Membrane ATPase Activity Research Articles

Regulation of Plasma Membrane H+-Pump Activity by 14-3-3 Proteins in Barley (Hordeum disticum) Roots under Salt Stress

Regulatory changes in the activity of the plasma membrane H+-ATPase in salt-stressed roots were investigated using seven-day-old seedlings of two cultivars of barley (Hordeum disticum L.) with different salt tolerances: Moskovskii-121 (salt-tolerant) and Elf (salt-sensitive). During the first hour of salt stress, the rate of proton extrusion from the excised roots increased in parallel with the ATP hydrolase activity and the amount of 14-3-3 proteins bound to H+-ATPase in isolated plasma membranes. Subsequently, all these parameters decreased and dropped after 3–6 h below the initial levels. The initial stimulation of proton extrusion from the detached barley roots was caused by osmotic stress, whereas the subsequent retardation of proton extrusion was probably caused by a toxic effect of excessive Na+ content in the cytoplasm. The salt-stress responses showed similar trends in both cultivars, with the exception that Moskovskii-121 responded faster than cv. Elf. The results indicate that 14-3-3 proteins regulate the H+-ATPase activity in the plasma membranes of barley root cells during salt stress; furthermore, the response time might be a useful indicator to discriminate cultivars with different salt tolerances.

Does ABCG2 Need a Heterodimer Partner? Expression and Functional Evaluation of ABCG2 (Arg 482)*

Accumulating evidence suggests that several ATP-binding cassette (ABC) transporters mediate the elimination of anticancer drugs from cancer cells and thereby confer drug resistance. SN-38-selected PC-6/SN2-5H human lung carcinoma cells were shown to overexpress ABCG2 with the reduced intracellular accumulation of SN-38, the active metabolite of irinotecan. We have recently demonstrated that plasma membrane vesicles prepared from those cells transported SN-38 in an ATP-dependent manner, and it was suggested that ABCG2 is involved in the active extrusion of SN-38 from cancer cells. In the present study, we have cloned the cDNA of ABCG2 from PC-6/SN2-5H human lung carcinoma cells, expressed ABCG2 in Sf9 insect cells, and characterized its function. Sequence analysis has revealed that the cloned ABCG2 has an arginine at the amino acid position 482, as does the wild type. Expression of the cloned ABCG2 in Sf9 cell membranes was detected by immunoblotting with the BXP-21 antibody. Contrary to our expectation, however, ATPase activity in the cell membranes expressing ABCG2 was stimulated by neither SN-38 nor rhodamine 123. It is suggested that there is a partner protein of ABCG2 required for heterodimer formation to exhibit transport activity toward SN-38.

Neurotensin effect on Na+, K+-ATPase is CNS area- and membrane-dependent and involves high affinity NT1 receptor.

We have previously shown that peptide neurotensin inhibits cerebral cortex synaptosomal membrane Na+, K+-ATPase, an effect fully prevented by blockade of neurotensin NT1 receptor by antagonist SR 48692. The work was extended to analyze neurotensin effect on Na+, K+-ATPase activity present in other synaptosomal membranes and in CNS myelin and mitochondrial fractions. Results indicated that, besides inhibiting cerebral cortex synaptosomal membrane Na+, K+-ATPase, neurotensin likewise decreased enzyme activity in homologous striatal membranes as well as in a commercial preparation obtained from porcine cerebral cortex. However, the peptide failed to alter either Na+, K+-ATPase activity in cerebellar synaptosomal and myelin membranes or ATPase activity in mitochondrial preparations. Whenever an effect was recorded with the peptide, it was blocked by antagonist SR 48692, indicating the involvement of the high affinity neurotensin receptor (NT1), as well as supporting the contention that, through inhibition of ion transport at synaptic membrane level, neurotensin plays a regulatory role in neurotransmission.

Inhibition of Na+, K+-ATPase activity by the metabolites accumulating in homocystinuria.

Homocystinuria is an inborn error of sulfur amino acid metabolism characterized predominantly by vascular and nervous system dysfunction. In this study we determined the in vitro effects of homocysteine and methionine, metabolites which accumulate in homocystinuria, on Na+, K+-ATPase, and Mg2+-ATPase activities in synaptic membranes from the hippocampus of rats. The results showed that both metabolites significantly inhibit Na+, K+-ATPase but not Mg2+-ATPase activity at concentrations usually observed in plasma of homocystinuric patients. Furthermore, incubation of hippocampal homogenates with homocysteine also elicited an inhibition of the enzyme activity which was however prevented by the simultaneous addition of cysteine to the medium. In addition, cysteine or methionine per se did not modify the two enzymatic activities. These findings indicate that oxidation of critical groups in the enzyme may possibly be involved in homocysteine inhibitory effect. Moreover, kinetic studies performed to investigate the interaction between homocysteine and methionine on Na+, K+-ATPase inhibition suggested a common site for the two amino acids in the enzyme. Considering the critical role exerted by Na+, K+-ATPase in brain, it is proposed that the inhibition provoked by homocysteine and methionine on the enzyme activity may be possibly related to the brain dysfunction characteristic of homocystinuria.

Effect of arsenic on bacterial growth and plasma membrane atpase activity

The effects of arsenic in the forms of arsenite and arsenate on bacterial growth and plasma membranes ATPase activity of was studied. Correlation of The rate of ATP hydrolysis was found to be correlated with bacterial resistance to toxic arsenic ions. Detoxification of arsenate by resistant cultures of bacteria was suggested to be related with an increase in bacterial ATPase activity and the degree of ATPase mobilization.

Characterization of the responses of cork oak (Quercus suber) to iron deficiency.

We studied responses of cork oak (Quercus suber L.) to iron (Fe) deficiency by comparing seedlings grown hydroponically in nutrient solution with and without Fe. Seedlings grown without Fe developed some responses typical of the Strategy I group of Fe-efficient plants, including two- and fourfold increases in plasma membrane ferric chelate reductase activity of root tips after 2 and 4 weeks of culture in the absence of Fe, respectively. Moreover, seedlings grown hydroponically for 2 weeks without Fe caused marked decreases in the pH of the nutrient solution, indicating that root plasma membrane ATPase activity was induced by Fe deficiency. Iron deficiency also caused marked decreases in leaf chlorophyll and carotenoid concentrations, and chlorophyll concentrations were decreased more than carotenoid concentrations. Iron deficiency resulted in an 8% decrease in the dark-adapted efficiency of photosystem II and a 43% decrease in efficiency of photosystem II at steady-state photosynthesis. No major root morphological changes were observed in seedlings grown without Fe, although seedlings grown in Fe-deficient nutrient solution had light-colored roots in contrast to the dark brown color of control roots.

Rh-deficiency syndrome

Jean-Pierre Cartron is Director of the Research Unit U76 of the Institut National de la Santé et de la Recherche Médicale, and Scientific Director of the Institut National de la Transfusion Sanguine in Paris.

New aspects of the glucose activation of the H(+)-ATPase in the yeast Saccharomyces cerevisiae.

The glucose-induced activation of plasma membrane ATPase from Saccharomyces cerevisiae was first described by Serrano in 1983. Many aspects of this signal transduction pathway are still obscure. In this paper, evidence is presented for the involvement of Snf3p as the glucose sensor related to this activation process. It is shown that, in addition to glucose detection by Snf3p, sugar transport is also necessary for activation of the ATPase. The participation of the G protein, Gpa2p, in transducing the internal signal (phosphorylated sugars) is also demonstrated. Moreover, the involvement of protein kinase C in the regulation of ATPase activity is confirmed. Finally, a model pathway is presented for sensing and transmission of the glucose activation signal of the yeast H(+)-ATPase.

In vivo and in vitro effect of imipramine and fluoxetine on Na+,K+-ATPase activity in synaptic plasma membranes from the cerebral cortex of rats

The effects of in vivo chronic treatment and in vitro addition of imipramine, a tricyclic antidepressant, or fluoxetine, a selective serotonin reuptake inhibitor, on the cortical membrane-bound Na+,K+-ATPase activity were studied. Adult Wistar rats received daily intraperitoneal injections of 10 mg/kg of imipramine or fluoxetine for 14 days. Twelve hours after the last injection rats were decapitated and synaptic plasma membranes (SPM) from cerebral cortex were prepared to determine Na+,K+-ATPase activity. There was a significant decrease (10%) in enzyme activity after imipramine but fluoxetine treatment caused a significant increase (27%) in Na+,K+-ATPase activity compared to control (P<0.05, ANOVA; N = 7 for each group). When assayed in vitro, the addition of both drugs to SPM of naive rats caused a dose-dependent decrease in enzyme activity, with the maximal inhibition (60-80%) occurring at 0.5 mM. We suggest that a) imipramine might decrease Na+,K+-ATPase activity by altering membrane fluidity, as previously proposed, and b) stimulation of this enzyme might contribute to the therapeutic efficacy of fluoxetine, since brain Na+,K+-ATPase activity is decreased in bipolar patients.

Osmoregulation in Plants: Implications for Agriculture

Drought and salinity stress are the major causes of historic and modern agricultural productivity losses throughout the world. Both drought and salinity result in osmotic stress that may lead to inhibition of growth. Salinity causes additional ion toxicity effects mainly through perturbations in protein and membrane structure. In contrast to animals, which rely on Na+/K+-ATPases for the expulsion of osmotica, plants rely on plasma membrane and endosomal ATPase activities to generate proton gradients to drive ion extrusion and intracellular sequestration. Consequently, most angiosperms, including all major crop species, have a diminished capacity for Na+ transport and tolerance to high salinity. New insights into the molecular mechanisms of Na+/K+ discrimination, Na+ extrusion and compartmentation, water transport, and osmolyte biosynthesis and function have led to genetically engineered plants with improved salt, drought, and cold tolerance. A deeper understanding of the complex signal transduction and regulatory responses to osmotic stress promises novel strategies for improving salinity and drought tolerance that will be of practical benefit to agriculture.

Osmoregulation in Plants: Implications for Agriculture1

Drought and salinity stress are the major causes of historic and modern agricultural productivity losses throughout the world. Both drought and salinity result in osmotic stress that may lead to inhibition of growth. Salinity causes additional ion toxicity effects mainly through perturbations in protein and membrane structure. In contrast to animals, which rely on Na /K -ATPases for the expulsion of osmotica, plants rely on plasma membrane and endosomal ATPase activities to generate proton gradients to drive ion extrusion and intracellular sequestration. Consequently, most angiosperms, including all major crop species, have a diminished capacity for Na transport and tolerance to high salinity. New insights into the molecular mechanisms of Na /K discrimination, Na extrusion and compartmentation, water transport, and osmolyte biosynthesis and function have led to genetically engineered plants with improved salt, drought, and cold tolerance. A deeper understanding of the complex signal transduction and regulatory responses to osmotic stress promises novel strategies for improving salinity and drought tolerance that will be of practical benefit to agriculture.

Similar effects of ether phospholipids, PAF and lyso-PAF on the Ca 2+-ATPase activity of rat brain synaptosomes and leukocyte membranes

The present study is an extension of our previous work with the antineoplastic ether phospholipid ET-18-OCH 3 (edelfosine), which was shown to affect the activity of the Ca 2+-ATPase of rat brain synaptosomes and peritoneal leukocyte membranes. The effect of ET-18-OCH 3 was compared with that of the 16-carbon chain analogue ET-16-OCH 3 as well as with the structurally related 16- and 18-carbon PAFs (platelet-activating factors) and lyso-PAFs. In addition, the two alkylphosphocholines D-20166 and D-21266 (perifosine) were included in the investigation. The influence of all of the compounds followed the same pattern, i.e., the Ca 2+-ATPase activity of the synaptosomes was increased over a relatively narrow concentration range (peak at 20–30 μM) and that of the leukocyte membranes was inhibited in a concentration-dependent manner by 10–50 μM concentrations of the drugs. Ether phospholipids with an 18-carbon chain at C-1 were more potent than those with a 16-carbon chain. All of the compounds increased the activity of the synaptosomal ATPase to the same extend (ca. 50%). With the exception of lyso-PAF, all inhibited the enzyme activity of leukocyte membranes by 60–70%, whereas lyso-PAF was less effective (ca. 50% inhibition). The concentration range of activity for PAF and lyso-PAF indicates that their effect on the enzyme activity was caused by receptor-independent mechanisms. The ether phospholipids and alkylphosphocholines are suggested to act by accumulating in the membranes and thereby altering the character of the lipid environment of the enzyme rather than by a direct interaction with the Ca 2+-ATPase.

The activity of plasma membrane H‐ATPase is strongly stimulated during Saccharomyces cerevisiae adaptation to growth under high copper stress, accompanying intracellular acidification

For the adaptation of cells of Saccharomyces cerevisiae, a period of latency is necessary before exponential growth is resumed in a medium supplemented with a highly inhibitory concentration of copper. In this work, we have examined some physiological responses occurring during this period of adaptation. The results revealed that plasma membrane H+-ATPase (PM-ATPase) activity is strongly stimulated (up to 24-fold) during copper-induced latency in growth medium with glucose, reaching maximal levels when the cells were about to start inhibited exponential growth. This in vivo activation of the ATPase activity by copper was accompanied by the stimulation of the H+-pumping activity of the enzyme in vivo and was essentially due to the increase of the apparent Vmax for MgATP. Although the exact molecular basis of the reported plasma membrane ATPase activation was not clarified, no increase in the mRNA levels from the encoding genes PMA1 and PMA2 was apparently detected during copper-induced latency. The physiological response reported here may allow the cells to cope with copper-induced lipid peroxidation and consequent decrease in plasma membrane lipid ordering and increase in the non-specific permeability to protons. The consequences of these copper deleterious effects were revealed by the decrease of the intracellular pH (pHi) of the yeast population, from approximately pHi 6 to pHi 5, during copper-induced latency in growth medium at pH 4.3. The time-dependent patterns of plasma membrane ATPase activation and of the decrease of pHi during the period of adaptation to growth with copper correlate, suggesting that the regulation of this membrane enzyme activity may be triggered by intracellular acidification. Consistent with this idea, when exponential growth under copper stress was resumed and the pHi of the yeast population recovered up to physiological values, plasma membrane ATPase activity simultaneously decreased from the highly stimulated level attained during the adaptation period of latency. Copyright © 2001 John Wiley & Sons, Ltd.

The activity of plasma membrane H(+)-ATPase is strongly stimulated during Saccharomyces cerevisiae adaptation to growth under high copper stress, accompanying intracellular acidification.

For the adaptation of cells of Saccharomyces cerevisiae, a period of latency is necessary before exponential growth is resumed in a medium supplemented with a highly inhibitory concentration of copper. In this work, we have examined some physiological responses occurring during this period of adaptation. The results revealed that plasma membrane H(+)-ATPase (PM-ATPase) activity is strongly stimulated (up to 24-fold) during copper-induced latency in growth medium with glucose, reaching maximal levels when the cells were about to start inhibited exponential growth. This in vivo activation of the ATPase activity by copper was accompanied by the stimulation of the H(+)-pumping activity of the enzyme in vivo and was essentially due to the increase of the apparent V(max) for MgATP. Although the exact molecular basis of the reported plasma membrane ATPase activation was not clarified, no increase in the mRNA levels from the encoding genes PMA1 and PMA2 was apparently detected during copper-induced latency. The physiological response reported here may allow the cells to cope with copper-induced lipid peroxidation and consequent decrease in plasma membrane lipid ordering and increase in the non-specific permeability to protons. The consequences of these copper deleterious effects were revealed by the decrease of the intracellular pH (pH(i)) of the yeast population, from approximately pH(i) 6 to pH(i) 5, during copper-induced latency in growth medium at pH 4.3. The time-dependent patterns of plasma membrane ATPase activation and of the decrease of pH(i) during the period of adaptation to growth with copper correlate, suggesting that the regulation of this membrane enzyme activity may be triggered by intracellular acidification. Consistent with this idea, when exponential growth under copper stress was resumed and the pH(i) of the yeast population recovered up to physiological values, plasma membrane ATPase activity simultaneously decreased from the highly stimulated level attained during the adaptation period of latency.

The Na+,K+-ATP-ase Activity and Lipids of Trout and Rat Brain Cell Membranes at the Pressure of 101 Atmospheres

The effect of elevated heliox pressure (101 ATA) on activity of Na+,K+-ATP-ase and some characteristics of fatty acid composition was studied in membrane phospholipids of trout and rat brain synaptosomal and mitochondrial fractions. The Na+,K+-ATP-ase activity was shown to decrease by 25% in both fractions of the rat brain; in mitochondrial fraction of the trout brain it decreased by 47%, while in synaptosomal fraction, only by 11%. It has also been established that under experimental conditions, the unsaturation index of fatty acids of phosphatidylcholine and phosphatidylethanolamine of trout synaptosomes decreased, with no changes in these lipids in mitochondrial fraction. The phosphatidylcholine unsaturation index in rats did not practically change in both fractions, while in rat phosphatidylethanolamine it increased in mitochondrial fraction and slightly decreased in synaptosomal fraction. Thus, under conditions of high pressure the reduction of the enzyme activity is also determined, specifically, by peculiarities of the phospholipid fatty acid composition in the subcellular fractions studied. A possibility of changes of the enzyme activity as a result of transition of its lipid component from the liquid crystalline to the gel state under effect of an enhancement of lipid peroxidation under conditions of elevated pressure is discussed.

Inhibition of rat brain Na+, K+-ATPase activity induced by homocysteine is probably mediated by oxidative stress.

The objective of the present study was to investigate the effects of preincubation of hippocampus homogenates in the presence of homocysteine or methionine on Na+, K+-ATPase and Mg2+-ATPase activities in synaptic membranes of rats. Homocysteine significantly inhibited Na+, K+-ATPase activity, whereas methionine had no effect. Mg2+-ATPase activity was not altered by the metabolites. We also evaluated the effect of incubating glutathione, cysteine, dithiothreitol, trolox, superoxide dismutase and GM1 ganglioside alone or incubation with homocysteine on Na+, K+-ATPase activity. Tested compounds did not alter Na+, K+-ATPase and Mg2+-ATPase activities, but except for trolox, prevented the inhibitory effect of homocysteine on Na+, K+-ATPase activity. These results suggest that inhibition of this enzyme activity by homocysteine is possibly mediated by free radicals and may contribute to the neurological dysfunction found in homocystinuric patients.

Glucose does not activate the plasma-membrane-bound H+-ATPase but affects pmaA transcript abundance in Aspergillus nidulans.

The addition of glucose to starved cells of Aspergillus nidulans increased the abundance of the pmaA transcript only transiently (15 min) and to a very low degree (1.3-fold), but strongly decreased its abundance during further incubation. This down-regulation was CreA (carbon catabolite repressor protein)-dependent. Glucose failed to stimulate the plasma membrane (PM)-ATPase activity of A. nidulans, whereas under the same experimental conditions the activity of the enzyme from Saccharomyces cerevisiae was enhanced four-fold within 5-10 min following glucose addition. Glucose stimulated the PM-ATPase of Neurospora crassa only 1.3-fold. Sequence comparison of the C-terminal end of the PM-ATPase from S. cerevisiae, N. crassa, A. nidulans, Fusarium sporotrichoides and Penicillium simplicissimum showed that the two regulatory sites necessary for glucose stimulation in S. cerevisiae are conserved in N. crassa and F. sporotrichoides but not in A. nidulans and P. simplicissimum, and their presence therefore does not correlate with glucose stimulation. We conclude that, in contrast to S. cerevisiae, which has become a paradigm of fungal glucose metabolism, glucose does not up-regulate the activity of the plasma membrane ATPase in the filamentous fungi examined.

Sensitivity of plasma membrane H+-ATPase of cucumber root system in response to low root temperature.

Cucumber plants (Cucumis sativus L.) were grown at different root temperatures and the H+-ATPase activities in the root plasma membranes were investigated. Electrolyte leakages and the amount of water transported from root to shoot indicated the sensitivity of cucumber root systems to low root temperature. The increase in H+-ATPase activity after 1 day of low root temperature treatment was reversed and gradually diminished as root temperatures of 10 °C continued for another 6 days. The H+-transport rate of the plasma membranes and the transcriptional activity of the corresponding gene expression closely followed the H+-ATPase activity. The role of H+-ATPase in root plasma membranes in terms of the root acclimation process at low root temperature is discussed.

Platelet Na+, K+-ATPase activity as a possible peripheral marker for the neurotoxic effects of phenylalanine in phenylketonuria.

The in vitro effects of phenylalanine or alanine alone or combined on Na+, K+-ATPase activity in membranes from human platelets were investigated. The enzyme activity was assayed in membranes prepared from platelet-rich plasma of healthy donors. Phenylalanine or alanine were added to the assay to final concentrations of 0.3 to 1.2 mM, similar to those found in plasma of phenylketonuric patients. Phenylalanine inhibited Na+, K+-ATPase activity by 20-50% [F(4,25)=11.47 ; p<0.001]. Alanine had no effect on Na+, K+-ATPase activity but when combined with phenylalanine prevented the enzyme inhibition. These results, allied to others previously reported on brain Na+, K+-ATPase activity, may reflect a general inhibitory effect of phenylalanine on this important enzyme activity. Therefore, it is possible that measurement of Na+, K+-ATPase activity in platelets from PKU patients may be a useful peripheral marker for the neurotoxic effects of phenylalanine.

Salinity Resistance of Citrus Seedlings in Relation to Hydraulic Conductance, Plasma Membrane ATPase and Anatomy of the Roots

Summary The evaluation of the response of three rootstocks to conditions of high salinity (NaCl 90 mmol/L) and the relationship with different parameters was the objective of this study. The three rootstocks were Cleopatra Mandarin, considered to be tolerant to salinity, Citrange Carrizo and Citrus Macrophylla, considered to be sensitive to salinity. We related the differences in salinity resistance to water and nutrient uptake, ATPase and anatomy of roots. For this, root hydraulic conductivity, ion concentrations in the xylem obtained under transpiration flow, root plasma membrane ATPase activity and anatomy of the root tips were determined in seedlings grown in a controlled environment. We found that under saline conditions, the Cleopatra M. rootstock showed less alteration of L 0 and nutrient uptake and better maintenance of the root anatomy when they were compared with controls. The increase observed in the ATPase activity of this rootstock could be closely related to the increase in Na + and Cl − uptake observed, indicating that a higher resistance to those ions must have occurred. Therefore, a whole succession of mechanisms and reactions can determine the degree of salt resistance in citrus plants.