Red blood cell pH,the Bohr effect,and other oxygenation‐linked phenomena in blood O2 and CO2 transport

Abstract The discovery of the S‐shaped O₂ equilibrium curve and the Bohr effect in 1904 stimulated a fertile and continued research into respiratory functions of blood and allosteric mechanisms in haemoglobin (Hb). The Bohr effect (influence of pH/CO₂ on Hb O₂ affinity) and the reciprocal Haldane effect (influence of HbO₂ saturation on H⁺/CO₂ binding) originate in the Hb oxy–deoxy conformational change and allosteric interactions between O₂ and H⁺/CO₂ binding sites. In steady state, H⁺ is passively distributed across the vertebrate red blood cell (RBC) membrane, and intracellular pH (pH_i) changes are related to changes in extracellular pH, Hb‐O₂ saturation and RBC organic phosphate content. As the Hb molecule shifts between the oxy and deoxy conformation in arterial‐venous gas transport, it delivers O₂ and takes up CO₂ and H⁺ in tissue capillaries (elegantly aided by the Bohr effect). Concomitantly, the RBC may sense local O₂ demand via the degree of Hb deoxygenation and release vasodilatory agents to match local blood flow with requirements. Three recent hypotheses suggest (1) release of NO from S‐nitroso‐Hb upon deoxygenation, (2) reduction of nitrite to vasoactive NO by deoxy haems, and (3) release of ATP. Inside RBCs, carbonic anhydrase (CA) provides fast hydration of metabolic CO₂ and ensures that the Bohr shift occurs during capillary transit. The formed H⁺ is bound to Hb (Haldane effect) while HCO₃^? is shifted to plasma via the anion exchanger (AE1). The magnitude of the oxylabile H⁺ binding shows characteristic differences among vertebrates. Alternative strategies for CO₂ transport include direct HCO₃^? binding to deoxyHb in crocodilians, and high intracellular free [HCO₃^?] (due to high pH_i) in lampreys. At the RBC membrane, CA, AE1 and other proteins may associate into what appears to be an integrated gas exchange metabolon. Oxygenation‐linked binding of Hb to the membrane may regulate glycolysis in mammals and perhaps also oxygen‐sensitive ion transport involved in RBC volume and pH_i regulation. Blood O₂ transport shows several adaptive changes during exposure to environmental hypoxia. The Bohr effect is involved via the respiratory alkalosis induced by hyperventilation, and also via the pH_i change that results from modulation of RBC organic phosphate content. In teleost fish, β‐adrenergic activation of Na⁺/H⁺ exchange rapidly elevates pH_i and O₂ affinity, particularly under low O₂ conditions.     

Increased reactive oxygen species and tumor necrosis factor-alpha production by monocytes are associated with elevated levels of uric acid in pre-eclamptic women

Citation Peraçoli MTS, Bannwart CF, Cristofalo R, Medeiros Borges VT, Araújo Costa RA, Witkin SS, Peraçoli JC. Increased reactive oxygen species and tumor necrosis factor‐alpha production by monocytes are associated with elevated levels of uric acid in pre‐eclamptic women. Am J Reprod Immunol 2011; 66: 460–467 Problem To evaluate associations between hyperuricemia and increases in production of reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF‐α) in pre‐eclamptic pregnancies. Method of study This study investigated serum uric acid levels, monocyte production of TNF‐α, superoxide anion (O₂^?) and hydrogen peroxide (H₂O₂), as well as superoxide dismutase (SOD) and catalase (CAT) activities in erythrocytes from 30 women with pre‐eclampsia (PE) compared with 30 normotensive (NT) pregnant women in the last trimester of pregnancy. Results Serum uric acid levels (6.1 versus 2.8 mg/dL) as well as endogenous O₂^? (2.2 versus 1.6 nm ), H₂O₂ (1.8 versus 1.4 nm ) and TNF‐α (91.6 versus 40.4 pg/mL) released from monocytes were significantly higher in the pre‐eclamptic group than in the NT group (P < 0.05). SOD activity in erythrocytes was also significantly elevated in the PE group (5969.2 versus 4834.7 U/g Hb). No significant difference between groups was observed in relation to CAT activity. Conclusions Elevated serum uric acid levels are correlated with higher O₂^? and TNF‐α production by monocytes in women with PE. This may contribute to the enhanced oxidative and inflammatory state characteristic of this disorder.     

Antigenotoxic potential of Gymnema montanum leaves on DNA damage in human peripheral blood lymphocytes and HL‐60 cell line

In this study we have evaluated the genoprotective effect of the ethanol extract of Gymnema montanum (GLEt) leaves in human peripheral blood lymphocytes and HL‐60 cell line in vitro using the comet assay. DNA damage was induced by treating the cells with H₂O₂ and methyl methane sulphonate (MMS). GLEt treatment effectively protected the lymphocytes and HL‐60 cell line from H₂O₂‐induced oxidative DNA damage in a dose‐dependent manner whereas it was not effective against alkylative DNA damage caused by MMS. The global percent repair efficiency also showed that both pre‐ and post‐ GLEt treatment provided effective protection against H₂O₂ induced DNA damage but not as effective against MMS. At 200 μg ml^?1 level, its repair capacity against H₂O₂ induced DNA damage was comparable to that of vitamin‐C (100 μM). Furthermore, exposure to GLEt reduced the formation of apoptotic cells caused by H₂O₂, which was demonstrated by the decreased sub‐G1‐DNA content in cell cycle analysis and apoptotic frequencies of lymphocytes in an annexin‐V binding assay. In addition, GLEt was found to have effective peroxide scavenging ability in dose‐dependent manner. The protective efficiency of the extract was found to be directly proportional to its total phenolic content. The present study indicates that G. montanum leaves are a significant source of phytochemicals with antigenotoxic and antioxidant activity, and thus has potential therapeutic use. Environ. Mol. Mutagen., 2010. © 2009 Wiley‐Liss, Inc.     

Synthesis of new polymers involving deltahedral carborane units

Aminocarboranes nido-7-NH₃⁺-(7-CB₁₀H₁₂)^? and closo-1-NH₃⁺-(CB₁₁H₁₁)^? were copolymerized with α,ω-dihalogenoalkanes to high-molecular-weight alternating copolymers. The nido-compound copolymer was crosslinked by means of sandwiching two open cages with Co^III ion yielding bis(monocarbollide)-cobaltate(III) units as the new type knot points. Metallacarboranes, 1,1′-diaminobis(monocarbollide)nickelate(IV) and two bridged bis(dicarbollide)cobaltates(III) (8,8′-μ-imino- and 8,8′-μ-oxyimino-), have provided hemipolymers of molecular weight up to a few thousands in the same type of copolymerization. A new methacrylic-type monomer was prepared by reaction of glycidyl methacrylate with 8,8′-(μ-imino)-(1,2-C₂B₉H₁₀)-3-Co^III and copolymerized with methyl methacrylate to high-molecular-weight statistical copolymers.     

Arsenate and dimethylarsinic acid in drinking water did not affect DNA damage repair in urinary bladder transitional cells or micronuclei in bone marrow

Arsenic is a human skin, lung, and urinary bladder carcinogen, and may act as a cocarcinogen in the skin and urinary bladder. Possible modes of action of arsenic carcinogenesis/cocarcinogenesis include oxidative stress induction and inhibition of DNA damage repair. We investigated the effects of arsenic in drinking water on DNA damage repair in urinary bladder transitional cells and on micronucleus formation in bone marrow. F344 rats were given 100 ppm arsenate [As(V)] or dimethylarsinic acid [DMA(V)] in drinking water for 1 week. The in vivo repair of cyclophosphamide (CP)‐induced DNA damage resulting from a single oral gavage of CP, and the in vitro repair of hydrogen peroxide (H₂O₂)‐ or formaldehyde‐induced DNA damage, resulting from adding H₂O₂ or formaldehyde into cell medium, were measured by the Comet assay. DMA(V) effects were not observed on either CP‐induced DNA damage induction or on DNA repair. Neither DMA(V) nor As(V) increased the H₂O₂‐ or formaldehyde‐induced DNA damage, and neither inhibited the repair of H₂O₂‐induced DNA damage. Neither DMA(V) nor As(V) increased the micronucleus frequency, nor did they elevate micronucleus frequency resulting from CP treatment above the level observed by the treatment with CP alone. These results suggest that arsenic carcinogenesis/cocarcinogenesis in the urinary bladder may not be via DNA damage repair inhibition. To our knowledge this is the first report of arsenic effects on DNA damage repair in the urinary bladder. Environ. Mol. Mutagen. 2009. Published 2009 by Wiley‐Liss, Inc.     

Effect of pre-breathing oxygen at different depth on oxidative status and calcium concentration in lymphocytes of scuba divers

Aim: In‐water pre‐breathing oxygen at various depths reduces decompression‐induced bubble formation and platelet activation, but it could induce side effects such as oxidative stress. The aim of this study was to investigate the effect of in‐water pre‐breathing oxygen, at different depths, on the oxidative status and intracellular calcium ([Ca²⁺]i) of peripheral blood lymphocytes isolated from six divers. They participated in a 4‐diving protocol. Two week recovery time was allowed between successive dives. Before diving, all divers, for 20 min, breathed normally at sea level (dive 1), 100% oxygen at sea level (dive 2), 100% oxygen at 6 msw (dive 3), 100% oxygen at 12 msw (dive 4). Then they dived to 30 msw for 20 min with air tank. Methods: Blood samples were collected before and after each dive. Hydrogen peroxide (H₂O₂) levels, catalase (CAT) activity, mRNA expression of CAT, glutathione peroxidase (GPx) and superoxide dismutase (SOD), and the [Ca²⁺]i in lymphocytes were measured. Results: The dives slightly decreased lymphocyte number and significantly reduced lymphocyte H₂O₂ levels. CAT activity was higher after scuba diving and, dive 3 enhanced mRNA gene expression of CAT, GPx and SOD. The [Ca²⁺]i was higher after dive 1 and 2 than pre‐diving, while was maintained at pre‐diving value after dive 3 and 4. Conclusion: Our results suggest that pre‐breathing oxygen, in particular at 12 msw, may enhance lymphocyte antioxidant activity and reduce reactive oxygen species levels. Pre‐breathing oxygen in water may also preserve calcium homeostasis, suggesting a protective role in the physiological lymphocyte cell functions.     

Detailed Kinetics of 1‐Hexene Oligomerization Reaction with (n‐Bu‐Cp)2ZrCl2–MAO Catalyst

The paper describes a kinetic study of 1‐hexene oligomerization reaction with the (n‐Bu‐Cp)₂ZrCl₂–MAO catalyst system at 70 °C. GC analysis of the monomer/oligomer mixtures at different reaction times in the course of a 5‐h reaction, provided information on the formation rates of individual oligomer molecules, from the 1‐hexene dimer, C₁₂H₂₄, to the tetradecamer, C₈₄H₁₆₈. Kinetic modeling of the oligomerization kinetics strongly supports the principal premise of polymerization kinetics, that the value of the propagation rate constant of the chain growth reaction does not depend on the number of monomer units in the growing polymer chain. The only exception from this rule is the insertion of a 1‐hexene molecule into the Zr⁺? C bond in the active center bearing a single monomer unit, (n‐Bu‐Cp)₂Zr⁺? CH₂? CH₂? C₄H₉. The rate constant of this reaction in one order of magnitude is higher than the rate constant of 1‐hexene insertion into active centers which are β‐branched with respect to the Zr atom, (n‐Bu‐Cp)₂Zr⁺? [CH₂? CH(C₄H₉)]_n? CH₂? CH₂? C₄H₉.

     

Ratio of myeloid and plasmacytoid dendritic cells and TH2 skew in CRS with nasal polyps

Background: The role of myeloid and plasmacytoid dendritic cells and its consequences for the T_H2 skew in chronic rhinosinusitis (CRS) with nasal polyps (CRSNP⁺) should be detailed. Methods: In 18 CRS patients without nasal polyps (CRSNP^?), 35 CRSNP⁺ patients and 22 patients with nasal structural abnormalities without rhinosinusitis (controls), dendritic cells (DC) were differentiated into myeloid (mDC) and plasmacytoid (pDC) subtypes using an antibody cocktail including CD1c (BDCA‐1) and CD303 (BDCA‐2) in peripheral blood mononuclear cells (PBMC) and single cell preparations of sinonasal mucosa by flow cytometry. Moreover, cells were analysed for expression of CD45, CD3, CD4, CXCR3 (T_H1) and CCR4 (T_H2) and IFN‐γ, IL‐5, TGF‐β1, TGF‐β2, ECP and total IgE in nasal secretions were determined. As a possible confounder, Staphylococcus aureus in nasal lavages was detected. Results: The tissue mDC/pDC‐ratio was 1.7 (1.0–2.4) in controls, 3.0 (1.8–4.0) in CRSNP^? and 0.8 (0.6–1.0) in CRSNP⁺ (P < 0.01). In tissue samples, the T_H1/T_H2 ratio was 12.6 (6.4–16.0) in controls, 12.5 (6.9–21.2) in CRSNP^? and 1.8 (1.3–3.6) in CRSNP⁺ (median and interquartile range, P < 0.001). Less pronounced differences were found in PBMC. S. aureus detection rates or TGF‐β levels did not differ between patient groups and S. aureus detection had no influence on the parameters investigated. Conclusion: A significant T_H2 skew in CRSNP⁺ could be confirmed on the cellular level. It was driven by low myeloid dendritic cell numbers. The T_H2 skew did not correlate with S. aureus detection. The data support the concept that CRSNP⁺ and CRSNP^? are pathophysiologically distinct.     

Anion exchanger 2 is critical for CD8+ T cells to maintain pHi homeostasis and modulate immune responses

Mitogenic stimulation of lymphocytes involves alkalinization of intracellular pH (pH_i). Subsequent pH_i regulation may involve HCO₃^? extrusion through Cl^?/HCO₃^? exchangers and/or Na⁺‐HCO₃^? co‐transporters with acid‐loading capability. Abnormalities in these mechanisms could result in immune dysfunctions, as suggested by the CD8⁺ T‐cell expansion encountered in mice lacking Ae2 (a widely expressed acid loader with electroneutral and Na⁺‐independent Cl^?/HCO₃^? anion‐exchange activity). Here we report that CD8⁺ T cells but not CD4⁺ T cells or other lymphocyte populations, are crucially dependent on Ae2 for pH_i regulation. While total lymphocytes (including isolated CD4⁺ T cells) exhibit Ae1 expression and Na⁺‐HCO₃^? co‐transport with acidifying potential, CD8⁺ T cells lack these acid‐loading mechanisms. In Ae2‐KO mice, CD4⁺ but not CD8⁺ T cells upregulate these potential Ae2 surrogates. As a consequence, Ae2‐KO CD8⁺ T cells exhibit alkalinized pH_i, and dramatically increase their pH_i upon CD3 stimulation. Moreover, stimulated Ae2‐deficient CD8⁺ T cells show enhanced intracellular production of IL‐2 and membrane expression of its receptor IL‐2Rα, together with increased cell proliferation and activation. These findings demonstrate that CD8⁺ T cells are critically dependent on Ae2 for pH_i homeostasis and tuning of cell proliferation and activation. Ae2 thus constitutes a novel target to modulate CD8⁺ T‐cell responses.     

Hyperpolarized δ‐[1‐13C]gluconolactone as a probe of the pentose phosphate pathway

The pentose phosphate pathway (PPP) is thought to be upregulated in trauma (to produce excess NADPH) and in cancer (to provide ribose for nucleotide biosynthesis), but simple methods for detecting changes in flux through this pathway are not available. MRI of hyperpolarized ¹³C–enriched metabolites offers considerable potential as a rapid, non‐invasive tool for detecting changes in metabolic fluxes. In this study, hyperpolarized δ‐[1‐¹³C]gluconolactone was used as a probe to detect flux through the oxidative portion of the pentose phosphate pathway (PPP_ox) in isolated perfused mouse livers. The appearance of hyperpolarized (HP) H¹³CO₃^? within seconds after exposure of livers to HP‐δ‐[1‐¹³C]gluconolactone demonstrates that this probe rapidly enters hepatocytes, becomes phosphorylated, and enters the PPP_ox pathway to produce HP‐H¹³CO₃^? after three enzyme catalyzed steps (6P–gluconolactonase, 6‐phosphogluconate dehydrogenase, and carbonic anhydrase). Livers perfused with octanoate as their sole energy source show no change in production of H¹³CO₃^? after exposure to low levels of H₂O₂, while livers perfused with glucose and insulin showed a twofold increase in H¹³CO₃^? after exposure to peroxide. This indicates that flux through the PPP_ox is stimulated by H₂O₂ in glucose perfused livers but not in livers perfused with octanoate alone. Subsequent perfusion of livers with non‐polarized [1,2‐¹³C]glucose followed by ¹H NMR analysis of lactate in the perfusate verified that flux through the PPP_ox is indeed low in healthy livers and modestly higher in peroxide damaged livers. We conclude that hyperpolarized δ‐[1‐¹³C]gluconolactone has the potential to serve as a metabolic imaging probe of this important biological pathway.     

Inhibitory effects of oxidants on n-type K+ channels in T lymphocytes and Xenopus oocytes

 Reactive oxygen species (ROS) appear to be involved in Fas-induced programmed cell death. We have previously demonstrated a tyrosine-kinase-dependent inhibition of the n-type K⁺ channels (K_n) by Fas stimulation. Thus, the effect of hydrogen peroxide (H₂O₂) on the function of K_n was examined using the patch-clamp technique. Incubation of Jurkat human T lymphocytes with 100 μM H₂O₂ resulted in a 46 ± 5% inhibition of the macroscopic whole-cell current. Experiments performed at the single-channel level using the cell-attached configuration revealed that the probability of the channel being open diminished upon incubation in H₂O₂. The effect was not dependent on src-like kinases, since H₂O₂ did not trigger tyrosine phosphorylation of the K_n channel protein and herbimycin A did not prevent channel inhibition. K_v1.3 channels underly the K_n of T lymphocytes and were expressed in Xenopus oocytes and subjected to electrophysiological analysis by the two-electrode voltage-clamp technique. Application of 1 mM H₂O₂ and 500 μM t-BOOH (tert, butylhydroperoxide) resulted in a marked inhibition of the K⁺ current within 20 min. Both the membrane-permeable thiol-group oxidizing agent DTNP [2,2′-dithiobis-(5-nitropyridine)] and the membrane-impermeable DTNB [5,5′-Dithiobis-(2-nitrobenzoic acid)] (50 μM) inhibited K_v1.3 channels, suggesting that extracellular domains of K_v1.3 are affected. These results point to a direct modulation of K_n by various oxidative agents. Received: 20 September 1996 / Received after revision and accepted: 29 November 1996     

c‐Rel is crucial for the induction of Foxp3+ regulatory CD4+ T cells but not TH17 cells

The NF‐κB/Rel family member c‐Rel was described to be required for the development of T_H1 responses. However, the role of c‐Rel in the differentiation of T_H17 and regulatory CD4⁺Foxp3⁺ T cells (Treg) remains obscure. Here, we show that in the absence of c‐Rel, in vitro differentiation of pro‐inflammatory T_H17 cells is normal. In contrast, generation of inducible Treg (iTreg) within c‐Rel‐deficient CD4⁺ T cells was severely hampered and correlated to reduced numbers of Foxp3⁺ T cells in vivo. Mechanistically, in vitro conversion of naive CD4⁺ T cells into iTreg was crucially dependent on c‐Rel‐mediated synthesis of endogenous IL‐2. The addition of exogenous IL‐2 was sufficient to rescue the development of c‐Rel‐deficient iTreg. Thus, c‐Rel is essential for the development of Foxp3⁺ Treg but not for T_H17 cells via regulating the production of IL‐2.     

Systemic hypoxia promotes lymphocyte apoptosis induced by oxidative stress during moderate exercise

Blood undergoes oxidative stress during severe hypoxia or intense exercise. Excessive exposure to oxidative stress induces replicative senescence and apoptosis of lymphocytes. This study determines how various exercises with/without hypoxia affect lymphocyte subset mobilization and oxidative stress-induced lymphocyte apoptosis. Eighteen sedentary males randomly engaged in two normoxic exercise bouts [severe exercise (SE) (up to VO_2max) and moderate-intensity exercise (ME) (50%VO_2max) while exposed to 21%O₂], two hypoxic exercise bouts (ME while exposed to 12%O₂ and 15%O₂) and two hypoxic resting conditions (resting while exposed to 12%O₂ and 15%O₂) in a normobaric hypoxia chamber. Under normoxic conditions, SE but not ME (1) increased the percentages of senescent (CD28⁻ and CD57⁺)/activated (CD62L⁻ and CD11a⁺)-form lymphocytes mobilized into the peripheral blood compartment; (2) decreased the levels of surface thiol and intracellular total (t-GSH) and reduced-form glutathione (r-GSH) of lymphocytes in blood; and (3) further enhanced the extents of H₂O₂-induced mitochondria trans-membrane potential diminishing, caspases 3/8/9 activation, poly(ADP-ribose) polymerase cleavage and phosphotidyl serine exposure in blood lymphocytes. However, no significant change occurred in the subset mobilization, antioxidant levels or apoptosis of lymphocytes following exposure to either 12%O₂ or 15%O₂. Although both 12%O₂ and 15%O₂ ME increased the mobilization of senescent/activated-form lymphocytes, only 12%O₂ ME enhanced H₂O₂-induced lymphocyte thiol, t-GSH and r-GSH consumption and apoptotic responses. Therefore, we conclude that the 12%O₂ exposure increases the mobilization of senescent/activated-form lymphocytes into the peripheral blood compartment and simultaneously enhances oxidative stress-induced lymphocyte apoptosis by diminishing cellular antioxidant levels during exercise.     

K+ as a vasodilator in resting human muscle: implications for exercise hyperaemia

Aim: Potassium (K⁺) released from contracting skeletal muscle is considered a vasodilatory agent. This concept is mainly based on experiments infusing non‐physiological doses of K⁺. The aim of the present study was to investigate the role of K⁺ in blood flow regulation. Methods: We measured leg blood flow (LBF) and arterio‐venous (A‐V) O₂ difference in 13 subjects while infusing K⁺ into the femoral artery at a rate of 0.2, 0.4, 0.6 and 0.8 mmol min^?1. Results: The lowest dose increased the calculated femoral artery plasma K⁺ concentration by approx.1 mmol L^?1. Graded K⁺ infusions increased LBF from 0.39 ± 0.06 to 0.56 ± 0.13, 0.58 ± 0.17, 0.61 ± 0.11 and 0.71 ± 0.17 L min^?1, respectively, whereas the leg A‐V O₂ difference decreased from 74 ± 9 to 60 ± 12, 52 ± 11, 53 ± 9 and 45 ± 7 mL L^?1, respectively (P < 0.05). Mean arterial pressure was unchanged, indicating that the increase in LBF was associated with vasodilatation. The effect of K⁺ was totally inhibited by infusion (27 μmol min^?1) of Ba²⁺, an inhibitor of Kir2.1 channels. Simultaneous infusion of ATP and K⁺ evoked an increase in LBF equalled to the sum of their effects. Conclusions: Physiological infusions of K⁺ induce significant increases in resting LBF, which are completely blunted by inhibition of the Kir2.1 channels. The present findings in resting skeletal muscle suggest that K⁺ released from contracting muscle might be involved in exercise hyperaemia. However, the magnitude of increase in LBF observed with K⁺ infusion suggests that K⁺ only accounts for a limited fraction of the hyperaemic response to exercise.     

Contribution of pH,diprotonated phosphate and potassium for the reflex increase in blood pressure during handgrip

The relative importance of pH, diprotonated phosphate (H₂PO₄^?) and potassium (K⁺) for the reflex increase in mean arterial pressure (MAP) during exercise was evaluated in seven subjects during rhythmic handgrip at 15 and 30% maximal voluntary contraction (MVC), followed by post-exercise muscle ischaemia (PEMI). During 15% MVC, MAP rose from 92 ± 1 to 103 ± 2 mmHg, [K⁺] from 4.1 ± 0.1 to 5.1 ± 0.1 mmol L^?1, while the intracellular (7.00 ± 0.01 to 6.80 ± 0.06) and venous pH fell (7.39 ± 0.01 to 7.30 ± 0.01) (P < 0.05). The intracellular [H₂PO₄^?] increased 8.4 ± 2 mmol kg^?1 and the venous [H₂PO₄^?] from 0.14 ± 0.01 to 0.16 ± 0.01 mmol L^?1 (P < 0.05). During PEMI, MAP remained elevated along with the intracellular [H₂PO₄^?] as well as a low intracellular and venous pH. However, venous [K⁺] and [H₂PO₄^?] returned to the level at rest. During 30% MVC handgrip, MAP rose to 130 ± 3 mmHg, [K⁺] to 5.8 ± 0.2 mmol L^?1, the intracellular and extracellular [H₂PO₄^?] by 20 ± 5 mmol kg^?1 and to 0.20 ± 0.02 mmol L^?1, respectively, while the intracellular (6.33 ± 0.06) and venous pH fell (7.23 ± 0.02) (P < 0.05). During post-exercise muscle ischaemia all variables remained close to the exercise levels. Analysis of each variable as a predictor of blood pressure indicated that only the intracellular pH and diprotonated phosphate were linked to the reflex elevation of blood pressure during handgrip.     

Mechanisms of hypotonic inhibition of the sodium, proton exchanger type 1 (NHE1) in a biliary epithelial cell line (Mz-Cha-1)

Aim: To elucidate the cellular events that results in inhibition of Na⁺, H⁺ exchanger type 1 (NHE1) by hypotonicity. Methods: Intracellular pH (pH_i) was measured in biliary epithelial cells, with the pH‐sensitive fluorochrome 2′,7′‐bis‐(carboxyethyl)‐5(6)‐carboxyfluorescein (BCECF) using a spectrophotometer. Regulatory volume decrease (RVD) was analysed from confocal images. Changes in NHE1 membrane content were visualized by confocal laser scanning microscopy after transfection of Mz‐Cha‐1 cells with a NHE1–cMyc fusion protein. Results: In Mz‐Cha‐1 cells hypotonicity (?80 mmol L^?1 NaCl) inhibited endogenous Na⁺, H⁺ exchange. Tyrosine and serine kinase inhibitors were incapable to prevent inhibition. As several signalling pathways influence Na⁺, H⁺ exchange, we tested the effect of the Ca⁺⁺, Calmodulin, protein kinase C or the cAMP, protein kinase A system on inhibition of Na⁺, H⁺ exchange by hypotonic challenge, but neither system was involved. In contrast, cytoskeleton did influence the effect of hypotonicity. Inhibition of microtubule polymerization by colchicine prevented inhibition of NHE1, and also restored Na⁺, H⁺ exchange kinetics. Specific inhibition of Src kinases with PP2, attenuated pH_i recovery rate from 1.93 ± 0.16 pH units min^?1 (normotonic environment) to 1.02 ± 0.50 pH units min^?1 (hypotonic environment). Membrane staining of NHE1–cMyc fusion protein was maintained after hypotonic exposure in colchicine pre‐treated cells as was RVD. Microfilament inhibition by cytochalasin preserved NHE1 activity. Inhibition of phosphatidylinositol‐3′‐kinase was unable to restore Na⁺, H⁺ exchange activity. Conclusion: We conclude that regulation of Na⁺, H⁺ exchange during RVD is mediated by cytoskeletal elements. This receptor independent pathway is regulated by Src.     

Murine experimental leprosy: Evaluation of immune response by analysis of peritoneal lavage cells and footpad histopathology

This study evaluated the immune response of nude and BALB/c mice inoculated in the footpads (FP) with Mycobacterium leprae after 3, 5 and 8 months. At each timepoint peritoneal cells, peripheral blood, FP and popliteal lymph nodes (PLN) were collected. Peritoneal cell cultures were performed to measure the H₂O₂, O₂^?, NO, IL‐2, IL‐4, IL‐10, IL‐12, IFN‐γ and TNF levels. Serum levels of anti‐PGL‐I antibodies were also quantified. The results showed that the infection was progressive in nude mice with bacterial multiplication, development of macroscopic lesions in the FP and presence of bacilli in the PLN at 8 months. In BALB/c mice, the infection reached a plateau of bacillary multiplication at 5 months and regressed at 8 months. Histopathological analysis of FP revealed a mononuclear inflammatory infiltrate with a large number of neutrophils at 5 months, with a higher number in nude mice. At 8 months, the number of neutrophils decreased and the infiltrate was predominantly mononuclear in both mouse strains. There was no H₂O_2, O₂^?, IL‐2, IL‐4, IL‐10 and IFN‐γ production in the course of infection in nude mice; however, in BALB/c, O₂^? and IL‐12 production was higher at 5 months and NO, IFN‐γ and TNF production was higher at 8 months when there was a decrease in the number of bacilli. The level of anti‐PGL‐I antibodies was higher in BALB/c mice. Thus, nude and BALB/c mice can be used as experimental models for the study of various aspects of leprosy.     

Ruthenium Ring‐Opening Metathesis Polymerization Catalysts Bearing o‐ Aryloxide‐N‐Heterocyclic Carbenes

A series of o‐aryloxide‐N‐heterocyclic carbene ruthenium complexes 2–4 is synthesized via sequential reactions of the o‐hydroxyaryl imidazolium proligands (2‐OH‐3,5‐^tBu₂C₆H₂)(R)(C₃H₃N₂)⁺Br^? (R = Me ( 1a ), ⁱPr ( 1b ), Mes ( 1c )) with Ag₂O and [(C₆H₆)RuCl₂]₂. All of the complexes are characterized by ¹H and ¹³C NMR spectroscopy, high‐resolution mass spectrometry (HRMS), and elemental analysis. The molecular structure of 2 is determined by single‐crystal X‐ray diffraction analysis. The ring‐opening metathesis polymerization (ROMP) of norbornene (NBE) with 2 – 4 is studied. Among them, complex 4 exhibits the highest activity and efficiency toward ROMP of NBE at 85 °C without any cocatalyst, and the resultant polymers have very high molecular weight (>10⁶ Da) and narrow molecular weight distributions. This complex can also efficiently catalyze the alternating copolymerization of NBE and cyclooctene.