Neuronal expression of sodium/bicarbonate cotransporter NBCn1 (SLC4A7) and its response to chronic metabolic acidosis - PubMed (original) (raw)

Neuronal expression of sodium/bicarbonate cotransporter NBCn1 (SLC4A7) and its response to chronic metabolic acidosis

Hae Jeong Park et al. Am J Physiol Cell Physiol. 2010 May.

Abstract

The sodium-bicarbonate cotransporter NBCn1 (SLC4A7) is an acid-base transporter that normally moves Na(+) and HCO(3)(-) into the cell. This membrane protein is sensitive to cellular and systemic pH changes. We examined NBCn1 expression and localization in the brain and its response to chronic metabolic acidosis. Two new NBCn1 antibodies were generated by immunizing a rabbit and a guinea pig. The antibodies stained neurons in a variety of rat brain regions, including hippocampal pyramidal neurons, dentate gyrus granular neurons, posterior cortical neurons, and cerebellar Purkinje neurons. Choroid plexus epithelia were also stained. Double immunofluorescence labeling showed that NBCn1 and the postsynaptic density protein PSD-95 were found in the same hippocampal CA3 neurons and partially colocalized in dendrites. PSD-95 was pulled down from rat brain lysates with the GST/NBCn1 fusion protein and was also coimmunoprecipitated with NBCn1. Chronic metabolic acidosis was induced by feeding rats with normal chow or 0.4 M HCl-containing chow for 7 days. Real-time PCR and immunoblot showed upregulation of NBCn1 mRNA and protein in the hippocampus of acidotic rats. NBCn1 immunostaining was enhanced in CA3 neurons, posterior cortical neurons, and cerebellar granular cells. Intraperitoneal administration of N-methyl-d-aspartate caused neuronal death determined by caspase-3 activity, and this effect was more severe in acidotic rats. Administering N-methyl-d-aspartate also inhibited NBCn1 upregulation in acidotic rats. We conclude that NBCn1 in neurons is upregulated by chronic acid loads, and this upregulation is associated with glutamate excitotoxicity.

PubMed Disclaimer

Figures

Fig. 1.

Fig. 1.

Characterization of new NBCn1 antibodies. A: immunoblot of membrane preparation from rat whole brain. Immunoblot was performed with the affinity-purified rabbit NBCn1 antibody. The antibody detected a major band of 130 kDa. The detection of the band of 250 kDa varied depending on the amount of SDS in the sample buffer. B: immunoblot of membrane preparation from rat hippocampus with the affinity-purified guinea pig NBCn1 antibody. The band in preserum is nonspecific. C: immunocytochemistry of HEK-293 cells transfected with NBCn1. Experiments were done with the guinea pig antibody. Controls were cells transfected with vector only. Bar: 50 μm.

Fig. 2.

Fig. 2.

NBCn1 localization in rat brain. A: immunoperoxidase immunochemistry for NBCn1 in rat brain. Brain slices were stained with the rabbit NBCn1 antibody. Images were taken from pyramidal CA1 and CA3 regions (CA1 and CA3), dentate gyrus (DG), posterior cortex (PC), cerebellum (CB), and choroid plexus (CP). The staining without the primary antibody served as a control (Con). SR, stratum radiatum; SO, stratum oriens; SL, stratum lucidum; SM, stratum moleculare; ML, molecular layer; GL, granular layer; P, Purkinje cells. Arrows are basolateral membranes of CP epithelia. Bars represent 50 μm. The bar for CA1 applies to the other 5 panels. B: immunofluorescence immunochemistry for NBCn1 in rat brain. Brain slices were labeled with the guinea pig NBCn1 antibody and then with the Texas red-conjugated goat anti-guinea pig IgG. Images were taken from CA3, CB, and olfactory bulb (OB). Bar: 100 μm.

Fig. 3.

Fig. 3.

Confocal immunofluorescence images of hippocampal CA3 neurons for NBCn1 and PSD-95. Hippocampal slices were labeled with the rabbit NBCn1 antibody (A, D, G) and the mouse PSD-95 antibody (B, E, H). NBCn1 was detected with the Alexa 488 anti-rabbit IgG (green) and Alexa 594 anti-mouse IgG (red). Images in A_–_C were taken in ×40 magnification (bar: 50 μm). Images of proximal dendrites (D_–_F) and cell bodies (G_–_I) were taken in ×100 magnification (bar: 10 μm). Asterisks (*) in F are examples for NBCn1/PSD-95 colocalization.

Fig. 4.

Fig. 4.

Interaction between NBCn1 and PSD-95. A: GST pull-down assay. Brain lysates were incubated with the GST/NBCn1 fusion protein containing the COOH-terminal 26 amino acids of rat NBCn1, or GST only. Proteins bound to GST/NBCn1 or GST were dissociated and examined with the PSD-95 antibody by immunoblot. Xenopus oocyte lysates were used as a negative control. B: coimmunoprecipitation of NBCn1 with the mouse PSD-95 antibody. Brain lysates were incubated with the PSD-95 antibody and immunoprecipitates were examined with the NBCn1 antibody by immunoblot. Mouse IgG was used as a control.

Fig. 5.

Fig. 5.

Effects of chronic metabolic acidosis on NBCn1 mRNA and protein levels in the hippocampus. A: real-time PCR. The PCR cycle threshold for NBCn1 or GAPDH was determined and fitted to the standard curve made from a serial dilution of NBCn1 and GAPDH templates. NBCn1 was then normalized to GAPDH (P = 0.03, n = 3 for each). B: immunoblot of hippocampal lysates. Left, immunoblots of membrane preparations from control and acidotic hippocampi were probed with the NBCn1 antibody. The blots were reprobed with the β-actin antibody. Right, mean pixel intensity of immunoreactive bands was quantitated, and NBCn1 was normalized to β-actin (P = 0.04, n = 5 for controls and 6 for HCl-fed rats).

Fig. 6.

Fig. 6.

NBCn1 expression in brains of control rats versus acidotic rats. A: immunohistochemistry for NBCn1. Brain slices were stained with the rabbit NBCn1 antibody. Arrows are proximal dendrites. Bar: 50 μm. B: quantitation of NBCn1 expression. The mean pixel intensity of NBCn1 was determined by collecting signals in cell bodies or dendrites. More than 20 signals were counted. *P < 0.05.

Fig. 7.

Fig. 7.

Caspase-3 activity in control versus acidotic rats treated with _N_-methyl-

d

-aspartate (NMDA). Control rats and acidotic rats (n = 4 for each) were intraperitoneally injected with NMDA (75 mg/kg body wt), and hippocampi were isolated 3 days later and lysed. Lysates were incubated with the caspase substrate Ac-DVED-_p_-nitroanilide at 37°C overnight. The production of _p_-nitroanilide was measured by the absorbance at 405 nm. The unit is pmole of _p_-nitroanilide per milligram of total protein. Positive and negative controls were incubations of purified caspase-3 without lysates in the absence/presence of the inhibitor Ac-DVED-CHO.

Fig. 8.

Fig. 8.

NBCn1, NR2A, PSD-95, and β-actin expression levels before and after NMDA administration. A_–_D: immunoblot of the hippocampus prepared from controls and acidotic rats 3 days after NMDA treatment (n = 4). The blots were probed with antibodies to NBCn1 (A), NR2A (B), PSD-95 (C), and β-actin (D). E: quantitative measurements of immunoreactive bands. The bands were quantitated and normalized to β-actin.

Similar articles

Cited by

References

    1. Baird NR, Orlowski J, Szabo EZ, Zaun HC, Schultheis PJ, Menon AG, Schull GE. Molecular cloning, genomic organization, and functional expression of Na+/H+ exchanger isoform 5 (NHE5) from human brain. J Biol Chem 274: 4377–4382, 1999 - PubMed
    1. Baxter KA, Church J. Characterization of acid extrusion mechanisms in cultured fetal rat hippocampal neurones. J Physiol 493: 457–470, 1996 - PMC - PubMed
    1. Bevensee MO, Boron WF. pH regulation in mammalian neurons. In: pH and Brain Function, edited by Kaila K, Ransom BR. New York: Wiley-Liss, 1998, p. 211–231
    1. Boedtkjer E, Praetorius J, Aalkjaer C. NBCn1 (slc4a7) mediates the Na+-dependent bicarbonate transport important for regulation of intracellular pH in mouse vascular smooth muscle cells. Circ Res 98: 515–523, 2006 - PubMed
    1. Boedtkjer E, Praetorius J, Fuchtbauer EM, Aalkjaer C. Antibody-independent localization of the electroneutral Na+-HCO3− cotransporter NBCn1 (Slc4a7) in mice. Am J Physiol Cell Physiol 294: C591–C603, 2008 - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources