Ac. Rego et al., OXIDATIVE STRESS IN ACIDIC CONDITIONS INCREASES THE PRODUCTION OF INOSITOL PHOSPHATES IN CHICK RETINAL CELLS IN CULTURE, Free radical biology & medicine, 20(2), 1996, pp. 175-187
The effect of oxidative stress on the production of [H-3]inositol phos
phates (InsP) by retinal cells in culture was analyzed. The process of
oxidation was induced by incubating the cells with ascorbic acid and
ferrous sulphate, and increased extent of oxidation was obtained by va
rying the pH from neutral to moderate acidosis (pH 6.5). The oxidative
process significantly reduced cell viability (about 15%) by decreasin
g the capacity of mitochondria dehydrogenases to reduce tetrazolium sa
lts, but had no effect on the leakage of lactate dehydrogenase. The pr
oduction of [H-3]InsP, in the absence of receptor activation, was incr
eased dose dependently by oxidative stress. Maximal increases to 189 /- 7%, 197 +/- 13%, and 329 +/- 22% were observed, respectively, for i
nositol monophosphates (InsP(1)), inositol bisphosphates (InsP(2)), an
d inositol trisphosphates (InsP(3)), at 2.5 nmol thiobarbituric acid r
eactive substances (TBARS)/mg protein. The response to cholinergic rec
eptor activation was slightly decreased in cells oxidized in acidic co
nditions. Antagonists of glutamate receptors failed to inhibit the enh
ancement in InsP that occurred upon cellular oxidation, suggesting tha
t the effect was not mediated by activation of glutamate receptors. Ce
llular oxidation increased by about two fold the uptake of Ca-45(2+) i
n the absence of agonist stimulation. However, stimulation of phosphol
ipase C by Ca2+ did not mediate the increase in [H-3]InsP upon cell ox
idation in acidic conditions, because the addition of 1-[6-[[17 10)-tr
ien-17-yl]amino]hexyl]-1-H-pyrrole-2,5-dione (U-73122), an inhibitor o
f phospholipase C-dependent processes, did not affect the production o
f [H-3]InsP in oxidized cells. Nevertheless, U-73122 significantly inh
ibited carbachol- and K+-stimulated accumulation of [H-3]InsP. Further
more, the enhancement of [H-3]InsP induced by ascorbate/Fe2+ was still
observed in the absence of external Ca2+. This increase in the produc
tion of InsP did not substantially induce the release of Ca2+ from int
ernal stores. The results suggest that both Ca2+-dependent and Ca2+-in
dependent pathways are involved in oxidative stress-mediated InsP incr
ement, and that the enzymes of the InsP metabolism may be affected by
oxidation.