Kang H Y, Kim N S, Lee C O, Lee J Y, Kang W H
Department of Dermatology, Ajou University School of Medicine, Suwon, Republic of Korea.
J Cell Physiol. 2000 Nov;185(2):200-6. doi: 10.1002/1097-4652(200011)185:2<200::AID-JCP4>3.0.CO;2-6.
We investigated the expression of ryanodine receptors (RyRs) in cultured human melanocytes with immunocytochemistry and reverse transcriptase-polymerase chain reaction. With the use of a monoclonal antibody, RyR immunoreactivity was detected in the cytoplasm of melanocytes, and was further confirmed by RT-PCR assay. The PCR products were cut with restriction enzymes specific for each RyR isoform. Using the RyR1-specific restriction enzyme SacI yielded fragments of 300, 100, and 130 base pairs, consistent with the expression of RyR1 isoforms. The function of RyR in Ca(2+) signaling was investigated using single-cell fura-2 imaging. Ryanodine (1 to approximately 100 microM) induced significant elevation of cytoplasmic Ca(2+) in single human melanocytes in a dose-dependent manner. The ryanodine-induced Ca(2+) increase was inhibited by neomycin. Furthermore, ryanodine inhibited proliferation and stimulated pigmentation of human melanocytes. This study demonstrates that the RyR1 isoform is expressed in cultured human melanocytes, and suggests that the RyR may be involved in regulating the intracellular Ca(2+) responses involved in proliferation and pigmentation of cultured human melanocytes.
我们采用免疫细胞化学和逆转录聚合酶链反应研究了培养的人黑素细胞中兰尼碱受体（RyRs）的表达。使用单克隆抗体，在黑素细胞的细胞质中检测到了RyR免疫反应性，并通过逆转录-聚合酶链反应（RT-PCR）分析进一步证实。用针对每种RyR亚型的限制性内切酶切割PCR产物。使用RyR1特异性限制性内切酶SacI产生了300、100和130个碱基对的片段，这与RyR1亚型的表达一致。使用单细胞fura-2成像研究了RyR在钙（Ca2+）信号传导中的功能。兰尼碱（1至约100 microM）以剂量依赖性方式诱导单个正常人黑素细胞中细胞质钙（Ca2+）显著升高。新霉素抑制了兰尼碱诱导的[Ca2+]i增加。此外，兰尼碱抑制人黑素细胞的增殖并刺激其色素沉着。本研究表明，RyR1亚型在培养的人黑素细胞中表达，并提示RyR可能参与调节培养的人黑素细胞增殖和色素沉着所涉及的细胞内钙（Ca2+）反应。
