Ebbe Boedtkjer

NBCn1 and NHE1 expression and activity in ΔNErbB2 receptor-expressing MCF-7 breast cancer cells: Contributions to pHi regulation and chemotherapy resistance

Altered pH-regulatory ion transport is characteristic of many cancers; however, the mechanisms and consequences are poorly understood. Here, we investigate how a truncated, constitutively active ErbB2 receptor (DeltaNErbB2) common in breast cancer impacts on the Na(+)/H(+)-exchanger NHE1 and the Na(+),HCO(3)(-)-cotransporter NBCn1 in MCF-7 human breast cancer cells and address the roles of these transporters in chemotherapy resistance. Upon DeltaNErbB2 expression, mRNA and protein levels of NBCn1, yet not of NHE1, increased several-fold, and the localization of both transporters was altered paralleling extensive morphological changes. The rate of pH(i) recovery after acid loading increased by 50% upon DeltaNErbB2 expression. Knockdown and pharmacological inhibition confirmed the involvement of both NHE1 and NBCn1 in acid extrusion. NHE1 inhibition or knockdown sensitized DeltaNErbB2-expressing cells to cisplatin-induced programmed cell death (PCD) in a caspase-, cathepsin-, and reactive oxygen species-dependent manner. NHE1 inhibition augmented cisplatin-induced caspase activity and lysosomal membrane permeability followed by cysteine cathepsin release. In contrast, NBCn1 inhibition attenuated cathepsin release and had no net effect on viability. These findings warrant studies of NHE1 as a potential target in breast cancer and demonstrate that in spite of their similar transport functions, NHE1 and NBCn1 serve different functions in MCF-7 cells.

Disruption of Na+,HCO3− Cotransporter NBCn1 (slc4a7) Inhibits NO-Mediated Vasorelaxation, Smooth Muscle Ca2+ Sensitivity, and Hypertension Development in Mice

Background— Disturbances in pH affect artery function, but the mechanistic background remains controversial. We investigated whether Na+,HCO3− cotransporter NBCn1, by regulating intracellular pH (pHi), influences artery function and blood pressure regulation. Methods and Results— Knockout of NBCn1 in mice eliminated Na+,HCO3− cotransport and caused a lower steady-state pHi in mesenteric artery smooth muscle and endothelial cells in situ evaluated by fluorescence microscopy. Using myography, arteries from NBCn1 knockout mice showed reduced acetylcholine-induced NO-mediated relaxations and lower rho-kinase-dependent norepinephrine-stimulated smooth muscle Ca2+ sensitivity. Acetylcholine-stimulated NO levels (electrode measurements) and N-nitro-l-arginine methyl ester–sensitive l-arginine conversion (radioisotope measurements) were reduced in arteries from NBCn1 knockout mice, whereas relaxation to NO-donor S-nitroso-N-acetylpenicillamine, acetylcholine-induced endothelial Ca2+ responses (fluorescence microscopy), and total and Ser-1177 phosphorylated endothelial NO-synthase expression (Western blot analyses) were unaffected. Reduced NO-mediated relaxations in arteries from NBCn1 knockout mice were not rescued by superoxide scavenging. Phosphorylation of myosin phosphatase targeting subunit at Thr-850 was reduced in arteries from NBCn1 knockout mice. Evaluated by an in vitro assay, rho-kinase activity was reduced at low pH. Without CO2/HCO3−, no differences in pHi, contraction or relaxation were observed between arteries from NBCn1 knockout and wild-type mice. Based on radiotelemetry and tail-cuff measurements, NBCn1 knockout mice were mildly hypertensive at rest, displayed attenuated blood pressure responses to NO-synthase and rho-kinase inhibition and were resistant to developing hypertension during angiotensin-II infusion. Conclusions— Intracellular acidification of smooth muscle and endothelial cells after knockout of NBCn1 inhibits NO-mediated and rho-kinase–dependent signaling in isolated arteries and perturbs blood pressure regulation.

NBCn1 (slc4a7) Mediates the Na+-Dependent Bicarbonate Transport Important for Regulation of Intracellular pH in Mouse Vascular Smooth Muscle Cells

The contribution of sodium-dependent bicarbonate transport to intracellular pH (pHi) regulation in vascular smooth muscle cells is controversial, partly because the molecular identity of the transporter(s) responsible has not been identified. Here, using the pH-sensitive fluorophore 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), we show that smooth muscle cells of intact mouse mesenteric, coronary, and cerebral small arteries all display a sodium- and bicarbonate-dependent pHi recovery after an NH4+-prepulse. The sodium-dependent bicarbonate flux was largely 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) sensitive (56% to 91%) and of a magnitude similar to the amiloride-sensitive flux. Additionally, steady-state pHi was lower (0.2 to 0.4 pH units magnitude) in all 3 vascular beds when CO2/bicarbonate was omitted. RT-PCR analyses showed that NBCn1 (slc4a7) is the only Na+-dependent bicarbonate transporter of the slc4 family detectable at the mRNA level in all 3 vascular beds investigated. Whole-mount immunolabeling and immunogold electron microscopy confirmed the presence of NBCn1 protein in the sarcolemma of mouse mesenteric small arterial smooth muscle cells. Intact mouse mesenteric small arteries were electropermeated to facilitate transfection with small interfering RNA targeting NBCn1, which resulted in an approximate 43% decrease in the ratio of NBCn1 to glyceraldehyde-3-phosphate dehydrogenase mRNA. After knock-down, we found a decreased steady-state pHi (0.21±0.08 pH units) as well as a 68±10% decrease in the net Na+-dependent, amiloride-insensitive base influx after acid load. Finally, omission of CO2/bicarbonate resulted in a decreased contractile response to norepinephrine after sustained exposure to the agonist, underlining the importance of CO2/bicarbonate for vascular contractility. We conclude that NBCn1 mediates the Na+-dependent bicarbonate transport important for pHi regulation in smooth muscle cells of mouse mesenteric, coronary, and cerebral small arteries.

Contribution of Na+,HCO3−‐cotransport to cellular pH control in human breast cancer: A role for the breast cancer susceptibility locus NBCn1 (SLC4A7)

Genome‐wide association studies recently linked the locus for Na+,HCO3−‐cotransporter NBCn1 (SLC4A7) to breast cancer susceptibility, yet functional insights have been lacking. To determine whether NBCn1, by transporting HCO3− into cells, may dispose of acid produced during high metabolic activity, we studied the expression of NBCn1 and the functional impact of Na+,HCO3−‐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+,HCO3−‐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 (pHi) levels >6.6, CO2/HCO3−‐dependent mechanisms accounted for >90% of total net acid extrusion. Na+/H+‐exchange activity was prominent only at lower pHi‐values. Furthermore, steady‐state pHi was 0.35 ± 0.06 units lower in the absence than in the presence of CO2/HCO3−. In conclusion, expression of NBCn1 is upregulated in human primary breast carcinomas and metastases compared to normal breast tissue. Na+,HCO3−‐cotransport is a major determinant of pHi 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.

Regulation and roles of bicarbonate transporters in cancer.

A unifying feature of solid tumors is a markedly altered pH profile compared to normal tissues. This reflects that solid tumors, despite completely different origins, often share several phenotypic properties with implications for intra- and extracellular pH. These include: a metabolic shift in most cancer cells toward more acid-producing pathways, reflecting both oncogenic signaling and the development of hypoxia in poorly perfused regions of the tumors; the poorly perfused and often highly dense tumor microenvironment, reducing the diffusive flux of acid equivalents compared to that in normal tissues; and the markedly altered regulation of the expression and activity of pH-regulatory transport proteins in cancer cells. While some of these properties of tumors have been well described in recent years, the great majority of the research in this clinically important area has focused on proton transport, in particular via the Na+/H+ exchanger 1 (SLC9A1, NHE1) and various H+ ATPases. We have, however, recently demonstrated that at least under some conditions, including in vitro models of HER2 positive breast cancer, and measurements obtained directly in freshly dissected human mammary carcinomas, bicarbonate transporters such as the electroneutral Na+, HCO−3 cotransporter (SLC4A7, NBCn1), are upregulated and play central roles in pH regulation. In this review, we summarize and discuss the current knowledge regarding the regulation and roles of bicarbonate transporters in cancer. Furthermore, we present new analyses of publicly available expression data demonstrating widely altered expression levels of SLC4- and SLC26 family transporters in breast-, lung-, and colon cancer patients, and we hypothesize that bicarbonate transporter dysregulation may have both diagnostic and therapeutic potential in cancer treatment.

Opening of Small and Intermediate Calcium-Activated Potassium Channels Induces Relaxation Mainly Mediated by Nitric-Oxide Release in Large Arteries and Endothelium-Derived Hyperpolarizing Factor in Small Arteries from Rat

This study was designed to investigate whether calcium-activated potassium channels of small (SKCa or KCa2) and intermediate (IKCa or KCa3.1) conductance activated by 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309) are involved in both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in large and small rat mesenteric arteries. Segments of rat superior and small mesenteric arteries were mounted in myographs for functional studies. NO was recorded using NO microsensors. SKCa and IKCa channel currents and mRNA expression were investigated in human umbilical vein endothelial cells (HUVECs), and calcium concentrations were investigated in both HUVECs and mesenteric arterial endothelial cells. In both superior (∼1093 μm) and small mesenteric (∼300 μm) arteries, NS309 evoked endothelium- and concentration-dependent relaxations. In superior mesenteric arteries, NS309 relaxations and NO release were inhibited by both NG,NG-asymmetric dimethyl-l-arginine (ADMA) (300 μM), an inhibitor of NO synthase, and apamin (0.5 μM) plus 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) (1 μM), blockers of SKCa and IKCa channels, respectively. In small mesenteric arteries, NS309 relaxations were reduced slightly by ADMA, whereas apamin plus an IKCa channel blocker almost abolished relaxation. Iberiotoxin did not change NS309 relaxation. HUVECs expressed mRNA for SKCa and IKCa channels, and NS309 induced increases in calcium, outward current, and NO release that were blocked by apamin and TRAM-34 or charybdotoxin. These findings suggest that opening of SKCa and IKCa channels leads to endothelium-dependent relaxation that is mediated mainly by NO in large mesenteric arteries and by EDHF-type relaxation in small mesenteric arteries. NS309-induced calcium influx appears to contribute to the formation of NO.

Vasomotion has chloride-dependency in rat mesenteric small arteries

The possibility that Ca2+-activated Cl− conductances (CaCCs) contribute to oscillations in vascular tone (vasomotion) is tested in isolated mesenteric small arteries from rats where cGMP independent (ICl(Ca)) and cGMP-dependent (ICl(Ca,cGMP)) chloride conductances are important. The effect of anion substitution and Cl− channel blockers on noradrenaline (NA)-stimulated tension in isometrically mounted mesenteric arteries and for chloride conductance of smooth muscle cells isolated from these arteries were assessed electrophysiologically. Cl−o replacement with aspartate blocked vasomotion while 36mM SCN−o (substituted for Cl−) was sufficient to inhibit vasomotion. Oscillations in tone, membrane potential, and [Ca2+]i disappeared with 36mM SCN−. DIDS and Zn2+ blocked ICl(Ca,cGMP) but not ICl(Ca). Vasomotion was not sensitive to DIDS and Zn2+, and DIDS and Zn2+ induce vasomotion in arteries without endothelium. The vasomotion in the presence of DIDS and Zn2+ was sensitive to 36mM SCN−o. The anion substitution data indicate that Cl− is crucial for the Vm and [Ca2+]i oscillations underlying vasomotion. The Cl− channel blocker data are consistent with both CaCCs being important.

Intracellular pH in the resistance vasculature: regulation and functional implications.

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.

NHE1 knockout reduces blood pressure and arterial media/lumen ratio with no effect on resting pHi in the vascular wall

Key points  •  Small arteries are important for regulation of blood pressure and local blood flow. •  Changes in intracellular pH alter artery tone although the mechanistic background has been unclear. •  Using knockout mice for Na+/H+ exchanger NHE1 with reduced acid extrusion from cells in the arterial wall and low intracellular pH in the absence of CO2/HCO3‐, we show that intracellular acidification alters enzymatic activity and consequently artery dilatation and contraction. •  Although lack of NHE1 does not affect intracellular pH in the arterial wall when CO2/HCO3− is present, arteries from NHE1 knockout mice have thinner walls and produce less active force due to reduced volume and cross‐sectional area of individual smooth muscle cells. •  These results highlight the interplay between intracellular pH and artery function, provide new targets to consider for modulating artery structure, and underscore the need to evaluate acid–base transport in conditions of vascular disease and blood pressure disturbances.

Disrupting Na + ,HCO 3 – -cotransporter NBCn1 (Slc4a7) delays murine breast cancer development

Increased metabolism and insufficient blood supply cause acidic waste product accumulation in solid cancers. During carcinogenesis, cellular acid extrusion is upregulated but the underlying molecular mechanisms and their consequences for cancer growth and progression have not been established. Genome-wide association studies have indicated a possible link between the Na+,HCO3–-cotransporter NBCn1 (SLC4A7) and breast cancer. We tested the functional consequences of NBCn1 knockout (KO) for breast cancer development. NBCn1 protein expression increased 2.5-fold during breast carcinogenesis and was responsible for the increased net acid extrusion and alkaline intracellular pH of breast cancer compared with normal breast tissue. Genetic disruption of NBCn1 delayed breast cancer development: tumor latency was ~50% increased while tumor growth rate was ~65% reduced in NBCn1 KO compared with wild-type (WT) mice. Breast cancer histopathology in NBCn1 KO mice differed from that in WT mice and included less aggressive tumor types. The extracellular tumor microenvironment in NBCn1 KO mice contained higher concentrations of glucose and lower concentrations of lactate than that in WT mice. Independently of NBCn1 genotype, the cleaved fraction of poly(ADP-ribose) polymerase (PARP)-1 and expression of monocarboxylate transporter (MCT)1 increased while phosphorylation of Akt and ERK1 decreased as functions of tumor volume. Cell proliferation, evaluated from Ki-67 and phospho-histone H3 staining, was ~60% lower in breast cancer of NBCn1 KO than that of WT mice when corrected for variations in tumor size. We conclude that NBCn1 facilitates acid extrusion from breast cancer tissue, maintains the alkaline intracellular environment and promotes aggressive cancer development and growth.