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

Abundant evidences demonstrate that deuterium oxide (D 2O) modulates various secretory activities, but specific mechanisms remain unclear. Using AtT20 cells, we examined effects of D 2O on physiological processes underlying β-endorphin release. Immunofluorescent confocal microscopy demonstrated that 90% D 2O buffer increased the amount of actin filament in cell somas and decreased it in cell processes, whereas β-tubulin was not affected. Ca 2+ imaging demonstrated that high-K +-induced Ca 2+ influx was not affected during D 2O treatment, but was completely inhibited upon D 2O washout. The H 2O/D 2O replacement in internal solutions of patch electrodes reduced Ca 2+ currents evoked by depolarizing voltage steps, whereas additional extracellular H 2O/D 2O replacement recovered the currents, suggesting that D 2O gradient across plasma membrane is critical for Ca 2+ channel kinetics. Radioimmunoassay of high-K +-induced β-endorphin release demonstrated an increase during D 2O treatment and a decrease upon D 2O washout. These results demonstrate that the H 2O-to-D 2O-induced increase in β-endorphin release corresponded with the redistribution of actin, and the D 2O-to-H 2O-induced decrease in β-endorphin release corresponded with the inhibition of voltage-sensitive Ca 2+ 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 D 2O washout and this causes the dysfunction of Ca 2+ channels.