating system. It seems probable that this rise in [Ca2+]i initiates a calcium-dependent cellular response, such as activation of calcium/calmodulin dependent kinase, and subsequently regulates the proliferation and differentiation of human epidermal keratinocytes.