Deuteration as a tool in investigating the role of protons in cell signaling

The mechanisms underlying the inhibitory effects of deuterium oxide (D₂O; heavy water) are likely to provide insight into the fundamental significance of hydrogen bonds in biological functions. Previously, to begin elucidating the effect of D₂O on physiological functions in living cells, we studied the effects of D₂O on voltage-sensitive Ca²(+) channels in AtT 20 cells and showed that actin distribution, Ca²(+) currents, and β-endorphin release were affected. However, the molecular mechanisms underlying the inhibitory effects of D₂O in whole animals and living cells remain obscure, especially in the effects of D₂O on the cell signaling.

We investigated the molecular mechanisms underlying the inhibitory effects of D₂O on the IP₃-mediated Ca²(+) signaling pathway using Ca²(+) imaging and micro-calorimetric measurements in mGluR1-expressing CHO cells.

DHPG-induced Ca²(+) elevations were markedly reduced in D₂O. Moreover, the Ca²(+) elevations were completely suppressed in H₂O after receptor activation with DHPG in D₂O, recovering gradually in H₂O medium. Without prior stimulation in D₂O, however, DHPG-induced Ca²(+) elevations in H₂O were not affected. Micro-calorimetric measurements showed reduced total DHPG-evoked heat generation in D₂O, while initial heat production and absorption associated with receptor activation were found to be larger. The reduction of DHPG-induced Ca²(+) elevation and heat generation in D₂O medium may be due to decreased amount of IP₃ by the reduced hydrolysis of PIP₂.

Protein structure changes due to the replacement of hydrogen with deuterium will induce the inhibitory effects of D₂O by reduction of the frequency of -OH bonds.