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Differential Effects of Iodoacetamide and Iodoacetate on Glycolysis and Glutathione Metabolism of Cultured Astrocytes

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Differential Effects of Iodoacetamide and Iodoacetate on Glycolysis and Glutathione Metabolism of Cultured Astrocytes

Maike M Schmidt et al. Front Neuroenergetics.

Abstract

Iodoacetamide (IAA) and iodoacetate (IA) have frequently been used to inhibit glycolysis, since these compounds are known for their ability to irreversibly inhibit the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). However, the consequences of a treatment with such thiol reagents on the glutathione (GSH) metabolism of brain cells have not been explored. Exposure of astroglia-rich primary cultures to IAA or IA in concentrations of up to 1 mM deprived the cells of GSH, inhibited cellular GAPDH activity, lowered cellular lactate production and caused a delayed cell death that was detectable after 90 min of incubation. However, the two thiol reagents differed substantially in their potential to deprive cellular GSH and to inhibit astrocytic glycolysis. IAA depleted the cellular GSH content more efficiently than IA as demonstrated by half-maximal effects for IAA and IA that were observed at concentrations of about 10 and 100 muM, respectively. In contrast, IA was highly efficient in inactivating GAPDH and lactate production with half-maximal effects observed already at a concentration below 100 muM, whereas IAA had to be applied in 10 times higher concentration to inhibit lactate production by 50%. These substantial differences of IAA and IA to affect GSH content and glycolysis of cultured astrocytes suggest that in order to inhibit astrocytic glycolysis without substantially compromising the cellular GSH metabolism, IA - and not IAA - should be used in low concentrations and/or for short incubation periods.

Keywords: GAPDH; GSH; alkylation; astrocytes; carboxymethylation; glycolysis; lactate; thiol reagents.

Figures

Figure 1
Figure 1
Structural formulas of iodoacetamide (IAA) and iodoacetate (IA).
Figure 2
Figure 2
Disappearance of GSH after exposure to IAA or IA. GSH in a concentration of 10 μM was incubated with the indicated concentrations of IAA (A) or IA (B) for up to 60 min. (C) shows the GSx content that was detected after 60 min incubation with the given concentrations of IAA or IA. The data shown represent mean ± SD of 6 values that were obtained in two independent experiments, each performed in triplicates with individually prepared solutions. The significance of the differences between the values obtained for IAA and IA was calculated by the unpaired t-test and is indicated by #p < 0.05, ##p < 0.01 or ###p < 0.001).
Figure 3
Figure 3
GAPDH activity in cell lysates of astrocyte cultures in the absence or the presence of IAA or IA. The absorbance at 340 nm that indicates the formation of NADH by GAPDH was monitored. The results are presented as mean ± SD of data that were obtained on cell lysates derived from three independently prepared cultures.
Figure 4
Figure 4
Consequences of an application of IAA (A,C) or IA (B,D) on cellular viability (A,B) and specific cellular GSx content (C,D) in astrocyte cultures. The cells were incubated for up to 120 min without or with IAA or IA in the concentrations indicated in (A) and (B). The results represent mean ± SD of data that were obtained on three independently prepared cultures. The cultures contained initial protein contents of 80 ± 8 μg protein per well and initial specific GSx contents (100%) of 46.2 ± 10.2 nmol/mg protein. The significance of differences to the data obtained for the control condition (absence of inhibitor) are indicated by *p < 0.05, **p < 0.01 or ***p < 0.001.
Figure 5
Figure 5
Concentration dependency of the GSH depletion by IAA or IA in cultured astrocytes. The cells were incubated for 60 min with IAA or IA in concentrations of up to 1 mM. The results represent mean ± SD of data that were obtained on three independently prepared cultures that contained 80 ± 8 μg protein per well. The initial specific GSx content (100%) corresponded to 46.2 ± 10.2 nmol/mg protein. The significance of differences to the data obtained for the control condition (absence of inhibitor) are indicated by *p < 0.05, **p < 0.01 or ***p < 0.001. The significance of differences between the data observed after treatment with identical concentrations of IAA and IA was calculated by the paired t-test and is indicated by ###p < 0.001.
Figure 6
Figure 6
Consequences of an application of IAA (A) or IA (B) on the extracellular lactate concentration in astrocyte cultures. The cells were incubated for up to 120 min without or with IAA or IA in the concentrations indicated in (A) and (B). The results represent mean ± SD of data that were obtained on three independently prepared cultures. The cultures contained initial protein contents of 80 ± 8 μg protein per well. The significance of differences to the data obtained for the control condition (absence of inhibitor) are indicated by *p < 0.05, **p < 0.01 or ***p < 0.001.
Figure 7
Figure 7
Consequences of a treatment of cultured astrocytes with IAA or IA on the extracellular lactate concentration (A) and on the specific cellular GAPDH activity (B). The cells were incubated for 60 min with IAA or IA in concentrations of up to 1 mM. The results represent mean ± SD of data that were obtained on three (A) or six (B) independently prepared cultures that contained 80 ± 8 μg total protein per well (A) or 37 ± 11 μg cytosolic protein per well (B). The significance of differences to the data obtained for the control condition (absence of inhibitor) are indicated by **p < 0.01 or ***p < 0.001. The significance of differences between the data observed after treatment with identical concentrations of IAA and IA was calculated by the paired t-test and is indicated by #p < 0.05 or ##p < 0.01.
Figure 8
Figure 8
Time dependency of the inhibition of GAPDH by IAA or IA in cultured astrocytes. The cells were incubated for up to 60 min without (circles) or with IAA (A) or IA (B) in concentrations of 0.1 mM (triangles), 0.3 mM (squares) or 1 mM (diamonds). The results represent mean ± SD of data that were obtained on three independently prepared cultures that contained 45 ± 5 μg cytosolic protein per well. The initial specific GAPDH activity (100%) corresponded to 492 ± 45 nmol/(min × mg) cytosolic protein. The significance of differences to the data obtained for the control condition (absence of inhibitor) are indicated by **p < 0.01 or ***p < 0.001.

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