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NBCn1 (SLC4A7) is one of the two major Na+-HCO3− cotransporters in the human colonic epithelium, expressed predominantly in the highly proliferating colonocytes at the cryptal base. Increased NBCn1 expression levels are reported in tumors, including colorectal cancer. The study explores its importance for maintenance of the intracellular pH (pHi), as well as the proliferative, adhesive, and migratory behavior of the self-differentiating Caco2BBe colonic tumor cell line. In the self-differentiating Caco2BBe cells, NBCn1 mRNA was highly expressed from the proliferative stage until full differentiation. The downregulation of NBCn1 expression by RNA interference affected proliferation and differentiation and decreased intracellular pH (pHi) of the cells in correlation with the degree of knockdown. In addition, a disturbed cell adhesion and reduced migratory speed were associated with NBCn1 knockdown. Murine colonic Nbcn1–/– enteroids also displayed reduced proliferative activity. In the migrating Caco2BBe cells, NBCn1 was found at the leading edge and in colocalization with the focal adhesion markers vinculin and paxillin, which suggests that NBCn1 is involved in the establishment of cell-matrix adhesion. Our data highlight the physiological significance of NBCn1 in modulating epithelial pH homeostasis and cell-matrix interactions in the proliferative region of the colonic epithelium and unravel the molecular mechanism behind pathological overexpression of this transporter in human colorectal cancers. NEW & NOTEWORTHY: The transporter NBCn1 plays a central role in maintaining homeostasis within Caco2BBe colonic epithelial cells through its regulation of intracellular pH, matrix adhesion, migration, and proliferation. These observations yield valuable insights into the molecular mechanism of the aberrant upregulation of this transporter in human colorectal cancers. [ABSTRACT FROM AUTHOR]

Background: Na+,HCO3–-cotransporter NBCn1/Slc4a7 accelerates murine breast carcinogenesis. Lack of specific pharmacological tools previously restricted therapeutic targeting of NBCn1 and identification of NBCn1-dependent functions in human breast cancer. Methods: We develop extracellularly-targeted anti-NBCn1 antibodies, screen for functional activity on cells, and evaluate (a) mechanisms of intracellular pH regulation in human primary breast carcinomas, (b) proliferation, cell death, and tumor growth consequences of NBCn1 in triple-negative breast cancer, and (c) association of NBCn1-mediated Na+,HCO3–-cotransport with human breast cancer metastasis. Results: We identify high-affinity (KD ≈ 0.14 nM) anti-NBCn1 antibodies that block human NBCn1-mediated Na+,HCO3–-cotransport in cells, without cross-reactivity towards human NBCe1 or murine NBCn1. These anti-NBCn1 antibodies abolish Na+,HCO3–-cotransport activity in freshly isolated primary organoids from human breast carcinomas and lower net acid extrusion effectively in primary breast cancer tissue from patients with macrometastases in axillary lymph nodes. Inhibitory anti-NBCn1 antibodies decelerate tumor growth in vivo by ~50% in a patient-derived xenograft model of triple-negative breast cancer and pH-dependently reduce colony formation, cause G2/M-phase cell cycle accumulation, and increase apoptosis of metastatic triple-negative breast cancer cells in vitro. Conclusions: Inhibitory anti-NBCn1 antibodies block net acid extrusion in human breast cancer tissue, particularly from patients with disseminated disease, and pH-dependently limit triple-negative breast cancer growth. [ABSTRACT FROM AUTHOR]

Intracellular Ca2+ dynamics shape malignant behaviors of cancer cells. Whereas previous studies focused on cultured cancer cells, we here used breast organoids and colonic crypts freshly isolated from human and murine surgical biopsies. We performed fluorescence microscopy to evaluate intracellular Ca2+ concentrations in breast and colon cancer tissue with preferential focus on intracellular Ca2+ release in response to purinergic and cholinergic stimuli. Inhibition of the sarco‐/endoplasmic reticulum Ca2+ ATPase with cyclopiazonic acid elicited larger Ca2+ responses in breast cancer tissue, but not in colon cancer tissue, relative to respective normal tissue. The resting intracellular Ca2+ concentration was elevated, and ATP, UTP and acetylcholine induced strongly augmented intracellular Ca2+ responses in breast cancer tissue compared with normal breast tissue. In contrast, resting intracellular Ca2+ levels and acetylcholine‐induced increases in intracellular Ca2+ concentrations were unaffected and ATP‐ and UTP‐induced Ca2+ responses were smaller in colon cancer tissue compared with normal colon tissue. In accordance with the amplified Ca2+ responses, ATP and UTP substantially increased proliferative activity—evaluated by bromodeoxyuridine incorporation—in breast cancer tissue, whereas the effect was minimal in normal breast tissue. ATP caused cell death—identified with ethidium homodimer‐1 staining—in breast cancer tissue only at concentrations above the expected pathophysiological range. We conclude that intracellular Ca2+ responses are amplified in breast cancer tissue, but not in colon cancer tissue, and that nucleotide signaling stimulates breast cancer cell proliferation within the extracellular concentration range typical for solid cancer tissue. [ABSTRACT FROM AUTHOR]

Cancer metabolism produces large fluxes of lactate and H+, which are extruded by membrane transporters. However, H+ production and extrusion must be coupled by diffusion, facilitated by mobile buffers. Yan et al. propose that carnosine, generated by CARNS2, provides this mobile buffering and enables lysosomal functions that block T cell surveillance. [ABSTRACT FROM AUTHOR]

Insulin signals through several intracellular pathways. Here, we tested the hypothesis that insulin modulates [Na.sup.+]/[H.sup.+] exchange (NHE) activity in vascular cells through [H.sub.2][O.sub.2]-mediated inhibition of tyrosine phosphatase Src homology 2 domain containing tyrosine phosphatase 2 (SHP-2). We measured intracellular pH ([pH.sub.i]) in isolated mouse mesenteric arteries using fluorescence confocal and wide-field microscopy. In the absence of C[O.sub.2]/HC[O.sub.3], removal of bath [Na.sup.+] produced endothelial acidification ([DELTA][pH.sub.i] = -0.71 [+ or -] 0.12) inhibited by cariporide. Cariporide reduced endothelial steady-state [pH.sub.i] ([DELTA][pH.sub.i] = -0.28 [+ or -] 0.08). Insulin and H202 acidified endothelial cells 0.2-0.3 pH units and reduced the acidification upon [Na.sup.+] removal by ~65%. Cariporide abolished the effect of insulin and [H.sub.2][O.sub.2]. In vascular smooth muscle cells, [H.sub.2][O.sub.2] produced intracellular acidification ([DELTA][pH.sub.i] = -0.48 [+ or -] 0.06) as did high concentrations of insulin ([DELTA][pH.sub.i] = -0.03 [+ or -] 0.01). NHE activity after an N[H.sup.+.sub.4] prepulse was ~80% attenuated by [H.sub.2][O.sub.2] and ~40% by high insulin concentrations. [H.sub.2][O.sub.2] had no effect on [Na.sup.+]-HC[O.sup.-.sub.3] cotransport activity. NHE1 (slc9a1) was the only plasma membrane NHE isoform detected in mouse mesenteric arteries by RT-PCR analyses. In both cell types, polyethylene glycol catalase abolished the effect of insulin on [pH.sub.i]. Exposure to insulin increased the intracellular concentration of reactive oxygen species estimated with the fluorophore 5-(6)chloromethyl-2',7'-dichlorodihydrofluorescein. The SHP-2 selective inhibitor NSC-87877 and protein tyrosine phosphatase (PTP) inhibitor IV reduced steady-state [pH.sub.i] up to 0.3 pH units and inhibited NHE activity 60-80%; when applied in combination with insulin or [H.sub.2][O.sub.2], no further effect was obtained. We conclude that NHE contributes to phi regulation in arterial endothelial and smooth muscle cells in situ and is inhibited by insulin and [H.sub.2][O.sub.2]. We propose that insulin signaling involves [H.sub.2][O.sub.2] and inhibition of PTP SHP-2. sodium/hydrogen exchanger 1; protein tyrosine phosphatase; hydrogen peroxide; intracellular pH; mesenteric artery

The expression pattern of the electroneutral [Na.sup.+]-HC [O.sup.-.sub.3]cotransporter NBCn1 (s1c4a7) was investigated by [beta]-galactosidase staining of mice with a LacZ insertion into the NBCn1 gene. This method is of particular value because it is independent of immunoreactivity. We find that the NBCn1 promoter is active in a number of tissues where NBCn1 has previously been functionally or immunohistochemically identified, including a broad range of blood vessels (vascular smooth muscle cells and endothelial cells), kidney thick ascending limb and medullary collecting duct epithelial cells, the epithelial lining of the kidney pelvis, duodenal enterocytes, choroid plexus epithelial cells, hippocampus, and retina. Kidney corpuscles, colonic mucosa, and nonvascular smooth muscle cells (from the urinary bladder, trachea, gastrointestinal wall, and uterus) were novel areas of promoter activity. Atrial but not ventricular cardiomyocytes were stained. In the brain, distinct layers of the cerebral cortex and cerebellar Purkinje cells were stained as was the dentate nucleus. No staining of skeletal muscle or cortical collecting ducts was observed. RT-PCR analyses confirmed the expression of NBCnl and [beta]-galactosidase in selected tissues. Disruption of the NBCn1 gene resulted in reduced NBCn 1 expression, and in bladder smooth muscle cells, reduced amiloride-insensitive [Na.sup.+]-dependent HC[O.sup.-.sub.3] influx was observed. Furthermore, disruption of the NBCn1 gene resulted in a lower intracellular steady-state pH of bladder smooth muscle cells in the presence of C[O.sub.2]/ HC[O.sup.-.sub.3] but not in its nominal absence. We conclude that NBCn1 has a broad expression profile, supporting previous findings based on immunoreactivity, and suggest several new tissues where NBCn1 may be important. sodium-dependent bicarbonate transport; intracellular pH; LacZ reporter gene; acid-base physiology; smooth muscle cells