The electroneutral Na+-HCO3- cotransporter NBCn1 (SLC4A7) modulates colonic enterocyte pHi, proliferation, and migration.

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.

Strengthening the basics: acids and bases influence vascular structure and function, tissue perfusion, blood pressure, and human cardiovascular disease.

Acids and their conjugate bases accumulate in or dissipate from the interstitial space when tissue perfusion does not match the metabolic demand. Extracellular acidosis dilates most arterial beds, but associated acid-base disturbances-e.g., intracellular acidification and decreases in HCO3- concentration-can also elicit pro-contractile influences that diminish vasodilation and even dominate in some vascular beds to cause vasoconstriction. The ensemble activities of the acid-base-sensitive reactions in vascular smooth muscle and endothelial cells optimize vascular resistance for blood pressure control and direct the perfusion towards active tissue. In this review, we describe the mechanisms of intracellular pH regulation in the vascular wall and discuss how vascular smooth muscle and endothelial cells sense acid-base disturbances. We further deliberate on the functional effects of local acid-base disturbances and their integrated cardiovascular consequences under physiological and pathophysiological conditions. Finally, we address how mutations and polymorphisms in the molecular machinery that regulates pH locally and senses acid-base disturbances in the vascular wall can result in cardiovascular disease. Based on the emerging molecular insight, we propose that targeting local pH-dependent effectors-rather than systemic acid-base disturbances-has therapeutic potential to interfere with the progression and reduce the severity of cardiovascular disease.

Antibodies toward Na+,HCO3--cotransporter NBCn1/SLC4A7 block net acid extrusion and cause pH-dependent growth inhibition and apoptosis in breast cancer.

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.

Ion Channels, Transporters, and Sensors Interact with the Acidic Tumor Microenvironment to Modify Cancer Progression.

Solid tumors, including breast carcinomas, are heterogeneous but typically characterized by elevated cellular turnover and metabolism, diffusion limitations based on the complex tumor architecture, and abnormal intra- and extracellular ion compositions particularly as regards acid-base equivalents. Carcinogenesis-related alterations in expression and function of ion channels and transporters, cellular energy levels, and organellar H+ sequestration further modify the acid-base composition within tumors and influence cancer cell functions, including cell proliferation, migration, and survival. Cancer cells defend their cytosolic pH and HCO3- concentrations better than normal cells when challenged with the marked deviations in extracellular H+, HCO3-, and lactate concentrations typical of the tumor microenvironment. Ionic gradients determine the driving forces for ion transporters and channels and influence the membrane potential. Cancer and stromal cells also sense abnormal ion concentrations via intra- and extracellular receptors that modify cancer progression and prognosis. With emphasis on breast cancer, the current review first addresses the altered ion composition and the changes in expression and functional activity of ion channels and transporters in solid cancer tissue. It then discusses how ion channels, transporters, and cellular sensors under influence of the acidic tumor microenvironment shape cancer development and progression and affect the potential of cancer therapies.

The Acidic Tumor Microenvironment as a Driver of Cancer.

Acidic metabolic waste products accumulate in the tumor microenvironment because of high metabolic activity and insufficient perfusion. In tumors, the acidity of the interstitial space and the relatively well-maintained intracellular pH influence cancer and stromal cell function, their mutual interplay, and their interactions with the extracellular matrix. Tumor pH is spatially and temporally heterogeneous, and the fitness advantage of cancer cells adapted to extracellular acidity is likely particularly evident when they encounter less acidic tumor regions, for instance, during invasion. Through complex effects on genetic stability, epigenetics, cellular metabolism, proliferation, and survival, the compartmentalized pH microenvironment favors cancer development. Cellular selection exacerbates the malignant phenotype, which is further enhanced by acid-induced cell motility, extracellular matrix degradation, attenuated immune responses, and modified cellular and intercellular signaling. In this review, we discuss how the acidity of the tumor microenvironment influences each stage in cancer development, from dysplasia to full-blown metastatic disease.

Carbonic anhydrases reduce the acidity of the tumor microenvironment, promote immune infiltration, decelerate tumor growth, and improve survival in ErbB2/HER2-enriched breast cancer.

BACKGROUND: Carbonic anhydrases catalyze CO2/HCO3- buffer reactions with implications for effective H+ mobility, pH dynamics, and cellular acid-base sensing. Yet, the integrated consequences of carbonic anhydrases for cancer and stromal cell functions, their interactions, and patient prognosis are not yet clear. METHODS: We combine (a) bioinformatic analyses of human proteomic data and bulk and single-cell transcriptomic data coupled to clinicopathologic and prognostic information; (b) ex vivo experimental studies of gene expression in breast tissue based on quantitative reverse transcription and polymerase chain reactions, intracellular and extracellular pH recordings based on fluorescence confocal microscopy, and immunohistochemical protein identification in human and murine breast cancer biopsies; and (c) in vivo tumor size measurements, pH-sensitive microelectrode recordings, and microdialysis-based metabolite analyses in mice with experimentally induced breast carcinomas. RESULTS: Carbonic anhydrases-particularly the extracellular isoforms CA4, CA6, CA9, CA12, and CA14-undergo potent expression changes during human and murine breast carcinogenesis. In patients with basal-like/triple-negative breast cancer, elevated expression of the extracellular carbonic anhydrases negatively predicts survival, whereas, surprisingly, the extracellular carbonic anhydrases positively predict patient survival in HER2/ErbB2-enriched breast cancer. Carbonic anhydrase inhibition attenuates cellular net acid extrusion and extracellular H+ elimination from diffusion-restricted to peripheral and well-perfused regions of human and murine breast cancer tissue. Supplied in vivo, the carbonic anhydrase inhibitor acetazolamide acidifies the microenvironment of ErbB2-induced murine breast carcinomas, limits tumor immune infiltration (CD3+ T cells, CD19+ B cells, F4/80+ macrophages), lowers inflammatory cytokine (Il1a, Il1b, Il6) and transcription factor (Nfkb1) expression, and accelerates tumor growth. Supporting the immunomodulatory influences of carbonic anhydrases, patient survival benefits associated with high extracellular carbonic anhydrase expression in HER2-enriched breast carcinomas depend on the tumor inflammatory profile. Acetazolamide lowers lactate levels in breast tissue and blood without influencing breast tumor perfusion, suggesting that carbonic anhydrase inhibition lowers fermentative glycolysis. CONCLUSIONS: We conclude that carbonic anhydrases (a) elevate pH in breast carcinomas by accelerating net H+ elimination from cancer cells and across the interstitial space and (b) raise immune infiltration and inflammation in ErbB2/HER2-driven breast carcinomas, restricting tumor growth and improving patient survival.

Ketone body 3-hydroxybutyrate elevates cardiac output through peripheral vasorelaxation and enhanced cardiac contractility.

The ketone body 3-hydroxybutyrate (3-OHB) increases cardiac output and myocardial perfusion without affecting blood pressure in humans, but the cardiovascular sites of action remain obscure. Here, we test the hypothesis in rats that 3-OHB acts directly on the heart to increase cardiac contractility and directly on blood vessels to lower systemic vascular resistance. We investigate effects of 3-OHB on (a) in vivo hemodynamics using echocardiography and invasive blood pressure measurements, (b) isolated perfused hearts in Langendorff systems, and (c) isolated arteries and veins in isometric myographs. We compare Na-3-OHB to equimolar NaCl added to physiological buffers or injection solutions. At plasma concentrations of 2-4 mM in vivo, 3-OHB increases cardiac output (by 28.3±7.8%), stroke volume (by 22.4±6.0%), left ventricular ejection fraction (by 13.3±4.6%), and arterial dP/dtmax (by 31.9±11.2%) and lowers systemic vascular resistance (by 30.6±11.2%) without substantially affecting heart rate or blood pressure. Applied to isolated perfused hearts at 3-10 mM, 3-OHB increases left ventricular developed pressure by up to 26.3±7.4 mmHg and coronary perfusion by up to 20.2±9.5%. Beginning at 1-3 mM, 3-OHB relaxes isolated coronary (EC50=12.4 mM), cerebral, femoral, mesenteric, and renal arteries as well as brachial, femoral, and mesenteric veins by up to 60% of pre-contraction within the pathophysiological concentration range. Of the two enantiomers that constitute racemic 3-OHB, D-3-OHB dominates endogenously; but tested separately, the enantiomers induce similar vasorelaxation. We conclude that increased cardiac contractility and generalized systemic vasorelaxation can explain the elevated cardiac output during 3-OHB administration. These actions strengthen the therapeutic rationale for 3-OHB in heart failure management.

Amplified Ca2+ dynamics and accelerated cell proliferation in breast cancer tissue during purinergic stimulation.

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.

Loss of RPTPγ primes breast tissue for acid extrusion, promotes malignant transformation and results in early tumour recurrence and shortened survival.

BACKGROUND: While cellular metabolism and acidic waste handling accelerate during breast carcinogenesis, temporal patterns of acid-base regulation and underlying molecular mechanisms responding to the tumour microenvironment remain unclear. METHODS: We explore data from human cohorts and experimentally investigate transgenic mice to evaluate the putative extracellular HCO3--sensor Receptor Protein Tyrosine Phosphatase (RPTP)γ during breast carcinogenesis. RESULTS: RPTPγ expression declines during human breast carcinogenesis and particularly in high-malignancy grade breast cancer. Low RPTPγ expression associates with poor prognosis in women with Luminal A or Basal-like breast cancer. RPTPγ knockout in mice favours premalignant changes in macroscopically normal breast tissue, accelerates primary breast cancer development, promotes malignant breast cancer histopathologies, and shortens recurrence-free survival. In RPTPγ knockout mice, expression of Na+,HCO3--cotransporter NBCn1-a breast cancer susceptibility protein-is upregulated in normal breast tissue but, contrary to wild-type mice, shows no further increase during breast carcinogenesis. Associated augmentation of Na+,HCO3--cotransport in normal breast tissue from RPTPγ knockout mice elevates steady-state intracellular pH, which has known pro-proliferative effects. CONCLUSIONS: Loss of RPTPγ accelerates cellular net acid extrusion and elevates NBCn1 expression in breast tissue. As these effects precede neoplastic manifestations in histopathology, we propose that RPTPγ-dependent enhancement of Na+,HCO3--cotransport primes breast tissue for cancer development.

NBCn1 Increases NH4 + Reabsorption Across Thick Ascending Limbs, the Capacity for Urinary NH4 + Excretion, and Early Recovery from Metabolic Acidosis.

BACKGROUND: The electroneutral Na+/HCO3 - cotransporter NBCn1 (Slc4a7) is expressed in basolateral membranes of renal medullary thick ascending limbs (mTALs). However, direct evidence that NBCn1 contributes to acid-base handling in mTALs, urinary net acid excretion, and systemic acid-base homeostasis has been lacking. METHODS: Metabolic acidosis was induced in wild-type and NBCn1 knockout mice. Fluorescence-based intracellular pH recordings were performed and NH4 + transport measured in isolated perfused mTALs. Quantitative RT-PCR and immunoblotting were used to evaluate NBCn1 expression. Tissue [NH4 +] was measured in renal biopsies, NH4 + excretion and titratable acid quantified in spot urine, and arterial blood gasses evaluated in normoventilated mice. RESULTS: Basolateral Na+/HCO3 - cotransport activity was similar in isolated perfused mTALs from wild-type and NBCn1 knockout mice under control conditions. During metabolic acidosis, basolateral Na+/HCO3 - cotransport activity increased four-fold in mTALs from wild-type mice, but remained unchanged in mTALs from NBCn1 knockout mice. Correspondingly, NBCn1 protein expression in wild-type mice increased ten-fold in the inner stripe of renal outer medulla during metabolic acidosis. During systemic acid loading, knockout of NBCn1 inhibited the net NH4 + reabsorption across mTALs by approximately 60%, abolished the renal corticomedullary NH4 + gradient, reduced the capacity for urinary NH4 + excretion by approximately 50%, and delayed recovery of arterial blood pH and standard [HCO3 -] from their initial decline. CONCLUSIONS: During metabolic acidosis, NBCn1 is required for the upregulated basolateral HCO3 - uptake and transepithelial NH4 + reabsorption in mTALs, renal medullary NH4 + accumulation, urinary NH4 + excretion, and early recovery of arterial blood pH and standard [HCO3 -]. These findings support that NBCn1 facilitates urinary net acid excretion by neutralizing intracellular H+ released during NH4 + reabsorption across mTALs.

Na+,HCO3- cotransporter NBCn1 accelerates breast carcinogenesis.

Cell metabolism increases during carcinogenesis. Yet, intracellular pH in solid cancer tissue is typically maintained equal to or above that of normal tissue. This is achieved through accelerated cellular acid extrusion that compensates for the enhanced metabolic acid production. Upregulated Na+,HCO3- cotransport is the predominant mechanism of net acid extrusion in human and murine breast cancer tissue, and in congruence, the protein expression of the electroneutral Na+,HCO3- cotransporter NBCn1 is increased in primary breast carcinomas and lymph node metastases compared to matched normal breast tissue. The capacity for net acid extrusion and level of steady-state intracellular pH are lower in carcinogen- and ErbB2-induced breast cancer tissue from NBCn1 knockout mice compared to wild-type mice. Consistent with importance of intracellular pH control for breast cancer development, tumor-free survival is prolonged and tumor growth rate decelerated in NBCn1 knockout mice compared to wild-type mice. Glycolytic activity increases as function of tumor size and in areas of poor oxygenation. Because cell proliferation in NBCn1 knockout mice is particularly reduced in larger-sized breast carcinomas and central tumor regions with expected hypoxia, current evidence supports that NBCn1 facilitates cancer progression by eliminating intracellular acidic waste products derived from cancer cell metabolism. The present review explores the mechanisms and consequences of acid-base regulation in breast cancer tissue. Emphasis is on the Na+,HCO3- cotransporter NBCn1 that accelerates net acid extrusion from breast cancer tissue and thereby maintains intracellular pH in a range permissive for cell proliferation and development of breast cancer.

Increased NBCn1 expression, Na+/HCO3- co-transport and intracellular pH in human vascular smooth muscle cells with a risk allele for hypertension.

Genome-wide association studies have revealed an association between variation at the SLC4A7 locus and blood pressure. SLC4A7 encodes the electroneutral Na+/HCO3- co-transporter NBCn1 which regulates intracellular pH (pHi). We conducted a functional study of variants at this locus in primary cultures of vascular smooth muscle and endothelial cells. In both cell types, we found genotype-dependent differences for rs13082711 in DNA-nuclear protein interactions, where the risk allele is associated with increased SLC4A7 expression level, NBCn1 availability and function as reflected in elevated steady-state pHi and accelerated recovery from intracellular acidosis. However, in the presence of Na+/H+ exchange activity, the SLC4A7 genotypic effect on net base uptake and steady-state pHi persisted only in vascular smooth muscle cells but not endothelial cells. We found no discernable effect of the missense polymorphism resulting in the amino acid substitution Glu326Lys. The finding of a genotypic influence on SLC4A7 expression and pHi regulation in vascular smooth muscle cells provides an insight into the molecular mechanism underlying the association of variation at the SLC4A7 locus with blood pressure.

Enhanced nitric oxide signaling amplifies vasorelaxation of human colon cancer feed arteries.

Inadequate perfusion of solid cancer tissue results in low local nutrient and oxygen levels and accumulation of acidic waste products. Previous investigations have focused primarily on tumor blood vessel architecture, and we lack information concerning functional differences between arteries that deliver blood to solid cancer tissue versus normal tissue. Here, we use isometric myography to study resistance-sized arteries from human primary colon adenocarcinomas and matched normal colon tissue. Vasocontraction of colon cancer feed arteries in response to endothelin-1 and thromboxane stimulation is attenuated compared with normal colon arteries despite similar wall dimensions and comparable contractions to arginine vasopressin and K+-induced depolarization. Acetylcholine-induced vasorelaxation and endothelial NO synthase expression are increased in colon cancer feed arteries compared with normal colon arteries, whereas vasorelaxation to exogenous NO donors is unaffected. In congruence, the differences in vasorelaxant and vasocontractile function between colon cancer feed arteries and normal colon arteries decrease after NO synthase inhibition. Rhythmic oscillations in vascular tone, known as vasomotion, are of lower amplitude but similar frequency in colon cancer feed arteries compared with normal colon arteries. In conclusion, higher NO synthase expression and elevated NO signaling amplify vasorelaxation and attenuate vasocontraction of human colon cancer feed arteries. We propose that enhanced endothelial function augments tumor perfusion and represents a potential therapeutic target. NEW & NOTEWORTHY Local vascular resistance influences tumor perfusion. Arteries supplying human colonic adenocarcinomas show enhanced vasorelaxation and reduced vasocontraction mainly due to elevated nitric oxide-mediated signaling. Rhythmic oscillations in tone, known as vasomotion, are attenuated in colon cancer feed arteries.

Murine breast cancer feed arteries are thin-walled with reduced α1A-adrenoceptor expression and attenuated sympathetic vasocontraction.

BACKGROUND: Perfusion of breast cancer tissue limits oxygen availability and metabolism but angiogenesis inhibitors have hitherto been unsuccessful for breast cancer therapy. In order to identify abnormalities and possible therapeutic targets in mature cancer arteries, we here characterize the structure and function of cancer feed arteries and corresponding control arteries from female FVB/N mice with ErbB2-induced breast cancer. METHODS: We investigated the contractile function of breast cancer feed arteries and matched control arteries by isometric myography and evaluated membrane potentials and intracellular [Ca2+] using sharp electrodes and fluorescence microscopy, respectively. Arterial wall structure is assessed by transmission light microscopy of arteries mounted in wire myographs and by evaluation of histological sections using the unbiased stereological disector technique. We determined the expression of messenger RNA by reverse transcription and quantitative polymerase chain reaction and studied receptor expression by confocal microscopy of arteries labelled with the BODIPY-tagged α1-adrenoceptor antagonist prazosin. RESULTS: Breast cancer feed arteries are thin-walled and produce lower tension than control arteries of similar diameter in response to norepinephrine, thromboxane-analog U46619, endothelin-1, and depolarization with elevated [K+]. Fewer layers of similarly-sized vascular smooth muscle cells explain the reduced media thickness of breast cancer arteries. Evidenced by lower media stress, norepinephrine-induced and thromboxane-induced tension development of breast cancer arteries is reduced more than is explained by the thinner media. Conversely, media stress during stimulation with endothelin-1 and elevated [K+] is similar between breast cancer and control arteries. Correspondingly, vascular smooth muscle cell depolarizations and intracellular Ca2+ responses are attenuated in breast cancer feed arteries during norepinephrine but not during endothelin-1 stimulation. Protein expression of α1-adrenoceptors and messenger RNA levels for α1A-adrenoceptors are lower in breast cancer arteries than control arteries. Sympathetic vasocontraction elicited by electrical field stimulation is inhibited by α1-adrenoceptor blockade and reduced in breast cancer feed arteries compared to control arteries. CONCLUSION: Thinner media and lower α1-adrenoceptor expression weaken contractions of breast cancer feed arteries in response to sympathetic activity. We propose that abnormalities in breast cancer arteries can be exploited to modify tumor perfusion and thereby either starve cancer cells or facilitate drug and oxygen delivery during chemotherapy or radiotherapy.

The net acid extruders NHE1, NBCn1 and MCT4 promote mammary tumor growth through distinct but overlapping mechanisms.

High metabolic and proliferative rates in cancer cells lead to production of large amounts of H+ and CO2 , and as a result, net acid extruding transporters are essential for the function and survival of cancer cells. We assessed protein expression of the Na+ /H+ exchanger NHE1, the Na+ - HCO3- cotransporter NBCn1, and the lactate-H+ cotransporters MCT1 and -4 by immunohistochemical analysis of a large cohort of breast cancer samples. We found robust expression of these transporters in 20, 10, 4 and 11% of samples, respectively. NHE1 and NBCn1 expression both correlated positively with progesterone receptor status, NHE1 correlated negatively and NBCn1 positively with HER2 status, whereas MCT4 expression correlated with lymph node status. Stable shRNA-mediated knockdown (KD) of either NHE1 or NBCn1 in the MDA-MB-231 triple-negative breast cancer (TNBC) cell line significantly reduced steady-state intracellular pH (pHi ) and capacity for pHi recovery after an acid load. Importantly, KD of any of the three transporters reduced in vivo primary tumor growth of MDA-MB-231 xenografts. However, whereas KD of NBCn1 or MCT4 increased tumor-free survival and decreased in vitro proliferation rate and colony growth in soft agar, KD of NHE1 did not have these effects. Moreover, only MCT4 KD reduced Akt kinase activity, PARP and CD147 expression and cell motility. This work reveals that different types of net acid extruding transporters, NHE1, NBCn1 and MCT4, are frequently expressed in patient mammary tumor tissue and demonstrates for the first time that they promote growth of TNBC human mammary tumors in vivo via distinct but overlapping mechanisms.

Negative News: Cl- and HCO3- in the Vascular Wall.

Cl(-) and HCO3 (-) are the most prevalent membrane-permeable anions in the intra- and extracellular spaces of the vascular wall. Outwardly directed electrochemical gradients for Cl(-) and HCO3 (-) permit anion channel opening to depolarize vascular smooth muscle and endothelial cells. Transporters and channels for Cl(-) and HCO3 (-) also modify vascular contractility and structure independently of membrane potential. Transport of HCO3 (-) regulates intracellular pH and thereby modifies the activity of enzymes, ion channels, and receptors. There is also evidence that Cl(-) and HCO3 (-) transport proteins affect gene expression and protein trafficking. Considering the extensive implications of Cl(-) and HCO3 (-) in the vascular wall, it is critical to understand how these ions are transported under physiological conditions and how disturbances in their transport can contribute to disease development. Recently, sensing mechanisms for Cl(-) and HCO3 (-) have been identified in the vascular wall where they modify ion transport and vasomotor function, for instance, during metabolic disturbances. This review discusses current evidence that transport (e.g., via NKCC1, NBCn1, Ca(2+)-activated Cl(-) channels, volume-regulated anion channels, and CFTR) and sensing (e.g., via WNK and RPTPγ) of Cl(-) and HCO3 (-) influence cardiovascular health and disease.

Perivascular tissue inhibits rho-kinase-dependent smooth muscle Ca(2+) sensitivity and endothelium-dependent H2 S signalling in rat coronary arteries.

Interactions between perivascular tissue (PVT) and the vascular wall modify artery tone and contribute to local blood flow regulation. Using isometric myography, fluorescence microscopy, membrane potential recordings and phosphospecific immunoblotting, we investigated the cellular mechanisms by which PVT affects constriction and relaxation of rat coronary septal arteries. PVT inhibited vasoconstriction to thromboxane, serotonin and α1 -adrenergic stimulation but not to depolarization with elevated extracellular [K(+) ]. When PVT was wrapped around isolated arteries or placed at the bottom of the myograph chamber, a smaller yet significant inhibition of vasoconstriction was observed. Resting membrane potential, depolarization to serotonin or thromboxane stimulation, and resting and serotonin-stimulated vascular smooth muscle [Ca(2+) ]-levels were unaffected by PVT. Serotonin-induced vasoconstriction was almost abolished by rho-kinase inhibitor Y-27632 and modestly reduced by protein kinase C inhibitor bisindolylmaleimide X. PVT reduced phosphorylation of myosin phosphatase targeting subunit (MYPT) at Thr850 by ∼40% in serotonin-stimulated arteries but had no effect on MYPT-phosphorylation in arteries depolarized with elevated extracellular [K(+) ]. The net anti-contractile effect of PVT was accentuated after endothelial denudation. PVT also impaired vasorelaxation and endothelial Ca(2+) responses to cholinergic stimulation. Methacholine-induced vasorelaxation was mediated by NO and H2 S, and particularly the H2 S-dependent (dl-propargylglycine- and XE991-sensitive) component was attenuated by PVT. Vasorelaxation to NO- and H2 S-donors was maintained in arteries with PVT. In conclusion, cardiomyocyte-rich PVT surrounding coronary arteries releases diffusible factors that reduce rho-kinase-dependent smooth muscle Ca(2+) sensitivity and endothelial Ca(2+) responses. These mechanisms inhibit agonist-induced vasoconstriction and endothelium-dependent vasorelaxation and suggest new signalling pathways for metabolic regulation of blood flow.

Na+,HCO3- -cotransport is functionally upregulated during human breast carcinogenesis and required for the inverted pH gradient across the plasma membrane.

Metabolic and biochemical changes during breast carcinogenesis enhance cellular acid production. Extrusion of the acid load from the cancer cells raises intracellular pH, while it decreases extracellular pH creating an inverted pH gradient across the plasma membrane compared to normal cells and promoting cancer cell metabolism, proliferation, migration, and invasion. We investigated the effects of breast carcinogenesis on the mechanisms of cellular pH control using multicellular epithelial organoids freshly isolated from human primary breast carcinomas and matched normal breast tissue. Intracellular pH was measured by fluorescence microscopy, while protein expression was investigated by immunofluorescence imaging and immunoblotting. We found that cellular net acid extrusion increased during human breast carcinogenesis due to enhanced Na(+),HCO3 (-)-cotransport, which created an alkaline shift (~0.3 units of magnitude) in steady-state intracellular pH of human primary breast carcinomas compared to normal breast tissue. Na(+)/H(+)-exchange activity and steady-state intracellular pH in the absence of CO2/HCO3 (-) were practically unaffected by breast carcinogenesis. These effects were evident under both acidic (pH 6.8, representative of the tumor microenvironment) and physiological (pH 7.4) extracellular conditions. Protein expression of the Na(+),HCO3 (-)-cotransporter NBCn1 (SLC4A7), which has been linked to breast cancer susceptibility in multiple genome-wide association studies, was twofold higher in human breast carcinomas compared to matched normal breast tissue. Protein expression of the Na(+)/H(+)-exchanger NHE1 (SLC9A1) was markedly less affected. We propose that upregulated NBCn1 during human breast carcinogenesis contributes to the characteristic acid distribution within human breast carcinomas and thereby plays a pathophysiological role for breast cancer development and progression.

Splice cassette II of Na+,HCO3(-) cotransporter NBCn1 (slc4a7) interacts with calcineurin A: implications for transporter activity and intracellular pH control during rat artery contractions.

Activation of Na(+),HCO3(-) cotransport in vascular smooth muscle cells (VSMCs) contributes to intracellular pH (pH(i)) control during artery contraction, but the signaling pathways involved have been unknown. We investigated whether physical and functional interactions between the Na(+),HCO3(-) cotransporter NBCn1 (slc4a7) and the Ca(2+)/calmodulin-activated serine/threonine phosphatase calcineurin exist and play a role for pHi control in VSMCs. Using a yeast two-hybrid screen, we found that splice cassette II from the N terminus of NBCn1 interacts with calcineurin Aß. When cassette II was truncated or mutated to disrupt the putative calcineurin binding motif PTVVIH, the interaction was abolished. Native NBCn1 and calcineurin Aß co-immunoprecipitated from A7r5 rat VSMCs. A peptide (acetyl-DDIPTVVIH-amide), which mimics the putative calcineurin binding motif, inhibited the co-immunoprecipitation whereas a mutated peptide (acetyl-DDIATAVAA-amide) did not. Na(+),HCO3(-) cotransport activity was investigated in VSMCs of mesenteric arteries after an NH4(+) prepulse. During depolarization with 50 mM extracellular K(+) to raise intracellular [Ca(2+)], Na(+),HCO3(-) cotransport activity was inhibited 20-30% by calcineurin inhibitors (FK506 and cyclosporine A). FK506 did not affect Na(+),HCO3(-) cotransport activity in VSMCs when cytosolic [Ca(2+)] was lowered by buffering, nor did it disrupt binding between NBCn1 and calcineurin Aß. FK506 augmented the intracellular acidification of VSMCs during norepinephrine-induced artery contractions. No physical or functional interactions between calcineurin Aß and the Na(+)/H(+) exchanger NHE1 were observed in VSMCs. In conclusion, we demonstrate a physical interaction between calcineurin Aß and cassette II of NBCn1. Intracellular Ca(2+) activates Na(+),HCO3(-) cotransport activity in VSMCs in a calcineurin-dependent manner which is important for protection against intracellular acidification.