Vector correlations in the F + HO → HF + O reaction and its isotopic variant

The F + H(D)O → HF(DF) + O reactions have been studied using quasi-classical trajectory (QCT) calculation method, based on the three different potential energy surfaces (PESs) of Gomez-Carrasco et al. (J Chem Phys 2004, 121:4605; J Chem Phys 2005, 123:114310; Chem Phys Lett 2007, 435:188). Facilitated with the analysis of the QCT results, the pictures for product scattering and product polarizations have been presented to investigate the vector correlations in the two reactions, with effects of isotope substitution and electronic state as well as collision energy being revealed at a chemical stereodynamical level.     

Differential regulation of Na+/H+ exchange and H+ -ATPase by pH and HCO 3 − in kidney proximal tubules

This study examines the effects of acute in vitro acid-base disorders on Na⁺/H⁺ and H⁺-ATPase transporters in rabbit kidney proximal tubules (PT). PT suspensions were incubated in solutions with varying acid base conditions for 45 min and utilized for brush border membrane (BBM) vesicles preparation. BBM vesicles were studied for Na⁺/H⁺ exchange activity (assayed by ²²Na⁺ influx) or abundance (using NHE-3 specific antibody) and H⁺-ATPase transporter abundance (using antibody against the 31 kDa subunit). The Na⁺/ H⁺ exchanger activity increased by 55% in metabolic acidosis (pH 6.5, HCO ₃ ^– 3 mm) and decreased by 41% in metabolic alkalosis (pH 8.0, HCO ₃ ^– 90 mm). The abundance of NHE-3 remained constant in acidic, control, and alkalotic groups. H⁺-ATPase abundance, however, decreased in metabolic acidosis and increased in metabolic alkalosis by 57% and 42%, respectively. In PT suspensions incubated in isohydric conditions (pH 7.4), Na⁺/H⁺ exchanger activity increased by 29% in high HCO ₃ ^– group (HCO ₃ ^– 96 mm) and decreased by 16% in the low HCO ₃ ^– groups (HCO ₃ ^– 7mm. The NHE-3 abundance remained constant in high, normal, and low [HCO ₃ ^– ] tubules. The abundance of H⁺-ATPase, however, increased by 82% in high [HCO ₃ ^– ] and decreased by 77% in the low [HCO ₃ ^– ] tubules. In PT suspensions incubated in varying pCO₂ and constant [HCO ₃ ^– ], Na⁺/H⁺ exchanger activity increased by 35% in high pCO₂ (20% pCO₂, respiratory acidosis) and decreased by 32% in low pCO₂ (1.5% pCO₂, respiratory alkalosis) tubules. The NHE-3 abundance remained unchanged in high, normal, and low pCO₂ tubules. However, the H⁺-ATPase abundance increased by 74% in high pCO₂ and decreased by 69% in low pCO₂ tubules.The results of these studies suggest that the luminal Na⁺/H⁺ exchanger is predominantly regulated by pH whereas H⁺-ATPase is mainly regulated by [HCO ₃ ^– ] and/ or pCO₂. They further suggest that the adaptive changes in H⁺-ATPase transporter are likely mediated via endocytic/exocytic pathway whereas the adaptive changes in Na⁺/H⁺ exchanger are via the nonendocytic/exocytic pathway.The excellent technical assistance of Yollanda J. Hattabaugh, Gwen L. Bizal, and L. Yang is greatly appreciated. Portions of these studies were presented at the annual meeting of the American Society of Nephrology, Boston, MA, November 1993, and published in abstract form (J.Am.Soc.Neph. 4:840A, 1993)These studies were supported by a Merit Review Grant from the Department of Veterans Affairs and a grant-in-aid from the American Heart Association (to M.S.), a Baxter Health Care Grant (to B.B.), and the National Institute of Health Grants DK 38510 (to E.B.C. and M.C.R.) and DK 42086 (to E.B.C.).     

Synergistic interactions between β-lapachone and fluconazole in the inhibition of CaCdr2p and CaMdr1p in Candida albicans

BackgroundMortality rate of invasive Candida infections is raising mainly amongst immunocompromised patients. These infections are hard-to-treat mainly due to the increasing incidence of resistance. The overexpression of ATP-binding cassette and major facilitator superfamily transporters is the main responsible for the failure of antifungal therapies. In a Saccharomyces cerevisiae model, β-lapachone inhibited Pdr5p, a transporter homologous to those found in Candida albicans.AimsTo determine whether β-lapachone reverses the resistance phenotype mediated by efflux transporters in C. albicans clinical isolates.MethodsThe antifungal activity of β-lapachone combined with fluconazole was measured by agarose chemosensitization and microdilution assays. CaCdr2p and CaMdr1p activities were evaluated through fluorescent dyes accumulation. ATPase activity was assessed using transporter-enriched plasma membranes.Resultsβ-lapachone reverted antifungal resistance of S. cerevisiae and C. albicans strains overexpressing CaCdr2p and CaMdr1p transporters by inhibiting these proteins activities. CaCdr2p ATPase activity was not impaired by the compound.Conclusionsβ-lapachone is a promising drug candidate to be used as an adjuvant in the treatment of candidiasis caused by fluconazole-resistant C. albicans strains.     

Role of Na+–H+ exchange in the modulation of L-type Ca2+ current during fluid pressure in rat ventricular myocytes

Application of fluid pressure (FP) using pressurized fluid flow suppresses the L-type Ca²⁺ current through both enhancement of Ca²⁺ release and intracellular acidosis in ventricular myocytes. As FP-induced intracellular acidosis is more severe during the inhibition of Na⁺–H⁺ exchange (NHE), we examined the possible role of NHE in the regulation of I_Ca during FP exposure using HOE642 (cariporide), a specific NHE inhibitor. A flow of pressurized (∼16 dyn/cm²) fluid was applied onto single rat ventricular myocytes, and the I_Ca was monitored using a whole-cell patch-clamp under HEPES-buffered conditions. In cells pre-exposed to FP, additional treatment with HOE642 dose-dependently suppressed the I_Ca (IC₅₀ = 0.97 ± 0.12 μM) without altering current–voltage relationships and inactivation time constants. In contrast, the I_Ca in control cells was not altered by HOE642. The HOE642 induced a left shift in the steady-state inactivation curve. The suppressive effect of HOE642 on the I_Ca under FP was not altered by intracellular high Ca²⁺ buffering. Replacement of external Cl⁻ with aspartate to inhibit the Cl⁻-dependent acid loader eliminated the inhibitory effect of HOE642 on I_Ca. These results suggest that NHE may attenuate FP-induced I_Ca suppression by preventing intracellular H⁺ accumulation in rat ventricular myocytes and that NHE activity may not be involved in the Ca²⁺-dependent inhibition of the I_Ca during FP exposure.     

Na+-dependent HCO 3 − transport and Na+/H+ exchange regulate pH i in human ciliary muscle cells

Summary We investigated intracellular pH (pH _i ) regulation in cultured human ciliary muscle cells by means of the pH-sensitive absorbance of 5(and 6)-carboxy-4,5-dimethylfluorescein (CDMF). The steady-state pH _i was 7.09±0.04 (n = 12) in CO₂/ HCO ₃ ^– -buffered and 6.86±0.03 (n = 12) in HEPES-buffered solution. Removal of extracellular sodium for 6 min acidified the cells by 1.11±0.06 pH units (n = 12) in the presence of CO₂/ HCO ₃ ^– and by 0.91±0.05 pH units (n = 8) in its absence. Readdition of external sodium resulted in a rapid pH _i recovery, which was almost completely amiloride-sensitive in the absence of CO₂/ HCO ₃ ^– but only slightly influenced by amiloride in its presence. Application of DIDS under steady-state conditions significantly acidified the ciliary muscle cells by 0.25±0.02 (n = 4) in 6 min, while amiloride had no effect. The pH _i recovery after an intracellular acid load was completely dependent on extracellular sodium. In HEPES-buffered solution the pH _i recovery was almost completely mediated by Na⁺/H⁺ exchange, since it was blocked by amiloride (1 mmol/liter). In contrast, a marked amilorideinsensitive pH _i recovery was observed in CO₂/HCO ₃ ^– -buffered solution which was mediated by chloride-independent and chloride-dependent Na⁺ HCO ₃ ^– cotransport. This recovery, inhibited by DIDS (0.2 mmol/liter). was also observed if the cells were preincubated in chloride-free solution for 4 hr. Analysis of the sodium dependence of the pH _i recovery after NH₄Cl prepulse revealed V _max = 0.57 pH units/min, K _m= 39.7 mmol/liter extracellular sodium for the amiloride-sensitive component and V _max = 0.19 pH units/min, K _m= 14.3 mmol/liter extracellular sodium for the arniloride-insensitive component. We conclude that Na⁺/H⁺ exchange and chloride-independent and chloride-dependent Na⁺HCO ₃ ^– cotransport are involved in the pH _i regulation of cultured human ciliary muscle cells.The expert technical assistance of Astrid Krolik is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft grant DFG Wi 328/11.     

The interrelationship between HCO 3 − , NO 3 − , and Cl− as effective anions in the photosynthetic electron transport

Thylakoids were isolated from the leaves of three different plants (Pisum sativium L., Lactuca sativa L., and Raphanus sativus L.). The addition of HCO ₃ ^? to a suspension of salt-and HCO ₃ ^? -epleted thylakoids (suspended in salt-free medium) raised the rate of O₂ evolution up to fourfold. This stimulation could be partially replaced by the addition of chloride or nitrate ions. However, the addition of HCO ₃ ^? in the presence of Cl^? or NO ₃ ^? resulted in a higher stimulation of O₂ evolution (sixfold in the presence of nitrate and sevenfold in the presence of chloride). On the other hand, the addition of HCO ₃ ^? to the thylakoids depleted from salt only raised the rate of O₂ evolution by 10–15%, whereas 40–70% was obtained by the addition of nitrate or chloride ions. The fluorescence induction studies indicated a significant decrease in the yield of the variable fluorescence of the salt- and HCO ₃ ^? -depleted thylakoids. A partial increase in the fluorescence yield was obtained by the addition of HCO ₃ ^? alone. A typical fluorescence induction curves were obtained by the addition of HCO ₃ ^? in the presence of Cl^? or NO ₃ ^? ions. The data obtained suggest a similar role for chloride and nitrate ions in O₂ evolution in the Hill-reaction, which is restricted at the donor side of photosystem II, whereas bicarbonate plays its role at both sides (acceptor and donor sides). The presented data are those obtained for the thylakoids of P. sativium, which were more or less similar to those obtained for L. sativa and R. sativus.     

Osmosensitivity of the 2H+−K+-exchange and the H+-ATPase complex F0F1 in anaerobically grownEscherichia coli

Escherichia coli grown anaerobically for osmotic studies upon increased osmolarity in alkaline medium carried out H⁺–K⁺-exchange in two steps, the first of which was DCCD¹ sensitive and osmo-dependent and had the 2H⁺/K⁺ stoichiometry. H⁺-efflux in the presence of protonophore (CCCP) upon increase of osmolarity was shown to be high and inhibited by DCCD, whereas H⁺-efflux induced by a decrease of osmolarity was small and not inhibited by DCCD. The 2H⁺/K⁺-exchange was absent intrkA anduncA mutants. InuncB mutant 2H⁺/K⁺-exchange was not DCCD-and osmosensitive. Competition between DCCD and osmoshock on inhibition of 2H⁺/K⁺-exchange was found. Osmosensitivity of this exchange disappeared in spheroplasts. Osmosensitivity of both 2H⁺/K⁺-exchange and the F₀F₁ and osmoregulation of the F₀F₁ via F₀ and a periplasmic space are postulated.Abbreviations F₀F₁ H⁺-ATPase complex - F₀ H⁺-channel, proteolipid - F₁ H⁺-ATPase - Trk constitutive system for K⁺ uptake - PV periplasmic protein valve - DCCD N,N-dicyclohexylcarbodiimide - CCCP carbonylcyanide-m-chlorophenylhydrazone - _H or _K transmembrane electrochemical gradient for H⁺ or K⁺ respectively - membrane potential - upshock or downshock increase or decrease of medium osmolarity, respectively - CGSC E. coli Genetic Stock Center, Yale University, USA     

Differences in the Large Extracellular Loop between the K+-Cl? Cotransporters KCC2 and KCC4

K⁺Cl⁻ cotransporters (KCCs) play fundamental physiological roles in processes such as inhibitory neurotransmission and cell volume regulation. Mammalian genomes encode four distinct KCC paralogs, which share basic transport characteristics but differ significantly in ion affinity, pharmacology, and relative sensitivity to cell volume. Studies to identify divergence in functional characteristics have thus far focused on the cytoplasmic termini. Here, we investigated sequence requirements of the large extracellular loop (LEL) for function in KCC2 and KCC4. Mutation of all four evolutionarily conserved cysteines abolished KCC2 transport activity. This behavior differs from that of its closest relative, KCC4, which is insensitive to this mutation. Chimeras supported the differences in the LEL of the two cotransporters, because swapping wild-type LEL resulted in functional KCC2 but rendered KCC4 inactive. Insertion of the quadruple cysteine substitution mutant of the KCC4 loop, which was functional in the parental isoform, abolished transport activity in KCC2. Dose-response curves of wild-type and chimeric KCCs revealed that the LEL contributes to the different sensitivity to loop diuretics; a KCC2 chimera containing the KCC4 LEL displayed an IC₅₀ of 396.5 μm for furosemide, which was closer to KCC4 (548.8 μm) than to KCC2 (184.4 μm). Cell surface labeling and immunocytochemistry indicated that mutations do not affect trafficking to the plasma membrane. Taken together, our results show a dramatic and unexpected difference in the sequence requirements of the LEL between the closely related KCC2 and KCC4. Furthermore, they demonstrate that evolutionarily highly conserved amino acids can have different functions within KCC members.     

Is a Ca2+ -ATPase involved in Ca2+ regulation during capacitation and the acrosome reaction of guinea-pig spermatozoa?

The Ca2+-ATPase antagonists quercetin and ethacrynic acid accelerated the onset of the acrosome reaction in guinea-pig spermatozoa incubated in the continuous presence of Ca2+, whereas furosemide had no effect, and sodium orthovanadate only affected sperm motility. When spermatozoa were preincubated in a 'Ca2+-free' medium, quercetin and ethacrynic acid shortened capacitation time: spermatozoa incubated for 1 h in 100-200 microM-ethacrynic acid showed 60-80% acrosome reactions when Ca2+ was added. Such spermatozoa were able to fertilize zona-free hamster eggs. Our results therefore point to the possible involvement of a Ca2+-ATPase in the regulation of intracellular Ca2+ in spermatozoa. Cysteine and dithiothreitol, both disulphide reducing agents, prevented the effects of quercetin and ethacrynic acid, suggesting that sulphydryl groups may be important for the expression of Ca2+-ATPase activity. Lysophosphatidylserine (LS) also prevented the stimulatory effect of ethacrynic acid, an effect similar to that shown by LS on lysophosphatidylcholine (LC). It is argued that both LS and LC could exert their action through an effect on the Ca2+-ATPase.     

Coupled electron and proton transfer reactions during the O→E transition in bovine cytochrome c oxidase

A combined DFT/electrostatic approach is employed to study the coupling of proton and electron transfer reactions in cytochrome c oxidase (CcO) and its proton pumping mechanism. The coupling of the chemical proton to the internal electron transfer within the binuclear center is examined for the O→E transition. The novel features of the His291 pumping model are proposed, which involve timely well-synchronized sequence of the proton-coupled electron transfer reactions. The obtained pK(a)s and E(m)s of the key ionizable and redox-active groups at the different stages of the O→E transition are consistent with available experimental data. The PT step from E242 to H291 is examined in detail for various redox states of the hemes and various conformations of E242 side-chain. Redox potential calculations of the successive steps in the reaction cycle during the O→E transition are able to explain a cascade of equilibria between the different intermediate states and electron redistribution between the metal centers during the course of the catalytic activity. All four electrometric phases are discussed in the light of the obtained results, providing a robust support for the His291 model of proton pumping in CcO.     

Na+−Ca2+ exchange and Ca2+ channel characteristics in bovine aorta and coronary artery smooth muscle sarcolemmal membranes

Tension generation and Ca²⁺ flux in smooth muscle varies depending upon the diameter of a vessel and its location. The purpose of the present investigation was to determine if the biochemical characteristics of the Na⁺–Ca²⁺ exchanger and the Ca²⁺ channel differ in sarcolemmal membrane preparations isolated from a large conduit vessel (thoracic aorta) or from large and small coronary arteries. We also investigated the possibility of differences between sarcolemmal membranes isolated from coronary arteries dissected from the right and left ventricles. The purification of the sarcolemmal membranes was of a similar magnitude amongst the different groups. Contamination of the sarcolemmal membranes with other membranous organelles was negligible and similar amongst the groups. The K_m and V_max of Na⁺-dependent Ca²⁺ uptake in sarcolemmal vesicles was similar amongst the groups. Calcium channel characteristics were examined by measuring [³H]PN200-110 binding to sarcolemmal vesicles. The right coronary artery membranes from both large and small caliber vessels exhibited a higher K_d and the small right coronary artery sarcolemmal preparation had a lower maximal binding density for [³H] PN200-110. The results suggest that the right coronary artery, and in particular the small diameter right coronary artery, possesses altered Ca²⁺ channel characteristics in isolated sarcolemmal membranes.