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Hydrogen-Deuterium Exchange Effects on β-Endorphin Release from AtT20 Murine Pituitary Tumor Cells |
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| Authors: | Masayuki Ikeda Shigeru Suzuki Masahiro Kishio Moritoshi Hirono Takashi Sugiyama Junko Matsuura Teppei Suzuki Takayuki Sota Charles N Allen Shiro Konishi and Tohru Yoshioka |
| Affiliation: | * Advanced Research Institute for Science and Engineering, Waseda University, 3-4-1 Okubo Shinjuku-ku, Tokyo 169-8555, Japan † Department of Molecular Behavioral Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan ‡ Department of Molecular Neurobiology, School of Human Sciences, Waseda University, 2-579-15 Mikajima, Tokorozawa, Saitama 359-1192, Japan § School of Science and Engineering, Waseda University, 3-4-1 Okubo Shinjuku-ku, Tokyo 169-8555, Japan ¶ Center for Research on Occupational and Environmental Toxicology, Oregon Health and Science University, Portland, Oregon 97201-3098, USA || Mitsubishi Kagaku Institute of Life Sciences, and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 11 Minamiooya, Machida-shi, Tokyo 194-8511, Japan |
| Abstract: | Abundant evidences demonstrate that deuterium oxide (D2O) modulates various secretory activities, but specific mechanisms remain unclear. Using AtT20 cells, we examined effects of D2O on physiological processes underlying β-endorphin release. Immunofluorescent confocal microscopy demonstrated that 90% D2O buffer increased the amount of actin filament in cell somas and decreased it in cell processes, whereas β-tubulin was not affected. Ca2+ imaging demonstrated that high-K+-induced Ca2+ influx was not affected during D2O treatment, but was completely inhibited upon D2O washout. The H2O/D2O replacement in internal solutions of patch electrodes reduced Ca2+ currents evoked by depolarizing voltage steps, whereas additional extracellular H2O/D2O replacement recovered the currents, suggesting that D2O gradient across plasma membrane is critical for Ca2+ channel kinetics. Radioimmunoassay of high-K+-induced β-endorphin release demonstrated an increase during D2O treatment and a decrease upon D2O washout. These results demonstrate that the H2O-to-D2O-induced increase in β-endorphin release corresponded with the redistribution of actin, and the D2O-to-H2O-induced decrease in β-endorphin release corresponded with the inhibition of voltage-sensitive Ca2+ channels. The computer modeling suggests that the differences in the zero-point vibrational energy between protonated and deuterated amino acids produce an asymmetric distribution of these amino acids upon D2O washout and this causes the dysfunction of Ca2+ channels. |
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