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

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 beta-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 beta-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 beta-endorphin release demonstrated an increase during D2O treatment and a decrease upon D(2)O washout. These results demonstrate that the H2O-to-D2O-induced increase in beta-endorphin release corresponded with the redistribution of actin, and the D2O-to-H2O-induced decrease in beta-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.

Functional Difference Between Deuterated and Protonated Macromolecules

“…This hypothesis can be expanded to various types of receptor dynamics, including mGluR1 in the CHO cells. A similar hysteresis effect of H/D exchanges on the voltage-dependent Ca 2+ channels was also reported previously (Ikeda et al, 2004). Thus we suggest that such hysteresis effect may be due to impairment of D/H exchanges.…”

“…Accordingly, the D 2 O exchange effect for both channels may be the same as each other. In order to examine the D 2 O effect on Na + and K + , high-K + -induced depolarization of membrane potential was measured using AtT-20 cells (Ikeda et al, 2004). As shown in Fig 1, when the cell is exposed to 30mM KCl, the membrane potential was depolarized from -69mV to -32mV (Fig.…”

“…An initial study on the D 2 O exchange effect of the Ca 2+ channel protein was carried out by Andjus et al using inter nodal cells of the fresh water alga, which was known as a unique system before the discovery of the patch clump method (Andjus et al, 1994 (Ikeda et al, 2004) As shown in Fig. 2, high-K + -induced depolarization made a reproducible and rapid increase in the intracellular Ca 2+ concentration ( Fig.…”

“…On the other hand, the decrease in the high K + -induced -endorphin release is in the phase of asymmetrically deuterated amino acids and the most probable cause is the complete inhibition of voltage-sensitive Ca 2+ channels. (Ikeda et al, 2004) (Ikeda et al, 2004). Therefore, it is likely that cells rapidly reach equilibrium after an exchange of H 2 O to D 2 O, but take a longer time to reach equilibrium after switching from D 2 O back to H 2 O.…”

Effects of Deuterium Oxide on Streptococcus mutans and Pseudomonas aeruginosa

“…At the cellular level, D 2 O affects mitosis and membrane function. Mice and rats which were forced to drink D 2 O instead of H 2 O died 7 days later 12,13) and higher concentrations (ca. 90%) of D 2 O rapidly killed fish, tadpoles and drosophila 20) .…”