Expression and function of ryanodine receptors in human melanocytes

Abstract

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+)](i) 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.