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Hydrogen-Deuterium Exchange Effects on ß-Endorphin Release from AtT20 Murine Pituitary Tumor Cells

Masayuki Ikeda ^* , Shigeru Suzuki , Masahiro Kishio , Moritoshi Hirono ^*, Takashi Sugiyama , Junko Matsuura , Teppei Suzuki , Takayuki Sota , Charles N. Allen ^¶, Shiro Konishi ^|| and Tohru Yoshioka ^* 

^* 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; and ^|| 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

Correspondence: Address reprint requests to Tohru Yoshioka, PhD, Tel.: +81-3-5286-2761; Fax: +81-3-3205-6419; E-mail: yoshioka{at}human.waseda.ac.jp.

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 ß-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 ß-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 ß-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 ß-endorphin release corresponded with the redistribution of actin, and the D₂O-to-H₂O-induced decrease in ß-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.