An anti-NH₂-terminal antibody localizes NBCn1 to heart endothelia and skeletal and vascular smooth muscle cells

Abstract

The electroneutral sodium bicarbonate cotransporter NBCn1 or NBC3 was originally cloned from rat aorta and from human skeletal muscle. NBCn1 (or NBC3) has been localized to the basolateral membrane of various epithelia, but thus far it has been impossible to detect the protein in these tissues by using anti-COOH-terminal antibodies. Hence an antibody was developed against the NH2-terminus of NBCn1 and was validated by peptide recognition and immunoblotting on positive control tissues and by binding of an ∼180-kDa protein in the rat kidney, cerebrum, cerebellum, and duodenum. In addition, an ∼180-kDa immunoreactive band appeared using samples from the aorta, heart ventricles and atria, mesenteric arteries, lung, spleen, liver, pancreas, and epididymis. Immunohistochemical analysis confirmed the previously described labeling in the kidney, duodenum, and the choroid plexus. The anti-NH2-terminal antibody localized NBCn1 to the plasma membrane domains of endothelia and smooth muscle cells in small mesenteric and renal arteries, as well as the capillaries of the heart ventricles, spleen, and salivary glands. NBCn1 was also detected in neuromuscular junctions and vasculature in skeletal muscle. Analysis of variable NBCn1 splicing by RT-PCR revealed that an NH2-terminal sequence, the cassette III, seems absent from cardiovascular NBCn1 and that both cassettes I and III are variable in most epithelia, whereas cassette II is absent from epithelial NBCn1. Thus the development of the NH2-terminal antibody allowed the localization of NBCn1 protein to major cardiovascular tissues where NBCn1 mRNA was previously detected. The NBCn1 is a likely candidate for mediating the reported electroneutral Na+-HCO3− cotransport in vascular smooth muscle.

Splice Cassette II of Na+,HCO3− Cotransporter NBCn1 (slc4a7) Interacts with Calcineurin A

“…Splice variants containing cassette II are expressed in VSMCs (21), heart (31), and skeletal muscle (22) but absent from most epithelia (24) and adult neuronal tissue (24,32). The location of a calcineurin binding site within cassette II provides a novel mechanism of tissue-specific regulation of NBCn1 activity and represents an important first step toward understanding the functional importance of the multiple splice variants described for NBCn1 (23).…”

“…A previously characterized (24) anti-N-terminal antibody against NBCn1 (generously provided by Dr. Jeppe Praeto-rius, Aarhus University, Denmark) was used. Additionally, commercially available antibodies (Santa Cruz Biotechnology) against calcineurin A␤ (sc-6124) and NHE1 (sc-136239) were employed in conjunction with species-matched horseradish peroxidase-coupled secondary antibodies (anti-rabbit, 7074P2, Cell Signaling; anti-goat, sc-2020, Santa Cruz Biotechnology; anti-mouse, 7076P2, Cell Signaling).…”

Constitutively Active ErbB2 Regulates Cisplatin-Induced Cell Death in Breast Cancer Cells via Pro- and Antiapoptotic Mechanisms

“…The polyclonal NBCn1 antibody (20) was a kind gift from Jeppe Praetorius, Aarhus University, Denmark. Antibodies against NHE1 (sc-136239), m-calpain (sc-7531), p21 (sc-397), Mcl-1 (sc-819), fodrin (sc-48382), and phospho Ser 15 p53 (sc-101762) were from Santa Cruz Biotechnology.…”

Modular structure of sodium-coupled bicarbonate transporters

“…NBCn1 mediates the apparent 1:1 cotransport of Na + and HCO 3 -and has an associated conductance, about 50% of which is carried by Na + . NBCn1 is widely expressed in mammalian organs, with especially high levels of transcript expression in muscular tissue Cooper et al, 2006;Damkier et al, 2006;Pushkin et al, 1999). Elsewhere in the body, NBCn1-mediated HCO 3 -transport may (1) influence neuronal activity (Cooper et al, 2005) by setting the resting pH i of neurons and contributing towards recovery of pH i after neuronal firing, (2) contribute to secretion of cerebrospinal fluid by mediating HCO 3 -influx across the basolateral membrane of the choroid plexus epithelium , (3) promote osteoclast survival by contributing to an anti-apoptotic alkaline pH i (Bouyer et al, 2007), and (4) neutralize the protons formed during the conversion of NH 4 + to NH 3 by mediating HCO 3 -influx across the basolateral membrane of mTAL epithelia .…”

“…4) have been amplified from cDNA. The distribution and individual character of these variants is still under investigation (Damkier et al, 2006).…”