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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Hirakura
Yutaka
Y
Advanced Research Institute for Bioscience, Waseda University, Nishitokyo-shi, Tokyo 202-0021, Japan.
Sugiyama
Takashi
T
Takeda
Masahiro
M
Ikeda
Masayuki
M
Yoshioka
Tohru
T
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects*; D002151:Calorimetry; D006224:Cricetinae; D003412:Cricetulus; D041961:Deuterium Exchange Measurement; D017666:Deuterium Oxide / Q000737:chemistry / Q000494:pharmacology*; D004963:Estrenes / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D008734:Methoxyhydroxyphenylglycol / Q000031:analogs & derivatives / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011522:Protons; D011760:Pyrrolidinones / Q000494:pharmacology; D018094:Receptors, Metabotropic Glutamate / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D004963:Estrenes; D010726:Phosphodiesterase Inhibitors; D011522:Protons; D011760:Pyrrolidinones; D018094:Receptors, Metabotropic Glutamate; C104077:metabotropic glutamate receptor type 1; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D008734:Methoxyhydroxyphenylglycol; D015544:Inositol 1,4,5-Trisphosphate; D017666:Deuterium Oxide; D002118:Calcium; C010117:3,4-dihydroxyphenylglycol