Molecular and functional characterization of sodium--hydrogen exchanger in skin as well as cultured keratinocytes and melanocytes.
Molecular and functional characterization of sodium--hydrogen exchanger in skin as well as cultured keratinocytes and melanocytes.
复制标题
皮肤以及培养的角质形成细胞和黑素细胞中钠-氢交换器的分子和功能表征。
DOI：
10.1016/s0005-2736(01)00273-5
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Boissy,RE
中科院分区：
文献类型：
--
作者：
Sarangarajan,R;Shumaker,H;Soleimani,M;LePoole,C;Boissy,RE
钠氢 (Na+/H+) 交换器是参与调节和维持大多数真核细胞类型的细胞内 pH 和细胞体积的少数转运蛋白之一。目前的研究通过逆转录聚合酶链式反应 (RT-PCR)、免疫组织化学分析和功能研究，研究了 Na+/H+ 交换子 (NHE) 异构体在人类皮肤和培养的角质形成细胞、黑色素细胞和黑色素瘤细胞中的表达。使用新生儿包皮从表皮和真皮中分离RNA，并启动角质形成细胞和黑素细胞的培养。使用 NHE-1 特异性 PCR 引物对从表皮、真皮、角质形成细胞、黑素细胞和黑素瘤细胞中分离的 RNA 进行 RT-PCR，产生 463 bp PCR 产物。使用 NHE 异构体 2、3、4 和 5 特异性引物进行 RT-PCR 未产生任何产物。使用 NHE-1 特异性多克隆抗体对新生儿包皮、角质细胞、黑素细胞和黑素瘤细胞进行蛋白质印迹分析（角质形成细胞和黑素细胞培养物）和间接免疫组织化学，证明 NHE-1 在蛋白质水平表达。使用 pH 敏感染料 BCECF 对细胞内 pH 进行生理调节，检测到人角质形成细胞、黑色素细胞和黑色素瘤细胞培养物中阿米洛利敏感的 NHE 活性。这些结果表明，从人类皮肤和黑色素瘤细胞建立的人类角质形成细胞和黑色素细胞培养物表达钠氢交换剂的 NHE-1 亚型。
The sodium–hydrogen (Na+/H+) exchanger is one of the few transporter proteins involved in the regulation and maintenance of intracellular pH and cell volume in most eukaryotic cell types. The current study investigates the expression of isoforms of the Na+/H+exchanger (NHE) in human skin and in cultured keratinocytes, melanocytes, and melanoma cells by reverse transcription–polymerase chain reaction (RT–PCR), immunohistochemical analysis and functional studies. Neonatal foreskins were used to isolate RNA from epidermis and dermis, and to initiate cultures of keratinocytes and melanocytes. RT–PCR on RNA isolated from epidermis, dermis, keratinocytes, melanocytes and melanoma cells using PCR primers specific for NHE-1 yielded a 463 bp PCR product. RT–PCR performed using primers specific for NHE isoforms 2, 3, 4 and 5 did not yield any products. Western blotting analysis (of keratinocyte and melanocyte cell cultures) and indirect immunohistochemistry on neonatal foreskin, keratinocytes, melanocytes and melanoma cells using a NHE-1-specific polyclonal antibody demonstrated NHE-1 expression at the protein level. Physiological regulation of intracellular pH using a pH-sensitive dye, BCECF, detected an amiloride-sensitive NHE activity in human keratinocyte, melanocyte and melanoma cell cultures. These results indicate that cultures of human keratinocytes and melanocytes established from human skin and melanoma cells express the NHE-1 isoform of the sodium–hydrogen exchanger.