Extracellular acidosis and very low [Na+ ] inhibit NBCn1- and NHE1-mediated net acid extrusion from mouse vascular smooth muscle cells.

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 ischaemia - 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 <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.

Crosstalk between cardiomyocyte-rich perivascular tissue and coronary arteries is reduced in the Zucker Diabetic Fatty rat model of type 2 diabetes mellitus.

AIM: We tested the hypothesis that crosstalk between cardiomyocyte-rich perivascular tissue (PVT) and coronary arteries is altered in diabetes. METHODS: We studied the vasoactive effects of PVT in arteries from the Zucker Diabetic Fatty (ZDF) rat model of type 2 diabetes, streptozotocin (STZ)-treated Wistar rats with type 1 diabetes, and corresponding - heterozygous Zucker Lean (ZL) or vehicle-treated Wistar - control rats. Vasocontractile and vasorelaxant functions of coronary septal arteries with and without PVT were investigated using wire myography. RESULTS: After careful removal of PVT, vasoconstriction in response to serotonin and thromboxane analogue U46619 was similar in arteries from ZDF and ZL rats, whereas depolarization-induced vasoconstriction - caused by elevating extracellular [K+ ] - was reduced in arteries from ZDF compared to ZL rats. PVT inhibited serotonin-, U46619- and depolarization-induced vasoconstriction in arteries from ZL rats, but this anticontractile influence of PVT was attenuated in arteries from ZDF rats. Methacholine-induced vasorelaxation was smaller in arteries from ZDF than ZL rats both with and without PVT, and the antirelaxant influence of PVT was comparable between arteries from ZDF and ZL rats. We observed no differences in vasoconstriction, vasorelaxation or PVT-dependent vasoactive effects between arteries from STZ- and vehicle-treated Wistar rats. CONCLUSION: Anticontractile influences of PVT are attenuated in coronary arteries from ZDF rats but unaffected in arteries from STZ-treated rats. Signs of endothelial dysfunction are evident in coronary septal arteries - with and without PVT - from ZDF rats but not STZ-treated rats. We propose that altered signalling between cardiomyocyte-rich PVT and coronary arteries can contribute to cardiovascular complications in type 2 diabetes mellitus.

Disrupting Na⁺, HCO3⁻-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 H3staining, 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.

Physiology, pharmacology and pathophysiology of the pH regulatory transport proteins NHE1 and NBCn1: similarities, differences, and implications for cancer therapy.

The Na⁺/H⁺-exchanger 1, NHE1 (SLC9A1) and the electroneutral Na⁺,HCO3⁻ cotransporter NBCn1 (SLC4A7) are coexpressed in a wide range of tissues. Under normal physiological conditions these transporters play an ostensibly similar role, namely that of net acid extrusion after cellular acidification. In addition, they have been implicated in multiple other cellular processes, including regulation of transepithelial transport, cell volume, cell death/survival balance, and cell motility. In spite of their apparent functional similarity, the two transporters also serve distinctly different functions and are differentially regulated. Here, we provide an update on the basic structure, function, regulation, physiology and pharmacology of NHE1 and NBCn1, with particular focus on the factors responsible for their functional similarities and differences. Finally, we highlight recent findings implicating these transporters in cancer development, and discuss issues relating to NHE1 and NBCn1 as potential targets in cancer treatment.

NBCn1 and NHE1 expression and activity in DeltaNErbB2 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.

Vasomotion has chloride-dependency in rat mesenteric small arteries.

The possibility that Ca(2+)-activated Cl(-) conductances (CaCCs) contribute to oscillations in vascular tone (vasomotion) is tested in isolated mesenteric small arteries from rats where cGMP independent (I (Cl(Ca))) and cGMP-dependent (I (Cl(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 [Ca(2+)](i) disappeared with 36mM SCN(-). DIDS and Zn(2+) blocked I (Cl(Ca,cGMP)) but not I (Cl(Ca)). Vasomotion was not sensitive to DIDS and Zn(2+), and DIDS and Zn(2+) induce vasomotion in arteries without endothelium. The vasomotion in the presence of DIDS and Zn(2+) was sensitive to 36mM SCN(-) (o). The anion substitution data indicate that Cl(-) is crucial for the V (m) and [Ca(2+)](i) oscillations underlying vasomotion. The Cl(-) channel blocker data are consistent with both CaCCs being important.