Ana Rosa Beltrán

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, βI 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 [3H]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 (p42/44mapk) 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 p42/44mapk and PKCα, βI 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.

Modulation of intracellular pH in human ovarian cancer.

To sustain tumor growth, the cancer cells need to adapt to low levels of oxygen (i.e., hypoxia) in the tumor tissue and to the tumor-associated acidic microenvironment. In this phenomenon, the activation of the sodium/proton exchanger 1 (NHE1) at the plasma membrane and the hypoxia-inducible factor (HIF) are critical for the control of the intracellular pH (pHi) and for hypoxia adaptation, respectively. Interestingly, both of these mechanisms end in sustaining cancer cell proliferation. However, regulatory mechanisms of pHi in human ovary tissue and in malignant ascites are unknown. Additionally, a potential role of NHE1 in the modulation of H(+) efflux in human ovarian cancer cells is unknown. In this review, we discussed the characteristics of tumor microenvironment of primary human ovarian tumors and tumor ascites, in terms of pHi regulatory mechanisms and oxygen level. The findings described in the literature suggest that NHE1 may likely play a role in pHi regulation and cell proliferation in human ovarian cancer, potentially involving HIF2α activation. Since ovarian cancer is the fifth cause of prevalence of women cancer in Chile and is usually of late diagnosis, i.e., when the disease jeopardizes peritoneal cavity and other organs, resulting in reduced patient survival, new efforts are required to improve patient-life span and for a better understanding of the pathophysiology of the disease. The potential advantage of the use of amiloride and amiloride-derivatives for cancer treatment in terms of NHE1 expression and activity is also discussed as a therapeutic approach in human ovarian cancer.

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.

Effects of general, specific and combined warm-up on explosive muscular performance

The purpose of this study was to compare the acute effects of general, specific and combined warm-up (WU) on explosive performance. Healthy male (n = 10) subjects participated in six WU protocols in a crossover randomized study design. Protocols were: passive rest (PR; 15 min of passive rest), running (Run; 5 min of running at 70% of maximum heart rate), stretching (STR; 5 min of static stretching exercise), jumping [Jump; 5 min of jumping exercises – 3x8 countermovement jumps (CMJ) and 3x8 drop jumps from 60 cm (DJ60)], and combined (COM; protocols Run+STR+Jump combined). Immediately before and after each WU, subjects were assessed for explosive concentric-only (i.e. squat jump – SJ), slow stretch-shortening cycle (i.e. CMJ), fast stretch-shortening cycle (i.e. DJ60) and contact time (CT) muscle performance. PR significantly reduced SJ performance (p =0.007). Run increased SJ (p =0.0001) and CMJ (p =0.002). STR increased CMJ (p =0.048). Specific WU (i.e. Jump) increased SJ (p =0.001), CMJ (p =0.028) and DJ60 (p =0.006) performance. COM increased CMJ performance (p =0.006). Jump was superior in SJ performance vs. PR (p =0.001). Jump reduced (p =0.03) CT in DJ60. In conclusion, general, specific and combined WU increase slow stretch-shortening cycle (SSC) muscle performance, but only specific WU increases fast SSC muscle performance. Therefore, to increase fast SSC performance, specific fast SSC muscle actions must be included during the WU.

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 (J H +) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and J H + (~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 J H + 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 J H + 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.

Bibliometric analysis of South American research in sports science from 1970 to 2012

Se estudio la produccion de articulos en ciencias del deporte en paises de America del Sur (n=11) entre 1970-2012, usando: todas las bases de datos de citaciones de ISI Web Knowledge. Se evaluo: numero de publicaciones de articulos; numero de publicaciones de articulos / numero de personas en ciencia y tecnologia (no.pub/no.peo); temas mas estudiados; predominancia de instituciones publicas y privadas; factor de impacto (IF); promedio de citas por documento por pais; frecuencia de publicaciones de articulos por revista por pais. Brasil tuvo mas publicaciones de articulos, seguido de Argentina. Bolivia muestra el mayor no.pub/no.peo, seguido por Peru. Los temas mas estudiados fueron fisiologia, ortopedia y rehabilitacion. Las instituciones publicas muestran un mayor numero de publicaciones de articulos. Peru y Bolivia son los unicos paises con una media de IF de 2 o mayor. El promedio de citaciones fue elevado en Peru (elevado coeficiente de variacion). En conclusion, nuestros resultados muestran que en la mayoria de los paises el rango de publicaciones de articulos se incremento con el tiempo. Esto puede ser relacionado con nuevas politicas gubernamentales e institucionales. Sin embargo, la produccion es baja comparado con otras areas.

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.

Kinematic and neuromuscular measures of intensity during plyometric jumps

The aim of this study was to assess jumping performance and neuromuscular activity in lower limb muscles after drop jumps (DJ) from different drop heights (intensity) and during continuous jumping (fatigue), using markers such as reactive strength, jump height, mechanical power and surface electromyography (sEMG). The eccentric (EC) and concentric (CON) sEMG from the medial gastrocnemius (MG), biceps femoris (BF) and rectus (R) muscles were assessed during all tests. In a cross-sectional, randomized study, eleven volleyball players (age 24.4±3.2 years) completed 20 to 90-cm (DJ20 to DJ90) drop jumps and a 60-s continuous jump test. A one-way ANOVA test was used for comparisons, with Sidak post-hoc. The α level was <0.05. Reactive strength was greater for DJ40 compared to DJ90 (p<0.05; ES: 1.27). Additionally jump height was greater for DJ40 and DJ60 compared to DJ20 (p<0.05; ES: 1.26 and 1.27, respectively). No clear pattern of neuromuscular activity appeared during DJ20 to DJ90: some muscles showed greater, lower, or no change with increasing heights for both agonist and antagonist muscles, as well as for eccentric and concentric activity. Mechanical power, but not reactive strength, was reduced in the 60-s jump test (p<0.05; ES: 3.46). No changes were observed in sEMG for any muscle during the eccentric phase nor for the R muscle during the concentric phase of the 60-s jump test. However, for both MG and BF, concentric sEMG was reduced during the 60-s jump test (p<0.05; ES: 5.10 and 4.61, respectively). In conclusion, jumping performance and neuromuscular markers are sensitive to DJ height (intensity), although not in a clear dose-response fashion. In addition, markers such as mechanical power and sEMG are especially sensitive to the effects of continuous jumping (fatigue). Therefore, increasing the drop height during DJ does not ensure a greater training intensity and a combination of different drop heights may be required to elicit adaptations.

Effects of Plyometric Training on Explosive and Endurance Performance at Sea Level and at High Altitude

Plyometric training performed at sea level enhance explosive and endurance performance at sea level. However, its effects on explosive and endurance performance at high altitude had not been studied. Therefore, the aim of this study was to determine the effects of a sea level short-term (i.e., 4-week) plyometric training program on explosive and endurance performance at sea level and at high altitude (i.e., 3,270 m above sea level). Participants were randomly assigned to a control group (n = 12) and a plyometric training group (n = 11). Neuromuscular (reactive strength index – RSI) and endurance (2-km time-trial; running economy [RE]; maximal oxygen uptake - VO2max) measurements were performed at sea level before, at sea level after intervention (SL +4 week), and at high altitude 24-h post SL +4 week. The ANOVA revealed that at SL +4 week the VO2max was not significantly changed in any group, although RE, RSI and 2-km time trial were significantly (p < 0.05) improved in the plyometric training group. After training, when both groups were exposed to high altitude, participants from the plyometric training group showed a greater RSI (p < 0.05) and were able to maintain their 2-km time trial (11.3 ± 0.5 min vs. 10.7 ± 0.6 min) compared to their pre-training sea level performance. In contrast, the control group showed no improvement in RSI, with a worse 2-km time trial performance (10.3 ± 0.8 min vs. 9.02 ± 0.64 min; p < 0.05; ES = 0.13). Moreover, after training, both at sea level and at high altitude the plyometric training group demonstrated a greater (p < 0.05) RSI and 2-km time trial performance compared to the control group. The oxygen saturation was significantly decreased after acute exposure to high altitude in the two groups (p < 0.05). These results confirm the beneficial effects of sea level short-term plyometric training on explosive and endurance performance at sea level. Moreover, current results indicates that plyometric training may also be of value for endurance athletes performing after an acute exposure to high altitude.

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 (v.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.