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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 HCO 3 –-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 +,HCO 3 –-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 +,HCO 3 –-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 +,HCO 3 –-cotransport primes breast tissue for cancer development.

Intracellular Ca 2+ 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 Ca 2+ concentrations in breast and colon cancer tissue with preferential focus on intracellular Ca 2+ release in response to purinergic and cholinergic stimuli. Inhibition of the sarco-/endoplasmic reticulum Ca 2+ ATPase with cyclopiazonic acid elicited larger Ca 2+ responses in breast cancer tissue, but not in colon cancer tissue, relative to respective normal tissue. The resting intracellular Ca 2+ concentration was elevated, and ATP, UTP and acetylcholine induced strongly augmented intracellular Ca 2+ responses in breast cancer tissue compared with normal breast tissue. In contrast, resting intracellular Ca 2+ levels and acetylcholine-induced increases in intracellular Ca 2+ concentrations were unaffected and ATP- and UTP-induced Ca 2+ responses were smaller in colon cancer tissue compared with normal colon tissue. In accordance with the amplified Ca 2+ 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 Ca 2+ 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.

Intracellular Ca

BACKGROUND: The electroneutral Na +/HCO 3 - 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 NH 4 + transport measured in isolated perfused mTALs. Quantitative RT-PCR and immunoblotting were used to evaluate NBCn1 expression. Tissue [NH 4 +] was measured in renal biopsies, NH 4 + excretion and titratable acid quantified in spot urine, and arterial blood gasses evaluated in normoventilated mice. RESULTS: Basolateral Na +/HCO 3 - cotransport activity was similar in isolated perfused mTALs from wild-type and NBCn1 knockout mice under control conditions. During metabolic acidosis, basolateral Na +/HCO 3 - 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 NH 4 + reabsorption across mTALs by approximately 60%, abolished the renal corticomedullary NH 4 + gradient, reduced the capacity for urinary NH 4 + excretion by approximately 50%, and delayed recovery of arterial blood pH and standard [HCO 3 -] from their initial decline. CONCLUSIONS: During metabolic acidosis, NBCn1 is required for the upregulated basolateral HCO 3 - uptake and transepithelial NH 4 + reabsorption in mTALs, renal medullary NH 4 + accumulation, urinary NH 4 + excretion, and early recovery of arterial blood pH and standard [HCO 3 -]. These findings support that NBCn1 facilitates urinary net acid excretion by neutralizing intracellular H + released during NH 4 + reabsorption across mTALs.

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 +,HCO 3 − 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 +,HCO 3 − 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 +,HCO 3 − 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.

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

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 HCO 3 - concentrations better than normal cells when challenged with the marked deviations in extracellular H +, HCO 3 -, 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.

Acid-base conditions modify artery tone and tissue perfusion but the involved vascular-sensing mechanisms and disease consequences remain unclear. We experimentally investigated transgenic mice and performed genetic studies in a UK-based human cohort. We show that endothelial cells express the putative HCO3–-sensor receptor-type tyrosine-protein phosphatase RPTPγ, which enhances endothelial intracellular Ca2+-responses in resistance arteries and facilitates endothelium-dependent vasorelaxation only when CO2/HCO3– is present. Consistent with waning RPTPγ-dependent vasorelaxation at low [HCO3–], RPTPγ limits increases in cerebral perfusion during neuronal activity and augments decreases in cerebral perfusion during hyperventilation. RPTPγ does not influence resting blood pressure but amplifies hyperventilation-induced blood pressure elevations. Loss-of-function variants in PTPRG, encoding RPTPγ, are associated with increased risk of cerebral infarction, heart attack, and reduced cardiac ejection fraction. We conclude that PTPRG is an ischemia susceptibility locus; and RPTPγ-dependent sensing of HCO3– adjusts endothelium-mediated vasorelaxation, microvascular perfusion, and blood pressure during acid-base disturbances and altered tissue metabolism., eLife digest Restricted blood flow in the heart or brain can deprive these vital organs of oxygen, thereby causing a heart attack or stroke. However, the body has compensatory mechanisms to mitigate damage: if the blood flow is reduced in one blood vessel, acidic waste accumulates locally. This causes nearby blood vessels to widen and increase the oxygen supply. Although scientists first observed this process 140 years ago, they have not yet devised a way to use it for treatment of heart attack or stroke. Now, Hansen et al. discovered that a protein called RPTPγ, which is found on the lining of blood vessels, could be a good target for drugs intended to reduce the consequences of heart attacks and strokes. The protein RPTPγ has a similar structure to other proteins that bind bicarbonate, an important ion that buffers acids in the body. RPTPγ can also trigger signals to nearby cells, which suggests that the protein can monitor bicarbonate levels in the blood and tissue and alert blood vessels of the need to widen. Hansen et al. found that the blood vessels of mice that lacked RPTPγ were unable to widen when needed. Moreover, mice without RPTPγ experienced abnormal changes in blood pressure and blood flow to the brain when oxygen consumption was elevated or pH was disrupted. Hansen et al. further analyzed genetic and health data from nearly 50,000 individuals in the UK Biobank. These analyses revealed that people with genetic changes that render RPTPγ ineffective are at higher risk of having a heart attack or stroke. People with these genetic variants also have reduced heart pumping ability. The experiments suggest that a lack of functional RPTPγ affects an individual’s ability to adjust local blood flow in response to acid-base disturbances and oxygen deficits, increasing the risk of a heart attack or stroke. This information may help scientists develop new ways to prevent or treat heart attacks and strokes, or to treat other conditions like cancer, where pH is disturbed.

BACKGROUND: The electroneutral Na(+)/HCO(3)(−) 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 NH(4)(+) transport measured in isolated perfused mTALs. Quantitative RT-PCR and immunoblotting were used to evaluate NBCn1 expression. Tissue [NH(4)(+)] was measured in renal biopsies, NH(4)(+) excretion and titratable acid quantified in spot urine, and arterial blood gasses evaluated in normoventilated mice. RESULTS: Basolateral Na(+)/HCO(3)(−) cotransport activity was similar in isolated perfused mTALs from wild-type and NBCn1 knockout mice under control conditions. During metabolic acidosis, basolateral Na(+)/HCO(3)(−) 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 NH(4)(+) reabsorption across mTALs by approximately 60%, abolished the renal corticomedullary NH(4)(+) gradient, reduced the capacity for urinary NH(4)(+) excretion by approximately 50%, and delayed recovery of arterial blood pH and standard [HCO(3)(−)] from their initial decline. CONCLUSIONS: During metabolic acidosis, NBCn1 is required for the upregulated basolateral HCO(3)(−) uptake and transepithelial NH(4)(+) reabsorption in mTALs, renal medullary NH(4)(+) accumulation, urinary NH(4)(+) excretion, and early recovery of arterial blood pH and standard [HCO(3)(−)]. These findings support that NBCn1 facilitates urinary net acid excretion by neutralizing intracellular H(+) released during NH(4)(+) reabsorption across mTALs.

Despite a widespread expression pattern in the central nervous system, the role of the sodium bicarbonate cotransporter NBCn1/Slc4a7 has not been investigated for locomotor activity, emotion and cognition. Here, we addressed the behavioral consequences of NBCn1 knockout and evaluated hearing and vision that are reportedly impaired in an earlier line of NBCn1 knockout mice and may contribute to behavioral changes. In a circular open field, the knockout mice traveled a shorter distance, especially in the periphery of the chamber, than wildtype littermates. The knockout mice also traveled a shorter total distance in a home cage-like open field. Rearing and grooming behaviors were reduced. The knockout and control mice displayed similar time spent and number of open and closed arms in the elevated plus maze test, indicating negligible change in anxiety. In the Morris water maze test, both groups of mice learned the location of an escape platform within comparable time on the training trials and showed similar platform identification on the probe trial. The knockout mice maintained normal visual responses in the optokinetic drum and produced evoked potentials in response to light stimuli. However, these mice failed to produce auditory evoked potentials. qPCR revealed a robust expression of an alternatively transcribed NBCn1 variant in the knockout mouse retina. These results indicate that NBCn1 deletion leads to reduced locomotor activity in mice by affecting their exploratory behaviors or emotionality. The deletion also causes hearing loss, but its effect on vision varies between different lines of knockout mice.

The acidic tumor microenvironment modifies malignant cell behavior. Here, we study consequences of the microenvironment in breast carcinomas. Beginning at carcinogen-based breast cancer induction, we supply either regular or NaHCO3-containing drinking water to female C57BL/6j mice. We evaluate urine and blood acid-base status, tumor metabolism (microdialysis sampling), and tumor pH (pH-sensitive microelectrodes) in vivo. Based on freshly isolated epithelial organoids from breast carcinomas and normal breast tissue, we assess protein expression (immunoblotting, mass spectrometry), intracellular pH (fluorescence microscopy), and cell proliferation (bromodeoxyuridine incorporation). Oral NaHCO3 therapy increases breast tumor pH in vivo from 6.68 &plusmn, 0.04 to 7.04 &plusmn, 0.09 and intracellular pH in breast epithelial organoids by ~0.15. Breast tumors develop with median latency of 85.5 &plusmn, 8.2 days in NaHCO3-treated mice vs. 82 &plusmn, 7.5 days in control mice. Oral NaHCO3 therapy does not affect tumor growth, histopathology or glycolytic metabolism. The capacity for cellular net acid extrusion is increased in NaHCO3-treated mice and correlates negatively with breast tumor latency. Oral NaHCO3 therapy elevates proliferative activity in organoids from breast carcinomas. Changes in protein expression patterns&mdash, observed by high-throughput proteomics analyses&mdash, between cancer and normal breast tissue and in response to oral NaHCO3 therapy reveal complex influences on metabolism, cytoskeleton, cell-cell and cell-matrix interaction, and cell signaling pathways. We conclude that oral NaHCO3 therapy neutralizes the microenvironment of breast carcinomas, elevates the cellular net acid extrusion capacity, and accelerates proliferation without net effect on breast cancer development or tumor growth. We demonstrate unexpected pro-neoplastic consequences of oral NaHCO3 therapy that in breast tissue cancel out previously reported anti-neoplastic effects.

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.

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.

The CO2/HCO3– buffer minimizes pH changes in response to acid–base loads, HCO3– provides substrate for Na+,HCO3–-cotransporters and Cl–/HCO3–-exchangers, and H+ and HCO3– modify vasomotor responses during acid–base disturbances. We show here that rat middle cerebral arteries express cytosolic, mitochondrial, extracellular, and secreted carbonic anhydrase isoforms that catalyze equilibration of the CO2/HCO3– buffer. Switching from CO2/HCO3–-free to CO2/HCO3–-containing extracellular solution results in initial intracellular acidification due to hydration of CO2 followed by gradual alkalinization due to cellular HCO3– uptake. Carbonic anhydrase inhibition decelerates the initial acidification and attenuates the associated transient vasoconstriction without affecting intracellular pH or artery tone at steady-state. Na+,HCO3–-cotransport and Na+/H+-exchange activity after NH4+-prepulse-induced intracellular acidification are unaffected by carbonic anhydrase inhibition. Extracellular surface pH transients induced by transmembrane NH3 flux are evident under CO2/HCO3–-free conditions but absent when the buffer capacity and apparent H+ mobility increase in the presence of CO2/HCO3– even after the inhibition of carbonic anhydrases. We conclude that (a) intracellular carbonic anhydrase activity accentuates pH transients and vasoconstriction in response to acute elevations of pCO2, (b) CO2/HCO3– minimizes extracellular surface pH transients without requiring carbonic anhydrase activity, and (c) carbonic anhydrases are not rate limiting for acid–base transport across cell membranes during recovery from intracellular acidification.

The previous reports on an addiction vulnerability marker in the human SLC4A7 gene encoding the Na/HCO3 transporter NBCn1 suggest that this pH-regulating protein may affect alcohol-related behavior and response. Here, we examined alcohol consumption and sensitivity to the sedative effects of alcohol in male NBCn1 knockout mice. These mice displayed lower pH in neurons than wildtype controls, determined by intracellular pH in hippocampal neuronal cultures. Neurons from knockout mice had a higher action potential threshold and a more depolarized membrane potential, thus reducing membrane excitability. In a two-bottle free choice procedure, knockout mice consumed more alcohol than controls and consistently increased alcohol consumption after repeated alcohol deprivation periods. Quinine and sucrose preference was similar between genotypes. Knockout mice showed increased propensity for alcohol-induced conditioned place preference. In loss of righting reflex assessment, knockout mice revealed increased sensitivity to alcohol-induced sedation and developed tolerance to the sedation after repeated alcohol administrations. Furthermore, chronic alcohol consumption caused NBCn1 downregulation in the hippocampus and striatum of mice and humans. These results demonstrate an important role of NBCn1 in regulation of alcohol consumption and sensitivity to alcohol-induced sedation.

Lymph vessels counteract edema by transporting interstitial fluid from peripheral tissues to the large veins and serve as conduits for immune cells, cancer cells, and pathogens. Because edema during inflammation and malignancies is frequently associated with acidosis, we tested the hypothesis that acid‐base disturbances affect human thoracic duct contractions. We studied, by isometric and isobaric myography, the contractile function of human thoracic duct segments harvested with written informed consent from patients undergoing esophageal cancer surgery. Human thoracic ducts produce complex contractile patterns consisting of tonic rises in tension (isometric myography) or decreases in diameter (isobaric myography) with superimposed phasic contractions. Active tone development decreases substantially (~90% at 30 vs. 7 mmHg) at elevated transmural pressure. Acidosis inhibits spontaneous as well as noradrenaline‐ and serotonin‐induced phasic contractions of human thoracic ducts by 70–90% at extracellular pH 6.8 compared to 7.4 with less pronounced effects observed at pH 7.1. Mean tension responses to noradrenaline and serotonin – averaged over the entire period of agonist exposure – decrease by ~50% at extracellular pH 6.8. Elevating extracellular [K+] from the normal resting level around 4 mmol/L increases overall tension development but reduces phasic activity to a level that is no different between human thoracic duct segments investigated at normal and low extracellular pH. In conclusion, we show that extracellular acidosis inhibits human thoracic duct contractions with more pronounced effects on phasic than tonic contractions. We propose that reduced phasic activity of lymph vessels at low pH attenuates lymph propulsion and increases the risk of edema formation.

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

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.

Increased metabolism accelerates local acid production in cancer tissue. The mechanisms eliminating acidic waste products from human colon cancer tissue represent promising therapeutic targets for pharmacological manipulation in order to improve prognosis for the increasing number of patients with colon cancer. We sampled biopsies of human colonic adenocarcinomas and matched normal colon tissue from patients undergoing colon cancer surgery. We measured steady-state intracellular pH and rates of net acid extrusion in freshly isolated human colonic crypts based on fluorescence microscopy. Net acid extrusion was almost entirely (>95%) Na+-dependent. The capacity for net acid extrusion was increased and steady-state intracellular pH elevated around 0.5 in crypts from colon cancer tissue compared with normal colon tissue irrespective of whether they were investigated in the presence or absence of CO2/HCO3–. The accelerated net acid extrusion from the human colon cancer tissue was sensitive to the Na+/H+-exchange inhibitor cariporide. We conclude that enhanced net acid extrusion via Na+/H+-exchange elevates intracellular pH in human colon cancer tissue.

Alterations in the retinal microvessel (RMV) compartment occurring in systemic disease states such as diabetes may eventually contribute to blindness. To specifically address the pathophysiological role of the microvasculature we developed a new method for RMV bulk isolation from individual rats. The extraction procedure performed in the cold throughout takes less than one hour. Slight modifications enable isolation of brain microvessels (BMVs) for comparison. Microscopically, RMVs and BMVs consisted mainly of capillaries of good structural integrity. The endothelial cell/pericyte ratio was approximately 1.8 in RMVs and 2.7 in BMVs, well in agreement with data from intact vascular beds. Total RNA extracted from individual rats amounted to approximately 7 ng in RMVs, 50 ng in BMVs, and 155 ng in pial arteries (which were also isolated) with highly preserved integrity throughout. Measurements using microfluidic card methodology revealed segregation of RMVs, BMVs, and pial arteries in distinct clusters based on principal component analysis. In all three vascular compartments endothelial cell-specific markers were significantly enriched. Similarly, pericyte-specific markers displayed accumulation in RMVs, BMVs, and pial arteries, the latter probably reflecting the common ontogenetic origin of pericytes and smooth muscle cells. Isolation of RMVs, BMVs, and pial arteries from rats suffering from 8-weeks hyperglycemia yielded expression patterns of endothelial cell- and pericyte-specific marker genes largely comparable to those obtained in control rats.Our newly developed protocols allow for selective studies of RMVs from individual rats to characterize reactive pathways, in comparison with the ontogenetically closely related BMVs. Moreover, our protocols with inclusion of pial arteries enable comparative studies of the macro- and microvasculature from the same organ.

Metabolic acid production challenges cellular pH homeostasis in solid cancer tissue, and mechanisms of net acid extrusion represent promising new targets for breast cancer therapy. Here, we used genetically engineered mice to investigate the contribution of the Na + ,HCO 3 – -cotransporter NBCn1 (Slc4a7) to intracellular acid–base regulation in ErbB2-induced breast cancer tissue and the consequences of NBCn1 knockout for breast tumor development and growth. We demonstrate an approximately 2-fold increase of NBCn1 protein abundance in ErbB2-induced breast cancer tissue compared to normal breast tissue despite a 4-fold decrease in the NBCn1 mRNA level. In congruence, we show that NBCn1 facilitates net acid extrusion and elevates steady-state intracellular pH in breast cancer tissue. Disruption of NBCn1 expression delayed ErbB2-induced breast carcinogenesis from a median tumor-free survival of 9.5 months in wild-type mice to 12 months in NBCn1-knockout mice and decelerated the tumor growth rate by approximately 1/3. Glycolytic metabolism—evaluated based on the interstitial concentrations of lactate and glucose measured in microdialysates—was increased in breast cancer tissue compared to normal breast tissue, but was unaffected by NBCn1 knockout. Disruption of NBCn1 expression inhibited cell proliferation—evaluated by staining for the proliferative marker Ki67—particularly in central tumor areas with predicted increase in acid loading from glycolytic metabolism. In conclusion, NBCn1 regulates intracellular pH in ErbB2-induced breast cancer tissue by providing a pathway for cellular uptake of HCO 3 – , which can neutralize metabolic acidic waste products. Disrupting NBCn1 expression delays ErbB2-induced breast cancer development, inhibits cancer cell proliferation, and decelerates tumor growth.

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.

Metabolic acid production challenges cellular pH homeostasis in solid cancer tissue, and mechanisms of net acid extrusion represent promising new targets for breast cancer therapy. Here, we used genetically engineered mice to investigate the contribution of the Na

MedChemComm 6(12), 2163-2169(2015). doi:10.1039/C5MD00367A, Published by RSC Publ., Cambridge

Metabolic regulation of cerebrovascular tone directs blood flow to areas of increased neuronal activity and during disease states partially compensates for insufficient perfusion by enhancing blood flow in collateral blood vessels. Acid–base disturbances frequently occur as result of enhanced metabolism or insufficient blood supply, but despite definitive evidence that acid–base disturbances alter arterial tone, effects of individual acid–base equivalents and the underlying signaling mechanisms are still being debated. H+is an important intra- and extracellular messenger that modifies cerebrovascular tone. In addition, low extracellular [HCO3–] promotes cerebrovascular contraction through an endothelium-dependent mechanism. CO2alters arterial tone development via changes in intra- and extracellular pH but it is still controversial whether CO2also has direct vasomotor effects. Vasocontractile responses to low extracellular [HCO3–] and acute CO2-induced decreases in intracellular pH can counteract H+-mediated vasorelaxation during metabolic and respiratory acidosis, respectively, and may thereby reduce the risk of capillary damage and cerebral edema that could be consequences of unopposed vasodilation. In this review, the signaling mechanisms for acid–base equivalents in cerebral arteries and the mechanisms of intracellular pH control in the arterial wall are discussed in the context of metabolic regulation of cerebrovascular tone and local perfusion.

Aim The electroneutral Na+,HCO3–-cotransporter NBCn1 and Na+/H+-exchanger NHE1 regulate acid-base balance in vascular smooth muscle cells (VSMCs) and modify artery function and structure. Pathological conditions—notably ischemia—can dramatically perturb intracellular (i) and extracellular (o) pH and [Na+]. We examined effects of low [Na+]o and pHo on NBCn1 and NHE1 activity in VSMCs of small arteries. Methods We measured pHi by 2’,7’-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-based fluorescence microscopy of mouse mesenteric arteries and induced intracellular acidification by NH4+-prepulse technique. Results NBCn1 activity—defined as Na+-dependent, amiloride-insensitive net base uptake with CO2/HCO3– present—was inhibited equally when pHo decreased from 7.4 (22 mM HCO3–/5% CO2) by metabolic (pHo 7.1/11 mM HCO3–: 22±8%; pHo 6.8/5.5 mM HCO3–: 61±7%) or respiratory (pHo 7.1/10% CO2: 35±11%; pHo 6.8/20% CO2: 56±7%) acidosis. Extracellular acidosis more prominently inhibited NHE1 activity—defined as Na+-dependent net acid extrusion without CO2/HCO3– present—at both pHo 7.1 (45±9%) and 6.8 (85±5%). Independently of pHo, lowering [Na+]o from 140 to 70 mM reduced NBCn1 and NHE1 activity less than 20% whereas transport activities declined markedly (25-50%) when [Na+]o was reduced to 35 mM. Steady-state pHi decreased more during respiratory (ΔpHi/ΔpHo=71±4%) than metabolic (ΔpHi/ΔpHo=30±7%) acidosis. Conclusion Extracellular acidification inhibits NBCn1 and NHE1 activity in VSMCs. NBCn1 is equivalently inhibited when pCO2 is raised or [HCO3–]o decreased. Lowering [Na+]o inhibits NBCn1 and NHE1 markedly only below the typical physiological and pathophysiological range. We propose that inhibition of Na+-dependent net acid extrusion at low pHo protects against cellular Na+ overload at the cost of intracellular acidification. This article is protected by copyright. All rights reserved.

The CO(2)/HCO(3)(–) buffer minimizes pH changes in response to acid–base loads, HCO(3)(–) provides substrate for Na(+),HCO(3)(–)-cotransporters and Cl(–)/HCO(3)(–)-exchangers, and H(+) and HCO(3)(–) modify vasomotor responses during acid–base disturbances. We show here that rat middle cerebral arteries express cytosolic, mitochondrial, extracellular, and secreted carbonic anhydrase isoforms that catalyze equilibration of the CO(2)/HCO(3)(–) buffer. Switching from CO(2)/HCO(3)(–)-free to CO(2)/HCO(3)(–)-containing extracellular solution results in initial intracellular acidification due to hydration of CO(2) followed by gradual alkalinization due to cellular HCO(3)(–) uptake. Carbonic anhydrase inhibition decelerates the initial acidification and attenuates the associated transient vasoconstriction without affecting intracellular pH or artery tone at steady-state. Na(+),HCO(3)(–)-cotransport and Na(+)/H(+)-exchange activity after NH(4)(+)-prepulse-induced intracellular acidification are unaffected by carbonic anhydrase inhibition. Extracellular surface pH transients induced by transmembrane NH(3) flux are evident under CO(2)/HCO(3)(–)-free conditions but absent when the buffer capacity and apparent H(+) mobility increase in the presence of CO(2)/HCO(3)(–) even after the inhibition of carbonic anhydrases. We conclude that (a) intracellular carbonic anhydrase activity accentuates pH transients and vasoconstriction in response to acute elevations of pCO(2), (b) CO(2)/HCO(3)(–) minimizes extracellular surface pH transients without requiring carbonic anhydrase activity, and (c) carbonic anhydrases are not rate limiting for acid–base transport across cell membranes during recovery from intracellular acidification.

Introduction: Healthy women of reproductive age have a vaginal pH around 4.5, whereas little is known about pH in the upper genital tract. A shift in the vaginal microbiota may result in an elevated pH in the upper genital tract. This might contribute to decreased fertility and increased risk of preterm birth. Therefore, we aimed to measure pH in different compartments of the female genital tract in both nonpregnant and pregnant women, stratifying into a normal and abnormal vaginal microbiota. Material and methods: In this descriptive study, we included 6 nonpregnant, 12 early-pregnant, and 8 term-pregnant women. A pH gradient was recorded with a flexible pH probe. An abnormal vaginal microbiota was diagnosed by a quantitative polymerase chain reaction technique for Atopobium vaginae; Sneathia sanguinegens; Leptotrichia amnionii; bacterial vaginosis-associated bacterium 1, 2, 3, and TM7; and Prevotella spp. among others. Results: In all participants we found the pH gradient in the lower reproductive canal to be most acidic in the lower vagina and most alkaline in the upper uterine cavity. Women with an abnormal vaginal microbiota had an increased pH in the lower vagina compared to the other groups. Conclusions: There is a pronounced pH gradient within the female genital tract. This gradient is not disrupted in women with an abnormal vaginal microbiota.

Activation of Na+,HCO3− cotransport in vascular smooth muscle cells (VSMCs) contributes to intracellular pH (pHi) 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 Ca2+/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 Aβ. When cassette II was truncated or mutated to disrupt the putative calcineurin binding motif PTVVIH, the interaction was abolished. Native NBCn1 and calcineurin Aβ 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 [Ca2+], 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 [Ca2+] was lowered by buffering, nor did it disrupt binding between NBCn1 and calcineurin Aβ. FK506 augmented the intracellular acidification of VSMCs during norepinephrine-induced artery contractions. No physical or functional interactions between calcineurin Aβ and the Na+/H+ exchanger NHE1 were observed in VSMCs. In conclusion, we demonstrate a physical interaction between calcineurin Aβ and cassette II of NBCn1. Intracellular Ca2+ activates Na+,HCO3− cotransport activity in VSMCs in a calcineurin-dependent manner which is important for protection against intracellular acidification.

Gap junctions mediate intercellular signalling in arteries and contribute to endothelium-dependent vasorelaxation, conducted vascular responses and vasomotion. Considering its putative role in vascular dysfunction, mechanistic insights regarding the control of gap junction conductivity are required. Here, we investigated the consequences of endothelial alkalinisation for gap junction communication and endothelium-dependent vasorelaxation in resistance arteries. We studied mesenteric arteries from NMRI mice by myography, confocal fluorescence microscopy and electrophysiological techniques. Removing CO2/HCO3(-), reducing extracellular [Cl(-)] or adding 4,4-diisothiocyanatostilbene-2,2-disulphonic acid inhibited or reversed Cl(-)/HCO3(-) exchange, alkalinised the endothelium by 0.2-0.3 pH units and inhibited acetylcholine-induced vasorelaxation. NO-synthase-dependent vasorelaxation was unaffected by endothelial alkalinisation whereas vasorelaxation dependent on small- and intermediate-conductance Ca(2+)-activated K(+) channels was attenuated by ∼75%. The difference in vasorelaxation between arteries with normal and elevated endothelial intracellular pH (pHi) was abolished by the gap junction inhibitors 18β-glycyrrhetinic acid and carbenoxolone while other putative modulators of endothelium-derived hyperpolarisations - Ba(2+), ouabain, iberiotoxin, 8Br-cAMP and polyethylene glycol catalase - had no effect. In the absence of CO2/HCO3(-), addition of the Na(+)/H(+)-exchange inhibitor cariporide normalised endothelial pHi and restored vasorelaxation to acetylcholine. Endothelial hyperpolarisations and Ca(2+) responses to acetylcholine were unaffected by omission of CO2/HCO3(-). By contrast, dye transfer between endothelial cells and endothelium-derived hyperpolarisations of vascular smooth muscle cells stimulated by acetylcholine or the proteinase-activated receptor 2 agonist SLIGRL-amide were inhibited in the absence of CO2/HCO3(-). We conclude that intracellular alkalinisation of endothelial cells attenuates endothelium-derived hyperpolarisations in resistance arteries due to inhibition of gap junction communication. These findings highlight the role of pHi in modulating vascular function.

BACKGROUND: Hypertension is a major risk factor for development of atherosclerotic cardiovascular disease (ASCVD). Although lowering blood pressure with antihypertensive drugs reduces the increased risk of ASCVD, residual increased risk still remains, suggesting that hypertension may cause chronic changes that promote atherosclerosis. Thus, we tested the hypothesis that hypertension increases the susceptibility to atherosclerosis in mice even after a period of re-established normotension.METHODS: We used the 2-kidney, 1-clip (2K1C) technique to induce angiotensin-driven renovascular hypertension, and overexpression of the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene to cause severe hypercholesterolemia and atherosclerosis.RESULTS: First, we performed 2K1C (n = 8) or sham surgery (n = 9) in PCSK9 transgenic mice before they were fed a high fat diet for 14 weeks. As expected, 2K1C did not affect cholesterol levels, but induced cardiac hypertrophy and significantly increased the atherosclerotic lesion area compared to sham mice (1.8 fold, p < 0.01). Next, we performed 2K1C (n = 13) or sham surgery (n = 14) in wild-type mice but removed the clipped/sham-operated kidney after 10 weeks to eliminate hypertension, and subsequently induced hypercholesterolemia by way of adeno-associated virus-mediated hepatic gene transfer of PCSK9 combined with high-fat diet. After 14 weeks of hypercholesterolemia, atherosclerotic lesion areas were not significantly different in mice with or without prior 2K1C hypertension (0.95 fold, p = 0.35).CONCLUSION: Renovascular hypertension in mice does not induce pro-atherogenic changes that persist beyond the hypertensive phase. These results indicate that hypertension only promotes atherogenesis when coinciding temporally with hypercholesterolemia.

Genome-wide association studies recently linked the locus for Na(+),HCO(3)(-)-cotransporter NBCn1 (SLC4A7) to breast cancer susceptibility, yet functional insights have been lacking. To determine whether NBCn1, by transporting HCO(3)(-) into cells, may dispose of acid produced during high metabolic activity, we studied the expression of NBCn1 and the functional impact of Na(+),HCO(3)(-)-cotransport in human breast cancer. We found that the plasmalemmal density of NBCn1 was 20-30% higher in primary breast carcinomas and metastases compared to matched normal breast tissue. The increase in NBCn1 density was similar in magnitude to that observed for Na(+)/H(+)-exchanger NHE1 (SLC9A1), a transporter previously implicated in cell migration, proliferation and malignancy. In primary breast carcinomas, the apparent molecular weight for NBCn1 was increased compared to normal tissue. Using pH-sensitive fluorophores, we showed that Na(+),HCO(3)(-)-cotransport is the predominant mechanism of acid extrusion and is inhibited 34 ± 9% by 200 μM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid in human primary breast carcinomas. At intracellular pH (pH(i) ) levels6.6, CO(2)/HCO(3)(-)-dependent mechanisms accounted for90% of total net acid extrusion. Na(+)/H(+)-exchange activity was prominent only at lower pH(i) -values. Furthermore, steady-state pH(i) was 0.35 ± 0.06 units lower in the absence than in the presence of CO(2)/HCO(3)(-). In conclusion, expression of NBCn1 is upregulated in human primary breast carcinomas and metastases compared to normal breast tissue. Na(+),HCO(3)(-)-cotransport is a major determinant of pH(i) in breast cancer and the modest DIDS-sensitivity is consistent with NBCn1 being predominantly responsible. Hence, our results suggest a major pathophysiological role for NBCn1 that may be clinically relevant.

Acid–base transport in the vascular wall remains incompletely understood. Here, we investigated (a) implications of Na(+)/H(+) exchanger NHE1 knockout for vascular smooth muscle (VSMC) and endothelial cell (EC) pH(i) regulation, mesenteric artery morphology, vasomotor function and blood pressure regulation, and (b) consequences of sustained EC and VSMC acidification for vasomotor function. Na(+)/H(+) exchange activity was abolished in VSMCs and ECs from NHE1 knockout mice, but with CO(2)/HCO(3)(−) present, steady-state pH(i) was unaffected. Active tension was 30% smaller in arteries from NHE1 knockout than wild-type mice, and media thickness equally reduced. Number of VSMCs per unit artery length was unchanged whereas volume and cross-sectional area of individual VSMCs were reduced. Media stress, force production per VSMC cross-sectional area and VSMC Ca(2+) responses were unaffected. Blood pressure was 25 mmHg lower in NHE1 knockout than wild-type mice. Omission of CO(2)/HCO(3)(−) caused VSMCs and ECs to acidify substantially more in NHE1 knockout (0.3–0.6 pH-units) than wild-type (0.02–0.1 pH units) mice. Removing CO(2)/HCO(3)(−) inhibited acetylcholine-induced NO-mediated relaxations in arteries from NHE1 knockout but not wild-type mice. Without CO(2)/HCO(3)(−), effects of NO synthase and rho kinase inhibition on noradrenaline-induced contractions were smaller in arteries from NHE1 knockout than wild-type mice whereas the EC Ca(2+) response to acetylcholine, VSMC Ca(2+) response to noradrenaline and vasorelaxation to S-nitroso-N-acetylpenicillamine were unaffected. In conclusion, NHE1 mediates the Na(+)/H(+) exchange in ECs and VSMCs. Under physiological conditions, CO(2)/HCO(3)(−)-dependent mechanisms mask the pH(i)-regulatory function of NHE1. NHE1 knockout causes hypotrophy of VSMCs, reduced artery tension and lower blood pressure. At acidic pH(i), NO-mediated vasorelaxation and rho kinase-dependent VSMC Ca(2+) sensitivity are reduced.

Net acid extrusion from vascular smooth muscle (VSMCs) and endothelial cells (ECs) in the wall of resistance arteries is mediated by the Na+,HCO3– cotransporter NBCn1 (SLC4A7) and the Na+/H+ exchanger NHE1 (SLC9A1) and is essential for intracellular pH (pHi) control. Experimental evidence suggests that the pHi of VSMCs and ECs modulates both vasocontractile and vasodilatory functions in resistance arteries with implications for blood pressure regulation. The connection between disturbed pHi and altered cardiovascular function has been substantiated by a genome-wide association study showing a link between NBCn1 and human hypertension. On this basis, we here review the current evidence regarding (a) molecular mechanisms involved in pHi control in VSMCs and ECs of resistance arteries at rest and during contractions, (b) implications of disturbed pHi for resistance artery function, and (c) involvement of disturbed pHi in the pathogenesis of vascular disease. The current evidence clearly implies that pHi of VSMCs and ECs modulates vascular function and suggests that disturbed pHi either consequent to disturbed regulation or due to metabolic challenges needs to be taken into consideration as a mechanistic component of artery dysfunction and disturbed blood pressure regulation.