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Comparative Study
. 2008 Oct 31:1238:85-92.
doi: 10.1016/j.brainres.2008.08.033. Epub 2008 Aug 23.

Effects of chronic continuous hypoxia on the expression of SLC4A8 (NDCBE) in neonatal versus adult mouse brain

Li-Ming Chen  1 , Gabriel G Haddad, Walter F Boron
Affiliations
Comparative Study

Effects of chronic continuous hypoxia on the expression of SLC4A8 (NDCBE) in neonatal versus adult mouse brain

Li-Ming Chen et al. Brain Res. .

Abstract

Na-coupled HCO(3) transporters (NCBTs) play important roles in brain pH regulation. One NCBT, the Na-driven Cl-HCO(3) exchanger (SLC4A8 or NDCBE), appears to be the major regulator of intracellular pH (pH(i)), at least in some hippocampal pyramidal neurons. NDCBE is widely expressed throughout the central nervous system in rodent brain. In a previous study, it has been demonstrated that CCH decreases the abundance of NBCn1 and NBCn2 proteins in four regions of the mouse brain: cerebral cortex (CX), subcortex (SCX), cerebellum (CB), and hippocampus (HC). Here we report the effect of CCH (11% O(2)) on the expression of NDCBE protein in mouse brain. Neonates (beginning at age P2) or adult mice (beginning at P90) were subjected to either normoxia or CCH for durations of 14 or 28 days. Membrane-protein levels were assessed by western blotting using our polyclonal antibody directed against NDCBE. In neonates, CCH significantly decreased NDCBE expression in HC after 14 days and SCX after 28 days, but had no significant effect for other combinations of region/duration. In adults, however, CCH significantly decreased (by 20-50%) the expression of NDCBE in all four brain regions, both with 14 and 28 day duration. Thus, the mouse brain exhibits marked developmental differences in the response of NDCBE protein expression to CCH. We hypothesize that decreases in adult NDCBE protein levels, which are probably out of proportion to the decreases in other proteins, may be part of an adaptive response that reduces energy consumption and/or stabilizes brain pH(i). The smaller or absent responses in the young animals could be related to neonatal hypoxia tolerance.

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Figures

Fig. 1
Fig. 1
Effect of chronic hypoxia (14 days’ duration) on the expression of NDCBE protein in the brains of neonatal mice. The mice were placed in the chamber at age P2. The upper panel shows typical western blots of NDCBE and actin in four brain regions—one pair of bands each for (from left to right) cerebral cortex (CX), subcortex (SCX), cerebellum (CB), and hippocampus (HC). In each pair of bands, the left one represents normoxia, and the right one, hypoxia. The arrowhead represents the expected molecular weight of the unglycosylated protein (i.e., ~116 kDa). We used actin (middle panel) only as a loading marker, and not to normalize the NDCBE data (see text). The lower panel shows a summary of densitometry data from experiments like that in the upper panel. Each bar represents 5 groups of mice (8 mice per group). We made statistical comparisons between adjacent bars using paired two-tailed student’s T-tests (* P<0.05).
Fig. 2
Fig. 2
Effect of chronic hypoxia (28 days’ duration) on the expression of NDCBE protein in the brains of neonatal mice. The mice were placed in the chamber at age P2. The upper panel shows typical western blots of NDCBE and actin in four brain regions—one pair of bands each for (from left to right) cerebral cortex (CX), subcortex (SCX), cerebellum (CB), and hippocampus (HC). In each pair of bands, the left one represents normoxia, and the right one, hypoxia. The arrowhead represents the expected molecular weight of the unglycosylated protein (i.e., ~116 kDa). We used actin (middle panel) only as a loading marker, and not to normalize the NDCBE data (see text). The lower panel shows a summary of densitometry data from experiments like that in the upper panel. Each bar represents 6 groups of mice (8 mice per group). We made statistical comparisons between adjacent bars using paired two-tailed student’s T-tests (** P<0.01).
Fig. 3
Fig. 3
Effect of chronic hypoxia (14 days’ duration) on the expression of NDCBE protein in the brains of adult mice. The mice were placed in the chamber at age P90. The upper panel shows typical western blots of NDCBE and actin in four brain regions—one pair of bands each for (from left to right) cerebral cortex (CX), subcortex (SCX), cerebellum (CB), and hippocampus (HC). In each pair of bands, the left one represents normoxia, and the right one, hypoxia. The arrowhead represents the expected molecular weight of the unglycosylated protein (i.e., ~116 kDa). We used actin (middle panel) only as a loading marker, and not to normalize the NDCBE data (see text). The lower panel shows a summary of densitometry data from experiments like that in the upper panel. Each bar represents 5 groups of mice (5 mice per group). We made statistical comparisons between adjacent bars using paired two-tailed student’s T-tests (* P<0.05, ** P<0.01).
Fig. 4
Fig. 4
Effect of chronic hypoxia (28 days’ duration) on the expression of NDCBE protein in the brains of adult mice. The mice were placed in the chamber at age P90. The upper panel shows typical western blots of NDCBE and actin in four brain regions—one pair of bands each for (from left to right) cerebral cortex (CX), subcortex (SCX), cerebellum (CB), and hippocampus (HC). In each pair of bands, the left one represents normoxia, and the right one, hypoxia. The arrowhead represents the expected molecular weight of the unglycosylated protein (i.e., ~116 kDa). We used actin (middle panel) only as a loading marker, and not to normalize the NDCBE data (see text). The lower panel shows a summary of densitometry data from experiments like that in the upper panel. Each bar represents 5 groups of mice (5 mice per group). We made statistical comparisons between adjacent bars using paired two-tailed student’s T-tests (* P<0.05, ** P<0.01).
Fig. 5
Fig. 5
Time course of the relative levels of NDCBE protein in four brain regions of normoxic mouse. A, NDCBE. In the case of P16, for each experiment summarized in Fig. 1, we summed the total densities of the four brain regions under normoxic conditions, and then determined the fraction of total density contributed by each region (totaling 100%). The four bars at P16 represent the means ± SE of these data. We similarly computed the values for P30 (from data that contributed to Fig. 2), P104 (Fig. 3), and P118 (Fig. 4). We also reproduce, from a previous paper (Chen et al., 2007), comparable bar graphs that show the relative levels of NBCn1 (panel B) and NBCn2 (panel C). Horizontal lines indicate a significant difference, as assessed by an ANOVA analysis (p<0.05), between bars in the same age group.
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