Arsenic trioxide-increased MDCK cells proliferation requires activator protein 1-mediated increase of the sodium/proton exchanger 1 activity.

The release of protons (H+) occurs via the Na+/H+ exchanger isoform 1 (NHE1) leading to a stable intracellular pH (pHi) in MDCK cells. Chronic intake of arsenic trioxide (ATO), in the drinking water, associated with higher morbidity and mortality in neoplastic tissues. ATO increased NHE1 expression and activity, resulting in intracellular alkalization and higher MDCK cells proliferation. Since the pro-proliferative transcription factor activator protein 1 (AP-1) gets activated by al alkaline intracellular pH, a phenomenon paralleled by higher NHEs activity, we asked whether ATO-increased MDCK cells proliferation involves AP-1-dependent NHE1 activation. Cells were exposed (48 h) to ATO (0.05 µmol/L), SR11302 (1 µmol/L, AP-1 inhibitor), HOE-694 (100 nmol/L, NHE1 inhibitor) and EIPA (50 µmol/L, NHE1/NHE3 inhibitor) in the presence of S3226 (10 µmol/L, NHE3 inhibitor), concanamycin A (0.1 µmol/L, V-ATPases inhibitor), and Schering (10 µmol/L, H+/K+-ATPase inhibitor). [3H]Thymidine incorporation, cell counting, wound healing assay, and AP-1 activity were determined. The pHi was measured in cells pre-loaded (10 min) with 2,7-bicarboxyethyl-5,6-carboxyfluorescein acetoxymethyl ester (12 mmol/L) and exposed to NH4Cl (20 mmol/L). Basal pHi and recovery rate (dpHi/dt), intracellular buffer capacity (ßi) and H+ flux (JH+) were determined. NHE1 protein abundance was measured by Western blotting and immunofluorescence. ATO increased the cell growth (1.5 fold), basal pHi (0.4 pHi units), dpHi/dt (1.8 fold), JH+ (1.4 fold), AP-1 activity and NHE1 protein abundance (1.3 fold). ATO also increased (1.5 fold) the nuclear/perinuclear NHE1 immunosignal. SR11302 and HOE-694 blocked ATO effects. Thus, ATO-increased proliferation resulted from AP-1-dependent NHE1 activation in MDCK cells.

Involvement of Intracellular pH in Vascular Insulin Resistance.

The maintenance of the pH homeostasis is maintained by several mechanisms including the efflux of protons (H+) via membrane transporters expressed in almost all mammalian cells. Along these membrane transporters the sodium/H+ exchangers (NHEs), mainly NHE isoform 1 (NHE1), plays a key role in this phenomenon. NHE1 is under modulation by several environmental conditions (e.g. hyperglycaemia, protein kinase C activity) as well as hormones, including insulin. NHE1 activation causes intracellular alkalization in human endothelial cells leading to activation of the endothelial Nitric Oxide Synthase (eNOS) to generate NO. Intracellular alkalization is a phenomenon that also results in upregulation of the glucose transporter GLUT4 in cells that are responsive to insulin. A reduction in the removal of the extracellular D-glucose is seen in states of insulin resistance, such as in diabetes mellitus and obesity. Since insulin is a potent activator of eNOS in human endothelium, therefore causing vasodilation, and its vascular effect is reduced in insulin resistance it is likely that a defective signal to activate NHE1 in insulin target cells is expected. This phenomenon results in lower redistribution and activation of GLUT4 leading to reduced uptake of D-glucose and hyperglycaemia. The general concept of a role for NHE1, and perhaps other NHEs isoforms, in insulin resistance in the human vasculature is proposed.

Intracellular acidification reduces l-arginine transport via system y+L but not via system y+/CATs and nitric oxide synthase activity in human umbilical vein endothelial cells.

l-Arginine is taken up via the cationic amino acid transporters (system y+/CATs) and system y+L in human umbilical vein endothelial cells (HUVECs). l-Arginine is the substrate for endothelial NO synthase (eNOS) which is activated by intracellular alkalization, but nothing is known regarding modulation of system y+/CATs and system y+L activity, and eNOS activity by the pHi in HUVECs. We studied whether an acidic pHi modulates l-arginine transport and eNOS activity in HUVECs. Cells loaded with a pH-sensitive probe were subjected to 0.1-20 mmol/L NH4Cl pulse assay to generate pHi 7.13-6.55. Before pHi started to recover, l-arginine transport (0-20 or 0-1000 µmol/L, 10 s, 37 °C) in the absence or presence of 200 µmol/L N-ethylmaleimide (NEM) (system y+/CATs inhibitor) or 2 mmol/L l-leucine (systemy+L substrate) was measured. Protein abundance for eNOS and serine1177 or threonine495 phosphorylated eNOS was determined. The results show that intracellular acidification reduced system y+L but not system y+/CATs mediated l-arginine maximal transport capacity due to reduced maximal velocity. Acidic pHi reduced NO synthesis and eNOS serine1177 phosphorylation. Thus, system y+L activity is downregulated by an acidic pHi, a phenomenon that may result in reduced NO synthesis in HUVECs.

Intracellular acidification increases adenosine transport in human umbilical vein endothelial cells.

INTRODUCTION: Adenosine is taken up via human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) at a physiological extracellular pH (pHo ∼7.4) in human umbilical vein endothelial cells (HUVECs). Acidic pHo increases the uptake of adenosine and 5-hydroxytryptamine (5HT) via hENT4 in this cell type. However, modulation of hENT1 and hENT2 transport activity by the pHi is unknown. We investigated whether hENT1 and hENT2-adenosine transport was regulated by acidic pHi. METHODS: HUVECs loaded with a pH sensitive probe were subjected to 0.1-20 mmol/L NH4Cl pulse assay to generate 6.9-6.2 pHi. Before pHi started to recover, adenosine transport kinetics (0-500 µmol/L, 37 °C) in the absence or presence 1 or 10 µmol/L S-(4-nitrobenzyl)-6-thio-inosine (NBTI), 2 mmol/L hypoxanthine, 2 mmol/L adenine, 100 µmol/L 5HT, or 500 µmol/L adenosine, was measured. RESULTS: Overall adenosine transport (i.e., hENT1+hENT2) was semisaturable and partially inhibited by 1 µmol/L, but abolished by 10 µmol/L NBTI in cells non-treated or treated with NH4Cl. The initial velocity and non-saturable, lineal component for overall transport were increased after NH4Cl pulse. hENT1 and hENT2-mediated adenosine transport maximal capacity was increased by acidic pHi. hENT1 activity was more sensitive than hENT2 activity to acidic pHi. DISCUSSION: hENT1 and hENT2-adenosine transport is differentially regulated by acidic pHi in HUVECs. These findings are important in pathologies associated with pHi alterations such as gestational diabetes mellitus.

Sodium/proton exchanger isoform 1 regulates intracellular pH and cell proliferation in human ovarian cancer.

Cancer cells generate protons (H+) that are extruded to the extracellular medium mainly via the Na+/H+ exchanger 1 (NHE1), which regulates intracellular pH (pHi) and cell proliferation. In primary cultures of human ascites-derived ovarian cancer cells (haOC) we assayed whether NHE1 was required for pHi modulation and cell proliferation. Human ovary expresses NHE1, which is higher in haOC and A2780 (ovarian cancer cells) compared with HOSE cells (normal ovarian cells). Basal pHi and pHi recovery (following a NH4Cl pulse) was higher in haOC and A2780, compared with HOSE cells. Zoniporide (NHE1 inhibitor) caused intracellular acidification and pHi recovery was independent of intracellular buffer capacity, but reduced in NHE1 knockdown A2780 cells. Zoniporide reduced the maximal proliferation capacity, cell number, thymidine incorporation, and ki67 (marker of proliferation) fluorescence in haOC cells. SLC9A1 (for NHE1) amplification associated with lower overall patient survival. In conclusion, NHE1 is expressed in human ovarian cancer where it has a pro-proliferative role. Increased NHE1 expression and activity constitute an unfavourable prognostic factor in these patients.

Escherichia coli Heat-Stable Enterotoxin Mediates Na+/H+ Exchanger 4 Inhibition Involving cAMP in T84 Human Intestinal Epithelial Cells.

The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa) enterotoxins. STa increases Cl-release in intestinal cells, including the human colonic carcinoma T84 cell line, involving increased cGMP and membrane alkalization due to reduced Na+/H+ exchangers (NHEs) activity. Since NHEs modulate intracellular pH (pHi), and NHE1, NHE2, and NHE4 are expressed in T84 cells, we characterized the STa role as modulator of these exchangers. pHi was assayed by the NH4Cl pulse technique and measured by fluorescence microscopy in BCECF-preloaded cells. pHi recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 µmol/L STa (30 minutes), 25 µmol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 µmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 µmol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 µmol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi) and H+ efflux (JH+) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and JH+ (~63%), without altering basal pHi (range 7.144-7.172). STa did not alter ßi value in a range of 1.6 pHi units. The dpHi/dt and JH+ was almost abolished (~94% inhibition) by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa-decreased dpHi/dt and JH+ was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T84 cells with STa results in reduced NHE4 activity leading to a lower capacity of pHi recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity) ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting human diarrhoea.

Potential role of sodium-proton exchangers in the low concentration arsenic trioxide-increased intracellular pH and cell proliferation.

Arsenic main inorganic compound is arsenic trioxide (ATO) presented in solution mainly as arsenite. ATO increases intracellular pH (pHi), cell proliferation and tumor growth. Sodium-proton exchangers (NHEs) modulate the pHi, with NHE1 playing significant roles. Whether ATO-increased cell proliferation results from altered NHEs expression and activity is unknown. We hypothesize that ATO increases cell proliferation by altering pHi due to increased NHEs-like transport activity. Madin-Darby canine kidney (MDCK) cells grown in 5 mmol/L D-glucose-containing DMEM were exposed to ATO (0.05, 0.5 or 5 µmol/L, 0-48 hours) in the absence or presence of 5-N,N-hexamethylene amiloride (HMA, 5-100 µmol/L, NHEs inhibitor), PD-98059 (30 µmol/L, MAPK1/2 inhibitor), Gö6976 (10 µmol/L, PKCα, ßI and µ inhibitor), or Schering 28080 (10 µmol/L, H(+)/K(+)ATPase inhibitor) plus concanamycin (0.1 µmol/L, V type ATPases inhibitor). Incorporation of [(3)H]thymidine was used to estimate cell proliferation, and counting cells with a hemocytometer to determine the cell number. The pHi was measured by fluorometry in 2,7-bicarboxyethyl-5,6-carboxyfluorescein loaded cells. The Na(+)-dependent HMA-sensitive NHEs-like mediated proton transport kinetics, NHE1 protein abundance in the total, cytoplasm and plasma membrane protein fractions, and phosphorylated and total p42/44 mitogen-activated protein kinases (p42/44(mapk)) were also determined. Lowest ATO (0.05 µmol/L, ~0.01 ppm) used in this study increased cell proliferation, pHi, NHEs-like transport and plasma membrane NHE1 protein abundance, effects blocked by HMA, PD-98059 or Gö6976. Cell-buffering capacity did not change by ATO. The results show that a low ATO concentration increases MDCK cells proliferation by NHEs (probably NHE1)-like transport dependent-increased pHi requiring p42/44(mapk) and PKCα, ßI and/or µ activity. This finding could be crucial in diseases where uncontrolled cell growth occurs, such as tumor growth, and in circumstances where ATO, likely arsenite, is available at the drinking-water at these levels.