Biological activity of quaternary ammonium salts and resistance of microorganisms to these compounds.

Quaternary ammonium salts (QASs) are ubiquitous in nature, being found in organisms ranging from microorganisms to vertebrates (e.g., glycine betaine, carnitine) where they have important cellular functions. QASs are also obtained by chemical synthesis. These compounds, due to their diverse chemical structure (e.g. monomeric QAS or gemini) and their biological properties, are widely used in medicine (as disinfectants, drugs, and DNA carriers), industry, environmental protection and agriculture (as preservatives, biocides, herbicides and fungicides). Discussed chemical compounds reduce the adhesion of microorganisms to various biotic and abiotic surfaces and cause the eradication of biofilms produced by pathogenic microorganisms. The properties of these chemicals depend on their chemical structure (length of the alkyl chain, linker and counterion), which has a direct impact on the physicochemical and biological activity of these compounds. QASs by incorporation into the membranes, inhibit the activity of proteins (H+-ATPase) and disrupt the transport of substances to the cell. Moreover, in the presence of QASs, changes in lipid composition (qualitative and quantitative) of plasma membrane are observed. The widespread use of disinfectants in commercial products can induce resistance in microorganisms to these surfactants and even to antibiotics. In this article we discuss the biological activity of QASs as cationic surfactants against microorganisms and their resistance to these compounds.

Trichoderma asperellum Induces Maize Seedling Growth by Activating the Plasma Membrane H+-ATPase.

Although Trichoderma spp. have beneficial effects on numerous plants, there is not enough knowledge about the mechanism by which they improves plant growth. In this study, we evaluated the participation of plasma membrane (PM) H+-ATPase, a key enzyme involved in promoting cell growth, in the elongation induced by T. asperellum and compared it with the effect of 10 µM indol acetic acid (IAA) because IAA promotes elongation and PM H+-ATPase activation. Two seed treatments were tested: biopriming and noncontact. In neither were the tissues colonized by T. asperellum; however, the seedlings were longer than the control seedlings, which also accumulated IAA and increased root acidification. An auxin transport inhibitor (2,3,5 triiodobenzoic acid) reduced the plant elongation induced by Trichoderma spp. T. asperellum seed treatment increased the PM H+-ATPase activity in plant roots and shoots. Additionally, the T. asperellum extracellular extract (TE) activated the PM H+-ATPase activity of microsomal fractions of control plants, although it contained 0.3 µM IAA. Furthermore, the mechanism of activation of PM H+-ATPase was different for IAA and TE; in the latter, the activation depends on the phosphorylation state of the enzyme, suggesting that, in addition to IAA, T. asperellum excretes other molecules that stimulate PM H+-ATPase to induce plant growth.

Ion transport in broad bean leaf mesophyll under saline conditions.

MAIN CONCLUSION: Salt stress reduces the ability of mesophyll tissue to respond to light. Potassium outward rectifying channels are responsible for 84 % of Na (+) induced potassium efflux from mesophyll cells. Modulation in ion transport of broad bean (Vicia faba L.) mesophyll to light under increased apoplastic salinity stress was investigated using vibrating ion-selective microelectrodes (the MIFE technique). Increased apoplastic Na(+) significantly affected mesophyll cells ability to respond to light by modulating ion transport across their membranes. Elevated apoplastic Na(+) also induced a significant K(+) efflux from mesophyll tissue. This efflux was mediated predominately by potassium outward rectifying channels (84 %) and the remainder of the efflux was through non-selective cation channels. NaCl treatment resulted in a reduction in photosystem II efficiency in a dose- and time-dependent manner. In particular, reductions in Fv'/Fm' were linked to K(+) homeostasis in the mesophyll tissue. Increased apoplastic Na(+) concentrations induced vanadate-sensitive net H(+) efflux, presumably mediated by the plasma membrane H(+)-ATPase. It is concluded that the observed pump's activation is essential for the maintenance of membrane potential and ion homeostasis in the cytoplasm of mesophyll under salt stress.

Proton pump inhibitors interfere with the anti-tumor potency of RC48ADC.

Antibody-drug conjugates (ADCs) are a promising modality for cancers, but the interaction between them and proton pump inhibitors (PPIs), the common adjuvant drugs for cancer treatment, has not been understood. Here, the interactions between PPIs and RC48ADC, a novel HER2-targeting ADC, were quantified in vitro. CCK-8 assay showed that RC48ADC displayed a significant inhibitory effect on the proliferation of SK-BR-3, NCI-N87 and SK-OV-3 cells with the IC50 values of 4.91 ± 1.15 ng/mL, 14.54 ± 0.85 ng/mL and 11.28 ± 0.68 ng/mL respectively. PPIs alone had no significant anti-tumor effect in the dose range of 1.37-1000 ng/mL. When used together, PPIs inhibited the anti-tumor activity of RC48ADC in a dose-dependent manner. And 1000 ng/mL (~Cmax) PPIs significantly recovered RC48ADC-inhibited cell proliferation by (32.85 ± 2.81) % (p < 0.05). However, cimetidine, a non-PPIs gastric acid secretion inhibitor, had no significant inhibitory effect on RC48ADC. Furthermore, omeprazole, rather than cimetidine, significantly reduced the activity of vacuolar H+-ATPase and Cathepsin B compared with the control cells. These results, if confirmed in vivo, indicate that PPIs are antagonists of RC48ADC, even all ADCs, appearing to be due to inhibition of vacuolar H+-ATPase activity. Moreover, cimetidine combined with ADCs instead of PPIs can prevent an adverse drug interaction.

Aluminum regulates oxalate secretion and plasma membrane H+-ATPase activity independently in tomato roots.

We demonstrated that aluminum (Al)-induced oxalate secretion and plasma membrane (PM) H(+)-ATPase activity in tomato (Lycopersicon esculentum 'Hezuo903') roots were poorly correlated. In addition, vanadate, an inhibitor of PM H(+)-ATPase, had no effect on Al-induced oxalate secretion, but significantly inhibited enzyme activity. An anion channel inhibitor phenylglyoxal inhibited oxalate secretion, but not PM H(+)-ATPase activity. Exposure of tomato roots to 10 µM LaCl(3) also stimulated PM H(+)-ATPase activity; however, La failed to induce oxalate secretion. Furthermore, Al-induced changes of PM H(+)-ATPase activity were not associated with oxalate secretion in two tomato cultivars differing in the ability to secrete oxalate under Al stress. These results indicate that Al independently regulates oxalate secretion and PM H(+)-ATPase activity in tomato roots. Analysis of expression levels of PM H(+)-ATPase genes by real-time reverse transcription-PCR and protein by Western blot and immunodetection revealed that the regulation of PM H(+)-ATPase in response to Al was subjected to transcriptional and posttranscriptional control. However, since neither transcriptional level of genes nor translational level of proteins directly relate to the enzyme activity, posttranslational modification of PM H(+)-ATPase under Al stress likely contributes to changes in activity of this protein.

cGMP regulates hydrogen peroxide accumulation in calcium-dependent salt resistance pathway in Arabidopsis thaliana roots.

3',5'-cyclic guanosine monophosphate (cGMP) is an important second messenger in plants. In the present study, roles of cGMP in salt resistance in Arabidopsis roots were investigated. Arabidopsis roots were sensitive to 100 mM NaCl treatment, displaying a great increase in electrolyte leakage and Na(+)/K(+) ratio and a decrease in gene expression of the plasma membrane (PM) H(+)-ATPase. However, application of exogenous 8Br-cGMP (an analog of cGMP), H(2)O(2) or CaCl(2) alleviated the NaCl-induced injury by maintaining a lower Na(+)/K(+) ratio and increasing the PM H(+)-ATPase gene expression. In addition, the inhibition of root elongation and seed germination under salt stress was removed by 8Br-cGMP. Further study indicated that 8Br-cGMP-induced higher NADPH levels for PM NADPH oxidase to generate H(2)O(2) by regulating glucose-6-phosphate dehydrogenase (G6PDH) activity. The effect of 8Br-cGMP and H(2)O(2) on ionic homeostasis was abolished when Ca(2+) was eliminated by glycol-bis-(2-amino ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, a Ca(2+) chelator) in Arabidopsis roots under salt stress. Taken together, cGMP could regulate H(2)O(2) accumulation in salt stress, and Ca(2+) was necessary in the cGMP-mediated signaling pathway. H(2)O(2), as the downstream component of cGMP signaling pathway, stimulated PM H(+)-ATPase gene expression. Thus, ion homeostasis was modulated for salt tolerance.

Effect of cadmium and lead on the membrane potential and photoelectric reaction of Nitellopsis obtusa cells.

The effects of Cd and Pb on membrane potential (E(m)) and photoelectric reaction of Nitellopsis obtusa cells were investigated. It was found that Cd and Pb at 1.0 mM caused a depolarization of the E(m), whereas both metals at lower concentrations changed the E(m) in a different way. Pb at 0.1 mM and 0.01 mM hyperpolarized the E(m), whereas Cd at the same concentrations depolarized and did not change the E(m), respectively. In the presence of 0.01 mM Pb, the light-induced hyperpolarization of the E(m) was by 18% higher as compared to the control, whereas at 1.0 mM Pb it was by 40% lower. Pb at 0.1 mM and Cd at 0.01 mM or 5 × 0.01 mM did not change the light-induced membrane hyperpolarization. However, in the presence of Cd at 0.1 mM and 1.0 mM this hyperpolarization was 2-fold lower or was completely abolished, respectively. These results suggest that at high Cd and Pb concentrations both depolarization of the E(m) and decrease of light-induced membrane hyperpolarization in Nitellopsis obtusa cells are probably due to inhibition of the plasma membrane H(+)-ATPase activity, whereas both metals at lower concentrations differ in mechanism of membrane potential changes.

Mitochondrial F1FO-ATPase and permeability transition pore response to sulfide in the midgut gland of Mytilus galloprovincialis.

The molecular mechanisms which rule the formation and opening of the mitochondrial permeability transition pore (mPTP), the lethal mechanism which permeabilizes mitochondria to water and solutes and drives the cell to death, are still unclear and particularly little investigated in invertebrates. Since Ca2+ increase in mitochondria is accompanied by mPTP opening and the participation of the mitochondrial F1FO-ATPase in the mPTP is increasingly sustained, the substitution of the natural cofactor Mg2+ by Ca2+ in the F1FO-ATPase activation has been involved in the mPTP mechanism. In mussel midgut gland mitochondria the similar kinetic properties of the Mg2+- or Ca2+-dependent F1FO-ATPase activities, namely the same affinity for ATP and bi-site activation kinetics by the ATP substrate, in spite of the higher enzyme activity and coupling efficiency of the Mg2+-dependent F1FO-ATPase, suggest that both enzyme activities are involved in the bioenergetic machinery. Other than being a mitochondrial poison and environmental contaminant, sulfide at low concentrations acts as gaseous mediator and can induce post-translational modifications of proteins. The sulfide donor NaHS, at micromolar concentrations, does not alter the two F1FO-ATPase activities, but desensitizes the mPTP to Ca2+ input. Unexpectedly, NaHS, under the conditions tested, points out a chemical refractoriness of both F1FO-ATPase activities and a failed relationship between the Ca2+-dependent F1FO-ATPase and the mPTP in mussels. The findings suggest that mPTP role and regulation may be different in different taxa and that the F1FO-ATPase insensitivity to NaHS may allow mussels to cope with environmental sulfide.

Alternating metabolic pathways in NGF-deprived sympathetic neurons affect caspase-independent death.

Mitochondrial release of cytochrome c in apoptotic cells activates caspases, which execute apoptotic cell death. However, the events themselves that culminate in caspase activation can have deleterious effects because caspase inhibitor-saved cells ultimately die in a caspase-independent manner. To determine what events may underlie this form of cell death, we examined bioenergetic changes in sympathetic neurons deprived of NGF in the presence of a broad-spectrum caspase inhibitor, boc-aspartyl-(OMe)-fluoromethylketone. Here, we report that NGF-deprived, boc-aspartyl-(OMe)-fluoromethylketone-saved neurons rely heavily on glycolysis for ATP generation and for survival. Second, the activity of F0F1 contributes to caspase-independent death, but has only a minor role in the maintenance of mitochondrial membrane potential, which is maintained primarily by electron transport. Third, permeability transition pore inhibition by cyclosporin A attenuates NGF deprivation-induced loss of mitochondrial proteins, suggesting that permeability transition pore opening may have a function in regulating the degradation of mitochondria after cytochrome c release. Identification of changes in caspase inhibitor-saved cells may provide the basis for rational strategies to augment the effectiveness of the therapeutic use of postmitochondrial interventions.

Coumarin enhances nitrate uptake in maize roots through modulation of plasma membrane H+ -ATPase activity.

Coumarin is one of the simplest plant secondary metabolites, widely distributed in the plant kingdom, affecting root form and function, including anatomy, morphology and nutrient uptake. Although, some plant responses to coumarin have been described, comprehensive knowledge of the physiological and molecular mechanisms is lacking. Maize seedlings exposed to different coumarin concentrations, alone or in combination with 200 µm nitrate (NO3- ), were analysed, through a physiological and molecular approach, to elucidate action of coumarin on net NO3- uptake rate (NNUR). In detail, the time course of NNUR, plasma membrane (PM) H+ -ATPase activity, proton pumping and related gene expression (ZmNPF6.3, ZmNRT2.1, ZmNAR2.1, ZmHA3 and ZmHA4) were evaluated. Coumarin alone did not affect nitrate uptake, PM H+ -ATPase activity or transcript levels of ZmNRT2.1 and ZmHA3. In contrast, coumarin alone increased ZmNPF6.3, ZmNAR2.1 and ZmHA4 expression in response to abiotic stress. When coumarin and NO3- were concurrently added to the nutrient solution, a significant increase in the NNUR, PM H+ -ATPase activity, together with ZmNAR2.1:ZmNRT2.1 and ZmHA4 expression was observed, suggesting that coumarin affected the inducible component of the high affinity transport system (iHATS), and this effect appeared to be mediated by nitrate. Moreover, results with vanadate, an inhibitor of the PM H+ -ATPase, suggested that this enzyme could be the main target of coumarin. Surprisingly, coumarin did not affect PM H+ -ATPase activity by direct contact with plasma membrane vesicles isolated from maize roots, indicating its possible elicitor role in gene transcription.

Regulation of collecting tubule adenosine triphosphatases by aldosterone and potassium.

To examine the precise role of potassium and aldosterone on acid-base composition and on collecting tubule ATPases, glucocorticoid-replete adrenalectomized rats were replaced with zero, physiological, or pharmacological doses of aldosterone and were fed varying potassium diets to produce hypokalemia, normokalemia, or hyperkalemia. Radiochemical measurement of ATPase activities showed that collecting tubule H/K-ATPase changed inversely with potassium and not with aldosterone whereas H-ATPase changed directly with aldosterone but not with potassium. When both enzymes changed in the same direction, alterations in acid-base composition were profound; however, when these two acidifying enzymes changed in opposite directions or when only one enzyme changed, the effect on acid-base balance was modest. Serum bicarbonate was approximately 45 meq/liter when aldosterone was high and potassium was low; it was only 29 meq/liter when aldosterone was high but potassium was normal or when aldosterone was normal and potassium was low. Our observations may help explain the metabolic alkalosis of primary aldosteronism in which aldosterone excess and hypokalemia are combined and the metabolic acidosis of aldosterone deficiency in which hypoaldosteronism and hyperkalemia are paired. The present study also demonstrated that aldosterone plays the major role in controlling Na/K-ATPase activity in cortical collecting tubule. Hypokalemia stimulates Na/K-ATPase activity in the medullary collecting tubule; this stimulatory effect of hypokalemia supports the hypothesis that the enzyme is present on the apical membrane at this site.

The role of brassinosteroids in the regulation of the plasma membrane H+-ATPase and NADPH oxidase under cadmium stress.

The present research aim was to define the role of brassinosteroids (BRs) in plant adaptation to cadmium stress. We observed a stimulating effect of exogenous BR on the activity of two plasma membrane enzymes which play a key role in plants adaptation to cadmium stress, H+-ATPase (EC 3.6.3.14) and NADPH oxidase (EC 1.6.3.1). Using anti-phosphothreonine antibody we showed that modification of PM H+-ATPase activity under BR action could result from phosphorylation of the enzyme protein. Also the relative expression of genes encoding both PM H+-ATPase and NADPH oxidase was affected by BR. To confirm the role of BR in the cadmium stimulating effect on activity of both studied plasma membrane enzymes, an assay in the presence of a BR biosynthesis inhibitor (propiconazole) was performed. Moreover, as a tool in our work we used commercially available plant mutants unable to BR biosynthesis or with dysfunctional BR signaling pathway, to further confirm participation of BR in plant adaptation to heavy metal stress. Presented results demonstrate some elements of the brassinosteroid-induced pathway activated under cadmium stress, wherein H+-ATPase and NADPH oxidase are key factors.

F1 -ATP synthase α-subunit: a potential target for RNAi-mediated pest management of Locusta migratoria manilensis.

BACKGROUND: The migratory locust is one of the most destructive agricultural pests worldwide. ATP synthase (F0 F1 -ATPase) uses proton or sodium motive force to produce 90% of the cellular ATP, and the α-subunit of F1 -ATP synthase (ATP5A) is vital for F1 -ATP synthase. Here, we tested whether ATP5A could be a potential target for RNAi-mediated pest management of L. migratoria. RESULTS: Lm-ATP5A was cloned and characterised. Lm-ATP5A is expressed in all tissues. Injection of 100 ng of the double-stranded RNA of ATP5A (dsATP5A) knocked down the transcription of the target gene and caused mortality in 1.5-5 days. The Lm-ATP5A protein level, the oligomycin-sensitive ATP synthetic and hydrolytic activities and the ATP content were correspondingly reduced following dsATP5A injection. CONCLUSION: These findings demonstrated the essential roles of Lm-ATP5A in L. migratoria and identified it as a potential target for insect pest control. © 2015 Society of Chemical Industry.

TIR1/AFB-Aux/IAA auxin perception mediates rapid cell wall acidification and growth of Arabidopsis hypocotyls.

Despite being composed of immobile cells, plants reorient along directional stimuli. The hormone auxin is redistributed in stimulated organs leading to differential growth and bending. Auxin application triggers rapid cell wall acidification and elongation of aerial organs of plants, but the molecular players mediating these effects are still controversial. Here we use genetically-encoded pH and auxin signaling sensors, pharmacological and genetic manipulations available for Arabidopsis etiolated hypocotyls to clarify how auxin is perceived and the downstream growth executed. We show that auxin-induced acidification occurs by local activation of H+-ATPases, which in the context of gravity response is restricted to the lower organ side. This auxin-stimulated acidification and growth require TIR1/AFB-Aux/IAA nuclear auxin perception. In addition, auxin-induced gene transcription and specifically SAUR proteins are crucial downstream mediators of this growth. Our study provides strong experimental support for the acid growth theory and clarified the contribution of the upstream auxin perception mechanisms.

Impairing effects of angiotensin-converting enzyme inhibitor Captopril on bone of normal mice.

There are contradicting results about the effects of angiotensin-converting enzyme inhibitors (ACEIs) on bones. This study was aimed to investigate the effect of ACEI, Captopril, on bone metabolism and histology as well as the action of Captopril on skeletal renin-angiotensin system (RAS) and bradykinin receptor pathway in normal male mice. The urine, serum, tibias and femurs from normal control mice and Captopril-treated (10mg/kg) mice were collected for biochemical, histological and molecular analyses after drug administration for eight weeks. The mice after the treatment with Captopril had a significant decrease of serum testosterone level. The histological measurements showed the loss of trabecular bone mass and trabecular bone number, and the breakage of trabecular bone network as well as the changes of chondrocyte zone at epiphyseal plate in Captopril-treated mice. The defect of Captopril on trabecular bone was reflected by the quantitative bio-parameters from micro-CT. The expression of renin receptor and bradykinin B2 receptor (B2R) was significantly up-regulated in tibia of mice upon to the Captopril treatment, which decreased the ratio of OPG/RANKL and the expression of osteoblastic factor RUNX2. Furthermore, Captopril treatment resulted in the increase of pAkt/Akt and pNFκB expression in tibia. The present study revealed the impairing effects of Captopril on bone via interfering with the circulating sex hormone level and B2R pathway, which suggests that the bone metabolism of patients need to be carefully monitored when being prescribed for ACEIs.

Sulfur dioxide inhibits the sucrose carrier of the plant plasma membrane.

Plasma membrane vesicles were prepared by phase partition from a microsomal fraction of broad bean (Vicia faba L.) leaf. In order to study the effects of sodium sulfite on active uptake of sucrose, the vesicles were artificially energized by a transmembrane pH gradient (delta pH) and/or a transmembrane electrical gradient (delta psi). At 1 mM, sulfite strongly inhibited sucrose uptake but did not affect the two components of the proton motive force, delta pH (measured by dimethyloxazolidine dione) and delta psi (measured by tetraphenylphosphonium). Moreover, sulfite did not inhibit the proton-pumping ATPase of the plasma membrane vesicles. These data demonstrate that sulfite may inhibit transport of photoassimilates in plant by a direct inhibition of the sucrose carrier of the plasma membrane.

The Escherichia coli F1-ATPase mutant beta Tyr-297-->Cys: functional studies and asymmetry of the enzyme under various nucleotide conditions based on reaction of the introduced Cys with N-ethylmaleimide and 7-chloro-4-nitrobenzofurazan.

Conversion of residue beta Tyr-297 of the Escherichia coli F1-ATPase (ECF1) to a Cys in the mutant beta Y297C led to impaired oxidative phosphorylation based on growth curves. The ATPase activity of ECF1 isolated from the mutant beta Y297C was only 1% of wild-type activity, but the residual activity involves cooperative multi-site enzyme turnover based on inhibition by DCCD and azide. ATPase activity could be increased to 8%, and 13% of wild-type by reaction of the introduced Cys with N-ethyl maleimide (NEM), and 7-chloro-4-nitrobenzofurazan (NbfCl), respectively, suggesting that enzymatic function is improved by an increased hydrophobicity of residue beta Cys-297. The mutation beta Tyr-297-->Cys had no effect on nucleotide binding in studies with the fluorescent analog lin-benzo-ADP. The asymmetry of ECF1 was investigated in the mutants beta Y297C and beta Y297C:E381C/epsilon S108C by examining the relative reactivity of Cys-297 in the three copies of the beta subunit under different nucleotide binding conditions. In agreement with a previous study (Haughton, M.A. and Capaldi, R.A. (1995) J. Biol. Chem., 270, 20568-20574), the asymmetry was maintained under all nucleotide conditions. The NbfCl reaction site was found to be beta free, which is also the site most reactive to NEM, beta epsilon is the second site which reacts with NbfCl or NEM, while the third site, beta gamma, is poorly reactive to either reagent.

Inhibition of Na(+)-independent H+ pump by Na(+)-induced changes in cell Ca2+.

Apical membrane H+ extrusion in the renal outer medullary collecting duct, inner stripe, is mediated by a Na(+)-independent H+ pump. To examine the regulation of this transporter, cell pH and cell Ca2+ were measured microfluorometrically in in vitro perfused tubules using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and fura-2, respectively. Apical membrane H+ pump activity, assayed as cell pH recovery from a series of acid loads (NH3/NH+4 prepulse) in the total absence of ambient Na+, initially occurred at a slow rate (0.06 +/- 0.02 pH units/min), which was not sufficient to account for physiologic rates of H+ extrusion. Over 15-20 min after the initial acid load, the rate of Na(+)-independent cell pH recovery increased to 0.63 +/- 0.09 pH units/min, associated with a steady-state cell pH greater than the initial pre-acid load cell pH. This pattern suggested an initial suppression followed by a delayed activation of the apical membrane H+ pump. Replacement of peritubular Na+ with choline or N-methyl-D-glucosamine resulted in an initial spike increase in cell Ca2+ followed by a sustained increase in cell Ca2+. The initial rate of Na(+)-independent cell pH recovery could be increased by elimination of the Na+ removal-induced sustained cell Ca2+ elevation by: (a) performing studies in the presence of 135 mM peritubular Na+ (1 mM peritubular amiloride used to inhibit basolateral membrane Na+/H+ antiport); (b) clamping cell Ca2+ low with dimethyl-BAPTA, an intracellular Ca2+ chelating agent; or (c) removal of extracellular Ca2+. Cell acidification induced a spike increase in cell Ca2+. The late acceleration of Na(+)-independent cell pH recovery was independent of Na+ removal and of the method used to acidify the cell, but was eliminated by prevention of the cell Ca2+ spike and markedly delayed by the microfilament-disrupting agent, cytochalasin B. This study demonstrates that peritubular Na+ removal results in a sustained elevation in cell Ca2+, which inhibits the apical membrane H+ pump. In addition, rapid cell acidification associated with a spike increase in cell Ca2+ leads to a delayed activation of the H+ pump. Thus, cell Ca2+ per se, or a Ca(2+)-activated pathway, can modulate H+ pump activity.

Ion tolerance of Saccharomyces cerevisiae lacking the Ca2+/CaM-dependent phosphatase (calcineurin) is improved by mutations in URE2 or PMA1.

Calcineurin is a conserved, Ca2+/CaM-stimulated protein phosphatase required for Ca2+-dependent signaling in many cell types. In yeast, calcineurin is essential for growth in high concentrations of Na+, Li+, Mn2+, and OH-, and for maintaining viability during prolonged treatment with mating pheromone. In contrast, the growth of calcineurin-mutant yeast is better than that of wild-type cells in the presence of high concentrations of Ca2+. We identified mutations that suppress multiple growth defects of calcineurin-deficient yeast (cnb1Delta or cna1Delta cna2Delta). Mutations in URE2 suppress the sensitivity of calcineurin mutants to Na+, Li+, and Mn2+, and increase their survival during treatment with mating pheromone. ure2 mutations require both the transcription factor Gln3p and the Na+ ATPase Pmr2p to confer Na+ and Li+ tolerance. Mutations in PMA1, which encodes the yeast plasma membrane H+-ATPase, also suppress many growth defects of calcineurin mutants. pma1 mutants display growth phenotypes that are opposite to those of calcineurin mutants; they are resistant to Na+, Li+, and Mn2+, and sensitive to Ca2+. We also show that calcineurin mutants are sensitive to aminoglycoside antibiotics such as hygromycin B while pma1 mutants are more resistant than wild type. Furthermore, pma1 and calcineurin mutations have antagonistic effects on intracellular [Na+] and [Ca2+]. Finally, we show that yeast expressing a constitutively active allele of calcineurin display pma1-like phenotypes, and that membranes from these yeast have decreased levels of Pma1p activity. These studies further characterize the roles that URE2 and PMA1 play in regulating intracellular ion homeostasis.

Effects of myristate phorbol ester on V-ATPase activity and Na(+)-H+ exchange in alveolar macrophages.

The roles of protein kinase C (PKC) in regulation of the plasmalemmal vacuolar-type H(+)-ATPase (V-ATPase) and Na(+)-H+ exchanger (NHE) of rabbit alveolar macrophages (m phi) were investigated using phorbol 12-myristate 13-acetate (PMA). At an extracellular pH (pHo) of 7.4 (nominal absence of CO2-HCO3-), PMA caused a dose-dependent increase in the rate of cellular H+ extrusion with little change in intracellular pH (pHi). PMA caused a prolonged cytosolic acidification at pHo < or = 6.8. PMA-induced changes in pHi were sensitive to bafilomycin A1, but were insensitive to amiloride. Studies of pHi recovery following intracellular acid challenge showed that both V-ATPase and the NHE were up-regulated by PMA. An inactive analog, 4 alpha-phorbol, had no detectable effects on pHi homeostasis. These data indicate that (a) PKC is involved in regulation of V-ATPase and the NHE of resident alveolar m phi and (b) V-ATPase is the predominant mechanism for pHi homeostasis in unstimulated and PMA-activated m phi.