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 D 2O 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 H 2O-to-D2O-induced increase in β-endorphin release corresponded with the redistribution of actin, and the D2O-to-H 2O-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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