Relationship of proton motive force and the F0F1-ATPase with bio-hydrogen production activity of Rhodobacter sphaeroides: effects of diphenylene iodonium, hydrogenase inhibitor, and its solvent dimethylsulphoxide

Rhodobacter sphaeroides MDC 6521 was able to produce bio-hydrogen (H(2)) in anaerobic conditions under illumination. In this study the effects of the hydrogenase inhibitor-diphenylene iodonium (Ph(2)I) and its solvent dimethylsulphoxide (DMSO) on growth characteristics and H(2) production by R. sphaeroides were investigated. The results point out the concentration dependent DMSO effect: in the presence of 10?mM DMSO H(2) yield was ~6 fold lower than that of the control. The bacterium was unable to produce H(2) in the presence of Ph(2)I. In order to examine the mediatory role of proton motive force (?p) or the F(0)F(1)-ATPase in H(2) production by R. sphaeroides, the effects of Ph(2)I and DMSO on ?p and its components (membrane potential (?ψ) and transmembrane pH gradient), and ATPase activity were determined. In these conditions ?ψ was of -98?mV and the reversed ?pH was +30?mV, resulting in ?p of -68?mV. Ph(2)I decreased ?ψ in concentrations of 20?μM and higher; lower concentrations of Ph(2)I as DMSO had no valuable effect on ?ψ. The R. sphaeroides membrane vesicles demonstrated significant ATPase activity sensitive to N,N'-dicyclohexylcarbodiimide. The 10-20?μM Ph(2)I did not affect the ATPase activity, whereas 40?μM Ph(2)I caused a marked inhibition (~2 fold) in ATPase activity. The obtained results provide novel evidence on the involvement of hydrogenase and the F(0)F(1)-ATPase in H(2) production by R. sphaeroides. Moreover, these data indicate the role of hydrogenase and the F(0)F(1)-ATPase in ?p generation. In addition, DMSO might increase an interaction of nitrogenase with CO(2), decreasing nitrogenase activity and affecting H(2) production.     

The viability of cryopreserved PBPC depends on the DMSO concentration and the concentration of nucleated cells in the graft

BACKGROUND: DMSO is widely used as a cryoprotectant for PBPC. It is desirable to reduce the amount of DMSO without jeopardizing the quality of the stem cell product. The present study was undertaken to investigate whether recovery and survival of CD34+ cells would be significantly altered when PBPC used for autologous transplantations were cryopreserved with four different DMSO concentrations. METHODS: Apheresis samples of PBPC from 20 consecutive patients were mixed in parallel with 2%, 4%, 5% and 10% DMSO, frozen with identical cell concentrations at a controlled rate, and stored in liquid nitrogen for 6-8 weeks. PBPC samples from 11 consecutive patients were also cryopreserved with two different cell concentrations (150 and 300 x 10(6) nucleated cells/mL) to investigate the effect of increasing the cell concentrations while decreasing the DMSO concentration. The flow cytometric absolute count method, based on ISHAGE guidelines, was used to measure the absolute count of total and viable CD34+ cells in the post-thaw samples. RESULTS: PBPC cryopreserved at 150 x 10(6) cells/mL with 2% DMSO yielded significantly inferior CD34+ cell recovery (P < 0.001) and survival (P < 0.001) compared with cryopreservation with 4% and 5% DMSO. This was also observed when comparing higher cell concentrations. However, a reduced cell survival (P = 0.02) was observed when the nucleated cell concentration was increased from 150 to 300 x 10(6) cells/mL in samples cryopreserved with 5% DMSO. DISCUSSION: We conclude that 5% DMSO may be the optimal dose for cryopreserving PBPC as long as the cells have not been concentrated at much more than 200 x 10(6) nucleated cells/mL.     

Participation of hyf-encoded hydrogenase 4 in molecular hydrogen release coupled with proton-potassium exchange in Escherichia coli

In a previous work (Trchounian et al., Biol. Membrany 16:416-428 (1999) (in Russian)) we reported the interrelations between production of H2 and H+-K+ exchange in fermenting Escherichia coli grown under anaerobic conditions at pH 7.5. The ion fluxes had stable stoichiometry 2H+/K+ and were N,N'-dicyclohexylcarbodiimide (DCC)-inhibitable at different external pH and K+ activity. In the present study, the H2 production was further studied in fermenting bacteria grown at pH 7.5 or 6.5. The H2 production was inhibited by DCC and did not occur if bacteria were grown at pH 7.5 in a medium containing formate or upon hypoosmotic stress. The H2 production was not sensitive to osmotic stress when bacteria were grown at pH 6.5. Formation of H2 and 2H+/K+ exchange were not observed in mutants with deletions of the hyfoperon genes, encoding membrane-associated hydrogenase 4. K+ influx in these mutants was not sensitive to valinomycin, in contrast to the K+ influx in the parental strain. If grown at pH 6.5, the mutants produced H2 and carried out 2H+/K+ exchange, when subjected to the hyperosmotic stress. The results suggest a participation of hydrogenase 4 in the production of H2 and proton-potassium exchange in fermenting E. coli grown at pH 7.5. In bacteria grown at pH 6.5 or in a medium containing formate, another membrane-bound hydrogenase, namely hydrogenase 3, may be responsible for the H2 production.     

The capacity of anaerobically grown bacteria to exchange the 2H+ of the cell for the K+ of the medium and to maintain a high K+ distribution between cell and medium

The N,N'-dicyclohexylcarbodiimide sensitive exchange of 2H+ of a cell for K+ of medium stable to pH, K+ activity and temperature changes has been discovered in anaerobically grown gram-negative Escherichia coli, Salmonella typhimurium. S. enteritidis, Proteus mirabilis, P. vulgaris, anaerobic gram-positive bacteria Streptococcus faecalis, Lactobacillus salivarius, L. lactis in the presence of exogenic energy source. This exchange in gram-negative bacteria is operating only at increase of medium osmolarity. The high K+ distribution between cell and medium has been reached during the exchange of 2H+ for one K+ and the corresponding potassium equilibrium potential is much more than the measured delta psi. In aerobically grown E. coli, S. typhimurium, Brevibacterium flavum and aerobic Micrococcus luteus exchange of 2H+ for K+ does not take place, the K+ distribution is lower and in good conformity with the measured delta psi. It is assumed that exchange of 2H+ for K+ in anaerobic bacteria is carried out by the H+-ATPase complex and the Trk (or Trk-like) system of K+ absorption united into the same membrane supercomplex which functions as the H+-K+-pump and supports the high K+ distribution between cell and medium.     

Radioprotective effects of dimethyl sulfoxide in golden hamster embryo cells exposed to gamma rays at 77 K. I. Radical formation as studied by electron spin resonance

Formation of free radicals in golden hamster embryo (GHE) cells in the frozen living state by gamma irradiation has been studied by electron spin resonance spectroscopy at 4.2 and 77 K. The relative yields of H atoms, OH radicals, and organic radicals trapped in the irradiated GHE cells are 12, 72, and 16%, respectively, of total radical yields. When dimethylsulfoxide (DMSO) is added to GHE cells at 77 K, a large quantity of CH2SOCH3 radicals (DMSO radicals) are formed after gamma irradiation. The yields of OH radicals are not affected by the addition of DMSO. When the GHE cell-DMSO mixtures are irradiated with gamma rays at 77 K and then warmed to 111 K, the OH radicals decay, whereas the DMSO radicals do not increase complementarily. Moreover, the decay rates of the OH radicals at 111 K do not depend upon the concentration of DMSO. Thus OH radicals do not react with DMSO during warming of the irradiated sample. When H atoms are produced by gamma irradiation of acid ice at 60 K, the decay rates of the H atoms at 77 K increase with increasing DMSO concentration, indicating that DMSO reacts with H atoms (CH3SOCH3 + H----.CH2SOCH3 + H2) at 77 K by quantum-mechanical tunneling. When the GHE cell-DMSO mixture is irradiated with gamma rays at 77 or 4.2 K in the dark, DMSO ions are produced in addition to DMSO radicals. Therefore it is concluded that DMSO does not scavenge OH radicals, but does capture H atoms, holes and/or electrons in the gamma-irradiated cells, resulting in the remarkable formation of DMSO radicals. This scavenger effect of DMSO may be related to the radioprotection of DMSO against cell killing described in the companion paper (Watanabe et al., Radiat. Res., this issue).     

The effect of ionophores and related agents on the induction of doming in a rat mammary epithelial cell line

Addition of dimethyl sulfoxide (DMSO) and the mammotropic hormones prolactin, hydrocortisone, insulin, and estradiol to confluent cultures of the epithelial cell line Rat Mammary (Rama) 25 increases dramatically the formation of domes in the cell monolayer after 48-72 hr. Associated with the increase in doming is an increase of 24% in the activity of the Na+/K+ ATPase. Both Ca2+ (A23187) and Na+ (monensin, gramicidin J, melittin) ionophores can replace DMSO in inducing domes, whilst the K+ ionophore valinomycin inhibits doming. However, there are no synergistic nor additive effects, respectively, with suboptimal or optimal concentrations of A23187 and melittin together. Ouabain, at concentrations which inhibit the Na/K ATPase in vitro, and amiloride, at concentrations reported to inhibit the passive transport of Na+, both inhibit completely the formation of domes induced by DMSO, A23187, and melittin. EGTA, however, inhibits only the induction of doming by DMSO and A23187; it is without effect with melittin. A23187 and melittin induce the major polypeptide changes that occur in doming cultures with DMSO, and most of these changes are also inhibited with ouabain. It is suggested that one possible interpretation of the findings is that the induction of doming by DMSO in Rama 25 cells occurs by means of sequential increases in Ca2+ and Na+ influxes into the cell, and that the increased intracellular concentration of Na+ so produced stimulates the Na+/K+ ATPase, with a net effect of pumping liquid beneath the cellular monolayer.     

Formation of an ion transport supercomplex in Escherichia coli. An experimental model of direct transduction of energy

Hydrogen gas production was observed to occur during ATP-driven H+/K+ exchange in anaerobically grown E. coli. Neither process was found in aerobically grown cells or anaerobic cells grown on nitrate medium or when the osmotic pressure was decreased or K+ removed, or finally when DCCD, arsenate or CCCP was applied. Dithiothreitol restored the process even in the presence of CCCP but not in other cases of inhibition. A model of a multienzyme transport super-complex is proposed. The supercomplex consists of three genetically independent mechanisms: F0F1 H+-ATPase to provide energy, the K+-transporting Trk system as energy sink and formate-hydrogen lyase as donor of reducing equivalents. Within this supercomplex direct transduction of energy is accomplished via oxidation of 2 SH to S-S.     

Dimethylsulfoxide modifies the sensitivity of BALB/c-3T3 cells to the activation of Na+/H+ exchange by phorbol esters

In a companion paper (Gillies et al.: J. Cell. Physiol. 139:124-129, 1989) we show that phorbol esters (PEs) are unable to stimulate Na+/H+ exchange in BALB/c-3T3 cells under a wide variety of conditions. The Na+/H+ exchangers of a number other cell types are also not responsive to PEs yet have been rendered responsive by treatment with agents such as dimethylsulfoxide (DMSO). We undertook the present study to evaluate whether or not the treatment of BALB/c-3T3 cells with DMSO will induce modifications in the sensitivity of these cells to activation by PEs. The present study indicates that a 3-5 day exposure of BALB/c-3T3 cells to 1.25% DMSO leads to changes in the sensitivity of these cells to the activation of Na+/H+ exchanger by PEs. These changes in sensitivity were apparent at day 3 and maximal at day 5. Non-tumor-promoting analogues of PEs do not activate Na+/H+ exchange, suggesting that the effect is mediated through kinase C. Sphingosine prevents PE-, but not serum-induced alkalinization. However, the half-time of the intracellular pH (pHin) response to serum was increased by sphingosine, suggesting that kinase C participates in, but is not required for, the serum induced activation. Since DMSO does not induce any apparent morphological change, the change in sensitivity of Na+/H+ exchange to PEs is not likely to be related to differentiation, but may be associated with structural changes in the Na+/H+ exchanger and/or changes in isoforms of kinase C which recognize the exchanger as a substrate.     

Inhibitory activity of 2-nitropropanol against select food-borne pathogens in vitro

AIMS: To test the inhibitory activity of 2-nitro-1-propanol (2NPOH) against Salmonella Typhimurium, Escherichia coli O157:H7 and Enterococcus faecalis. METHODS AND RESULTS: Specific growth rates (h(-1)) of S. Typhimurium, E. coli O157:H7 and Ent. faecalis were determined during culture in tryptic soya broth (TSB) supplemented with 0-10 mm 2NPOH. Growth rates were inhibited by 2NPOH, with nearly complete inhibition observed with 10 mm. Studies with S. Typhimurium revealed that its survivability during culture in TSB containing 5 or 10 mm 2NPOH was lower (P < 0.05) under aerobic than anaerobic conditions. The survivability of Salmonella during anaerobic culture in TSB containing 2.5 mm 2NPOH was less at pH 5.6 than at pH 7.0 and 8.0. No Salmonella survived anaerobic incubation in TSB supplemented with 10 mm 2NPOH regardless of pH. When incubated in suspensions of freshly collected populations of ruminal and faecal bacteria, Salmonella concentrations were lower (P < 0.05) in suspensions containing 10 mm 2NPOH than in suspensions containing no 2NPOH. CONCLUSIONS: 2NPOH inhibited S. Typhimurium, E. coli O157:H7 and Ent. faecalis. SIGNIFICANCE AND IMPACT OF THE STUDY: Results suggest that 2NPOH may be a useful antimicrobial supplement to reduce carriage of certain food-borne pathogens in food animals.     

Proton dissociation from nigericin at the membrane-water interface, the rate-limiting step of K+/H+ exchange on the bilayer lipid membrane.

The rate of K+/H+ exchange through bilayer lipid membranes (BLM) induced by nigericin was measured by the method of pH gradient offset according to Antonenko, Yu.N. and Yaguzhinsky L.S. [(1990) Biochim. Biophys. Acta 1026, 236-240]. It was shown that under the conditions of high potassium ion concentration the rate of nigericin-mediated K+/H+ exchange increased with an increase in the concentrations of such buffer compounds as citric acid and MES. The concentration dependence was different for citrate and MES. The buffer concentration effect was absent at low potassium ion concentrations. Citrate increased the rate of K+/H+ exchange being added to the side of BLM where the K+ concentration was higher and had no effect at the opposite side. At high KCl and citrate concentrations, the rate of K+/H+ exchange was about 6 times lower in D2O when compared to H2O solutions. It is concluded that under certain experimental conditions the overall rate of the K+/H+ exchange induced by nigericin is determined by the rate of proton dissociation from nigericin at the membrane-water interface.     

Thermal sweat lactate in cystic fibrosis and in normal children

We attempt to determine whether the decrease in Na+ reabsorption and the increase in K+ secretion in sweat of cystic fibrosis patients (CF) were associated with changes in glandular anaerobic metabolism evaluated by forehead sweat lactate excretion rate. 6 CF and 11 normal (C) children, 5 months to 14 years old, were exposed to external thermal load (45 degrees C). The data showed that: 1) Na+, K+ and Cl- concentrations in CF are constant at any flow rate (Qsw); 2) In both groups the excretion rates of Na+, K+ and Cl- increased linearly with Qsw but the slopes in CF were significantly higher than in C (p less than 0.001); 3) Lactate excretion rate increased with Qsw as in CF and C with the same slope. We suggest that an increase in energy expenditure of Na+ - K+ exchange and an active secretion of K+ by the duct could explain the normal energy metabolism that we observed in CF sweat glands.     

Effect of temperature on H+-K+ exchange in Escherichia coli bacteria during their anaerobic growth

The H(+)-K(+)-exchange in E.coli grown under anaerobic conditions at temperatures from 17 to 37 degrees C was studied. The Arrhenius plots for both the N,N'-dicyclohexylcarbodiimide-sensitive release of H+ and K+ uptake by cells transferred into a fresh medium containing a carbon source (glucose) are nonlinear. The activation energy values for the transport of these cations at different temperatures significantly differ. It is shown that as the temperature decreases, the accumulation of K+ by cells is reduced. In this process, the initial rate of K+ absorption through the TrkA system, the time of accumulation of these cations by cells and the osmosensitivity of K+ uptake substantially decrease. At temperatures below 20 degrees C, the absorption becomes insensitive to the secondary osmoshock. However, the stoichiometry of N,N'-dicyclohexylcar-bodiimide-sensitive cation fluxes remains unchanged and is equal to 2H+:K+. It is assumed that the H(+)-K(+)-exchange proceeds by the operation of an ensemble of oligomers, formed from the protomers of F0F1 and TrkA, which rearrange by the action of temperature, whereas F0F1 and TrkA in each protomer do not change.     

Inactivation of xanthine oxidase by hydrogen peroxide involves site-directed hydroxyl radical formation.

The mechanism of xanthine oxidase (XO) inactivation by hydrogen peroxide (H2O2) and its biologic significance are unclear. We found that addition of increasing concentrations of H2O2 progressively decreased xanthine oxidase activity in the presence but not the absence of xanthine in vitro. Inactivation of XO by H2O2 was also enhanced by anaerobic reduction of XO by xanthine. Inactivation of XO by H2O2 was accompanied by production of hydroxyl radical (.OH), measured as formation of formaldehyde from dimethylsulfoxide (DMSO). In contrast, addition of H2O2 to deflavo XO did not produce .OH. Inactivation of XO by H2O2 was decreased by simultaneous addition of the .OH scavenger, DMSO. However, inactivation of XO by H2O2 and formation of .OH were not decreased following addition of the metal chelator. DETAPAC, and/or the O2 scavenger, superoxide dismutase. The results suggest that inactivation of XO by H2O2 occurs by production of .OH following direct reduction of H2O2 by XO at the flavin site.     

Escherichia coli membrane proton conductance and proton efflux depend on growth pH and are sensitive to osmotic stress

The dependence of Escherichia coli membrane H+ conductance (Gm H+) with a steady-state pH in the presence and absence of an external source of energy (glucose) was studied, when cells were grown under anaerobic and aerobic conditions, with an assay pH of 7.0. Energy-dependent H+ efflux by intact cells growing at pH of 4.5-7.5 was also measured. The elevated H+ conductance and lowered H+ flux were shown for cells growing in acidic pH and under anaerobic conditions, when bacteria were fermenting glucose. The atp mutant, which is deprived of the F0F1- adenosine triphosphatase, had less Gm H+ independent of growth conditions. In contrast with wild-type or precursor strain, a remarkable difference in Gm H+ for atp mutant was observed between aerobic and anaerobic conditions; such a difference was significant at pH 4.5. These results could indicate distinguishing pathways determining Gm H+ under anaerobic conditions after the fermentation of glucose at different pH and an input of the F0F1-adenosine triphosphatase in Gm H+. In addition, the effect of osmotic stress was demonstrated with grown cells. Gm H+ and H+ efflux both were increased after hyperosmotic stress at pH 7.5, and these changes were inhibited by N,N\'-dicyclohexylcarbodiimide, whereas these changes were lower in atp mutant. A role of the F0F1-adenosine triphosphatase in osmo-sensitivity of bacteria was confirmed under fermentative conditions.     

Effects of dimethyl sulfoxide on catalysis in Escherichia coli F1-ATPase.

(1) Dimethyl sulfoxide (DMSO) markedly inhibited the Vmax of multisite ATPase activity in Escherichia coli F1-ATPase at concentrations greater than 30% (v/v). Vmax/KM was reduced by 2 orders of magnitude in 40% (v/v) DMSO at pH 7.5, primarily due to reduction of Vmax. The inhibition was rapidly reversed on dilution into aqueous buffer. (2) KdATP at the first, high-affinity catalytic site was increased 1500-fold from 2.3 x 10(-10) to 3.4 x 10(-7) M in 40% DMSO at pH 7.5, whereas KdADP was increased 3.2-fold from 8.8 to 28 microM. This suggests that the high-affinity catalytic site presents a hydrophobic environment for ATP binding in native enzyme, that there is a significant difference between the conformation for ADP binding as opposed to ATP binding, and that the ADP-binding conformation is more hydrophilic. (3) Rate constants for hydrolysis and resynthesis of bound ATP in unisite catalysis were slowed approximately 10-fold by 40% DMSO; however, the equilibrium between bound Pi/bound ATP was little changed. The reduction in catalysis rates may well be related to the large increase in KdATP (less constrained site). (4) Significant Pi binding to E. coli F1 could not be detected either in 40% DMSO or in aqueous buffer using a centrifuge column procedure. (5) We infer, on the basis of the measured constants KaATP, K2 (hydrolysis/resynthesis of ATP), k+3 (Pi release), and KdADP and from estimates of k-3 (Pi binding) that delta G for ATP hydrolysis in 40% DMSO-containing pH 7.5 buffer is between -9.2 and -16.8 kJ/mol.     

Increased alkali stability in Trichoderma reesei endo-1, 4-beta-xylanase II by site directed mutagenesis

A number of engineered Trichoderma reesei endo-beta-1,4-xylanase (Xyn II) mutants were created and activity tests were performed for increased stability. The stability of the earlier characterized mutant Y5 (T2C, T28C, K58R, +191D) was further increased by the mutations creating the constructs P9 (N97R+F93W+H144K), P12 (H144C+N92C), P15 (F180Q+H144C+N92C) and P21 (H22K+F180Q+H144C+N92C). The resistance towards thermal inactivation at alkaline pH was increased in all of the mutants. Residual activity T(50%) was increased 4-5 degrees C for P9 at pH 9. The performance of the P9 mutant in sulphate pulp bleaching was also tested and was shown to increase brightness markedly compared to the reference. The bleaching results showed the industrial potential of the obtained mutant.     

Effect of extreme salt concentrations on the physiology and biochemistry of Halobacteroides acetoethylicus.

Halobacteroides acetoethylicus grew in media with 6 to 20% NaCl and displayed optimal growth at 10% NaCl. When grown in medium with an [NaCl] of 1.7 M, the internal cytoplasmic [Na+] and [Cl-] were 0.92 and 1.2 M, respectively, while K+ and Mg2+ concentrations in cells were 0.24 and 0.02 M, respectively. Intracellular [Na+] was fourfold higher than intracellular [K+]. Since Na+ and Cl- ions were not excluded from the cell, the influence of high salt concentrations on key enzyme activities was investigated in crude cell extracts. Activities greater than 60% of the maximal activity of the following key catabolic enzymes occurred at the following [NaCl] ranges: glyceraldehyde-3-phosphate dehydrogenase, 1 to 2 M; alcohol dehydrogenase (NAD linked), 2 to 4 M; pyruvate dehydrogenase, 0.5 to 1 M; and hydrogenase (methyl viologen linked), 0.5 to 3 M. These studies support the hypothesis that obligately halophilic, anaerobic eubacteria adapt to extreme salt concentrations differently than do halophilic, aerobic eubacteria, because they do not produce osmoregulants or exclude Cl-. This study also demonstrated that these halophilic, anaerobic eubacteria have a physiological similarity to archaebacterial halophiles, since Na+ and Cl- are present in high concentrations and are required for enzymatic activity.     

Endogenous energy supply to the plasma membrane of dark aerobic cyanobacterium Anacystis nidulans: ATPase-independent efflux of H+ and Na+ from respiring cells

The ejection of protons from oxygen-pulsed cells and the gradients of Na+ concentration (Na+o/Na+i at 150 mM external NaCl) and proton electrochemical potential (delta mu H+) across the plasma membrane of Anacystis nidulans were studied in response to dark endogenous energy supply. Saturating concentrations of the F0F1-ATPase inhibitors dicyclohexylcarbodiimide (F0) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (F1) eliminated oxidative phosphorylation and lowered the ATP level from 2.6 +/- 0.15 to 0.7 +/- 0.1 nmol/mg dry wt while overall O2 uptake and delta mu H+ were much less affected. H+ efflux was inhibited only 60 to 75%. Aerobic Na+o/Na+i ratios (5.9 +/- 0.6) under these conditions remained 50% above the anaerobic level (2.1 +/- 0.2). Increasing concentrations of the electron transport inhibitors CO and KCN depressed H+ efflux and O2 uptake in parallel, with a pronounced discontinuity of the former at inhibitor concentrations, which reduced ATP levels from 2.6 to 0.8 nmol/mg dry wt, resulting in an abrupt shift of the apparent H+/O ratios from 4.0 +/- 0.3 to 1.9 +/- 0.2. Similarly, with KCN and CO the Na+o/Na+i ratios paralleled decreasing respiration rates more closely than decreasing ATP pool sizes. Ejection of protons also was observed when intact spheroplasts were pulsed with horse heart ferrocytochrome c or ferricyanide; the former reaction was inhibited, the latter was increased, by 1 mM KCN. Measurements of the proton motive force (delta mu H+) across the plasma membrane showed a strong correlation with respiration rates rather than ATP levels. It is concluded that the plasma membrane of intact A. nidulans can be directly energized by proton-translocating respiratory electron transport in the membrane and that part of this energy may be used by a Na+/H+ antiporter for the active exclusion of Na+ from the cell interior.     

Ionic responses and growth stimulation induced by nerve growth factor and epidermal growth factor in rat pheochromocytoma (PC12) cells

Rat pheochromocytoma cells (clone PC12) respond to nerve growth factor (NGF) by the acquirement of a phenotype resembling neuronal cells. In an earlier study we showed that NGF causes an increase in Na+,K+ pump activity, as monitored by ouabain-sensitive Rb+ influx. Here we show that addition of epidermal growth factor (EGF) to PC12 cells resulted in a stimulation of Na+,K+ pump activity as well. The increase of Na+,K+ pump activity by NGF or EGF was due to increased Na+ influx. This increased Na+ influx was sensitive to amiloride, an inhibitor of Na+,H+ exchange. Furthermore, no changes in membrane potential were observed upon addition of NGF or EGF. Amiloride-sensitive Na+,H+ exchange in PC12 cells was demonstrated by H+ efflux measurements and the effects of weak acids on Na+ influx. These observations suggest that both NGF and EGF activate an amiloride-sensitive, electroneutral Na+,H+ exchange mechanism in PC12 cells. These findings were surprising in view of the opposite ultimate biological effects of NGF and EGF, e.g., growth arrest vs. growth stimulation. However, within 24 h after addition, NGF was found to stimulate growth of PC12 cells, comparable to EGF. In the presence of amiloride, this stimulated growth by NGF and EGF was abolished. In contrast, amiloride did not affect NGF-induced neurite outgrowth of PC12 cells. From these observations it is concluded that in PC12 cells: (a) NGF has an initial growth stimulating effect; (b) neurite outgrowth is independent of increased amiloride-sensitive Na+ influx; and (c) growth stimulation by NGF and EGF is associated with increased amiloride-sensitive Na+ influx.     

Photosensitized irreversible binding of estrone to protein via a hydroperoxide intermediate: an explanation of (photo-) allergic side-effects of estrogens

Recent studies have established that polypeptide growth factors cause an elevation of the cytoplasmic pH (pHi) in cultured mammalian cells by stimulating Na+/H+ exchange. We show that vanadate, previously found to act as a mitogen for a number of cells, reversibly activates Na+/H+ exchange at micromolar concentrations in A431 cells, leading to a large increase of pHi. The stimulation of Na+/H+ exchange by vanadate is not due to inhibition of the Na+/K+ ATPase and is unrelated to possible effects of vanadate on cAMP levels. Elevation of pHi by vanadate and by epidermal growth factor (EGF) both display similar kinetics, and both EGF and vanadate stimulate the rate of pHi recovery following an acute acid load, suggesting that vanadate stimulates Na+/H+ exchange by a mechanism similar to that of polypeptide growth factor stimulation. Thus, stimulation of Na+/H+ exchange may be a common property not only of polypeptide growth factors but also of other, chemically unrelated mitogens.