Pressure adaptation of teleost gill Na+/K+-adenosine triphosphatase: role of the lipid and protein moieties

Summary The effects of temperature and pressure on Na⁺/K⁺-adenosine triphosphatases (Na⁺/K⁺-ATPases) from gills of marine teleost fishes were examined over a range of temperatures (10–25°C) and pressures (1–680 atm). The relationship between gill membrane fluidity and Na⁺/K⁺-ATPase activity was studied using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The increase in temperature required to offset the membrane ordering effects of high pressure was 0.015–0.025°C·atm^-1, the same coefficient that applied to Na⁺/K⁺-ATPase activities. Thus, temperature-pressure combinations yielding the same Na⁺/K⁺-ATPase activity also gave similar estimates of membrane fluidity. Substituion of endogenous lipids with lipids of different composition altered the pressure responses of Na⁺/K⁺-ATPase. Na⁺/K⁺-adenosine triphosphatase became more sensitive to pressure in the presence of chicken egg phosphatidylcholine, but phospholipids isolated from fish gills reduced the inhibition by pressure of Na⁺/K⁺-ATPase. Cholesterol increased enzyme pressure sensitivity. Membrane fluidity and pressure sensitivity of Na⁺/K⁺-ATPase were correlated, but the effects of pressure also dependent on the source of the enzyme. Our results suggest that pressure adaptation of Na⁺/K⁺-ATPase is the result of both changes in the primary structure of the protein and homeoviscous adaptation of the lipid environment.Abbreviations EDTA; DPH 1,6-diphenyl-1,3,5-hexatriene - PC phosphatidylcholine - PL phospholipid - SDH succinate dehydrogenase     

Distinct Na+/K+/2Cl- cotransporter localization in kidneys and gills of two euryhaline species, rainbow trout and killifish

The kidney is an organ playing an important role in ion regulation in both freshwater (FW) and seawater (SW) fish. The mechanisms of ion regulation in the fish kidney are less well studied than that of their gills, especially at the level of transporter proteins. We have found striking differences in the pattern of Na⁺/K⁺/2Cl^- cotransporter (NKCC) expression between species. In the killifish kidney, NKCC is apically localized in the distal and collecting tubules and basolaterally localized in the proximal tubules. However, in the SW killifish gill, NKCC is basolaterally co-localized with Na⁺/K⁺-ATPase, whereas in FW, NKCC immunoreactivity is primarily apical, although still colocalized within the same mitochondria-rich cell with basolateral Na⁺/K⁺-ATPase. Rainbow trout kidney has NKCC only in the apical membrane of the distal and collecting tubules in both environments, with no signal being detected in the proximal tubule. On the other hand, in the trout gill, NKCC is found basolaterally in both FW and SW environments. An important observation is that, in the gills of rainbow trout, the trailing edge of the filament possesses mostly Na⁺/K⁺-ATPase-positive but NKCC-negative mitochondria-rich cells, whereas in the region between and at the roots of the gill lamellae, most mitochondria-rich cells exhibit both Na⁺/K⁺-ATPase- and NKCC-positive immunoreactivity. These results suggest that the differential localization of transporters between the two species represents differences in function between these two euryhaline fishes with different life histories and strategies. Funding for this research was provided by NSERC Discovery Grants to G.G.G. and W.S.M., an Alberta Ingenuity Fund PDF, and a fellowship from the NSERC Research Capacity Development Grant to F.K.     

Freshwater to seawater acclimation of juvenile bull sharks (<Emphasis Type="Italic">Carcharhinus leucas</Emphasis>): plasma osmolytes and Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase activity in gill,rectal gland,kidney and intestine

This study examined the osmoregulatory status of the euryhaline elasmobranch Carcharhinus leucas acclimated to freshwater (FW) and seawater (SW). Juvenile C. leucas captured in FW (3 mOsm l^–1 kg^–1) were acclimated to SW (980–1,000 mOsm l^–1 kg^–1) over 16 days. A FW group was maintained in captivity over a similar time period. In FW, bull sharks were hyper-osmotic regulators, having a plasma osmolarity of 595 mOsm l^–1 kg^–1. In SW, bull sharks had significantly higher plasma osmolarities (940 mOsm l^–1 kg^–1) than FW-acclimated animals and were slightly hypo-osmotic to the environment. Plasma Na⁺, Cl^–, K⁺, Mg²⁺, Ca²⁺, urea and trimethylamine oxide (TMAO) concentrations were all significantly higher in bull sharks acclimated to SW, with urea and TMAO showing the greatest increase. Gill, rectal gland, kidney and intestinal tissue were taken from animals acclimated to FW and SW and analysed for maximal Na⁺/K⁺-ATPase activity. Na⁺/K⁺-ATPase activity in the gills and intestine was less than 1 mmol Pi mg^–1 protein h^–1 and there was no difference in activity between FW- and SW-acclimated animals. In contrast Na⁺/K⁺-ATPase activity in the rectal gland and kidney were significantly higher than gill and intestine and showed significant differences between the FW- and SW-acclimated groups. In FW and SW, rectal gland Na⁺/K⁺-ATPase activity was 5.6±0.8 and 9.2±0.6 mmol Pi mg^–1 protein h^–1, respectively. Na⁺/K⁺-ATPase activity in the kidney of FW and SW acclimated animals was 8.4±1.1 and 3.3±1.1 Pi mg^–1 protein h^–1, respectively. Thus juvenile bull sharks have the osmoregulatory plasticity to acclimate to SW; their preference for the upper reaches of rivers where salinity is low is therefore likely to be for predator avoidance and/or increased food abundance rather than because of a physiological constraint.     

Sulfatide and Na<Superscript>+</Superscript>-K<Superscript>+</Superscript>-ATPase: A Salinity-sensitive Relationship in the Gill Basolateral Membrane of Rainbow Trout

We investigated the effect of salinity on the relationship between Na⁺-K⁺-ATPase and sulfogalactosyl ceramide (SGC) in the basolateral membrane of rainbow trout (Oncorhynchus mykiss) gill epithelium. SGC has been implicated as a cofactor in Na⁺-K⁺-ATPase activity, especially in Na⁺-K⁺-ATPase rich tissues. However, whole-tissue studies have questioned this role in the fish gill. We re-examined SGC cofactor function from a gill basolateral membrane perspective. Nine SGC fatty acid species were quantified by tandem mass spectrometry (MS/MS) and related to Na⁺-K⁺-ATPase activity in trout acclimated to freshwater or brackish water (20 ppt). While Na⁺-K⁺-ATPase activity increased, the total concentration and relative proportion of SGC isoforms remained constant between salinities. However, we noted a negative correlation between SGC concentration and Na⁺-K⁺-ATPase activity in fish exposed to brackish water, whereas no correlation existed in fish acclimated to freshwater. Differential Na⁺-K⁺-ATPase/SGC sensitivity is discussed in relation to enzyme isoform switching, the SGC cofactor site model and saltwater adaptation.This revised version was published online in June 2005 with a corrected cover date.     

Effects of spironolactone and RU486 on gene expression and cell proliferation after freshwater transfer in the euryhaline killifish

We have explored the possible mechanisms by which mineralocorticoid (MR) and glucocorticoid (GR) receptors regulate the response to freshwater transfer in the gills of the euryhaline killifish Fundulus heteroclitus. Killifish were implanted with RU486 (GR antagonist) or spironolactone (MR antagonist) at doses of 0.1–1.0 mg g⁻¹, and subsequently transferred from 10‰ brackish water to freshwater. Compared to brackish water sham fish, mRNA expression of CFTR and NKCC1 decreased in the gills of sham fish transferred to freshwater, whereas Na⁺,K⁺–ATPase α_1a mRNA expression and α protein abundance, as well as cell proliferation (detected using BrdU) increased. Spironolactone inhibited the normal increase in cell proliferation and Na⁺,K⁺-ATPase expression after freshwater transfer. RU486 increased plasma cortisol levels and may have slightly inhibited Na⁺,K⁺–ATPase activity, but did not change α _1a expression. RU486 had no effect on cell proliferation in the non-lamellar region of the gills, but increased proliferation in the lamellar region. Neither antagonist inhibited the suppression of CFTR or NKCC1 expression after freshwater transfer. Glucocorticoid receptor expression was reduced in all sham and antagonist treatments compared to untreated controls, but no other consistent differences were observed. The effects of spironolactone suggest that MR is important for regulating ion transport in killifish gills after freshwater transfer.     

Branchial ammonia excretion in the Asian weatherloach Misgurnus anguillicaudatus

The weatherloach, Misgurnus anguillicaudatus, is a freshwater, facultative air-breathing fish that lives in streams and rice paddy fields, where it may experience drought and/or high environmental ammonia (HEA) conditions. The aim of this study was to determine what roles branchial Na⁺/K⁺-ATPase, H⁺-ATPase, and Rhcg have in ammonia tolerance and how the weatherloach copes with ammonia loading conditions. The loach's high ammonia tolerance was confirmed as was evident from its high 96 h LC₅₀ value and high tissue tolerance to ammonia. The weatherloach does not appear to make use of Na⁺/NH₄⁺-ATPase facilitated transport to excrete ammonia when exposed to HEA or to high environmental pH since no changes in activity were observed. Using immunofluorescence microscopy, distinct populations of vacuolar (V)-type H⁺-ATPase and Na⁺/K⁺-ATPase immunoreactive cells were identified in branchial epithelia, with apical and basolateral staining patterns, respectively. Rhesus C glycoprotein (Rhcg1), an ammonia transport protein, immunoreactivity was also found in a similar pattern as H⁺-ATPase. Rhcg1 (Slc42a3) mRNA expression also increased significantly during aerial exposure, although not significantly under ammonia loading conditions. The colocalization of H⁺-ATPase and Rhcg1 to the similar non-Na⁺/K⁺-ATPase immunoreactive cell type would support a role for H⁺-ATPase in ammonia excretion via Rhcg by NH₄⁺ trapping. The importance of gill boundary layer acidification in net ammonia excretion was confirmed in this fish; however, it was not associated with an increase in H⁺-ATPase expression, since tissue activity and protein levels did not increase with high environmental pH and/or HEA. However the V-ATPase inhibitor, bafilomycin, did decrease net ammonia flux whereas other ion transport inhibitors (amiloride, SITS) had no effect. H⁺-ATPase inhibition also resulted in a consequent elevation in plasma ammonia levels and a decrease in the net acid flux. In gill, aerial exposure was also associated with a significant increase in membrane fluidity (or increase in permeability) which would presumably enhance NH₃ permeation through the plasma membrane. Taken together, these results indicate the gill of the weatherloach is responsive to aerial conditions that would aid ammonia excretion.     

Changes in Na+/K+-ATPase activity, unsaturated fatty acids and metallothioneins in gills of the shore crab Carcinus aestuarii after dilute seawater acclimation

We have assessed the activity of Na⁺/K⁺-ATPase, cAMP, free fatty acids (FFA) and metallothionein (MT) in the posterior gills of the brackish water shore crab Carcinus aestuarii during acclimation to 10 ppt dilute seawater (DSW). Following 3–18 days acclimation in DSW specific activity of Na⁺/K⁺-ATPase in native gill homogenates and partially purified membrane vesicles was progressively increased, from 1.7- to 3.9-fold. After short-term acclimation of crabs in DSW with added sucrose to make media isosmotic with the haemolymph the specific Na⁺/K⁺-ATPase activity in homogenates was not increased, relative to SW enzyme activity. Moreover, hyposmotic conditions led to depletion of cAMP in gills.In partially purified membrane vesicles isolated from posterior gills, fatty acids with compositions 16:0, 18:0, 18:1, 20:4 and 20:5 dominated in both SW- and DSW-acclimated Carcinus. During a year in which the metabolic activity of crabs was increased, the arachidonic/linoleic acids ratio (ARA/LA) for DSW-acclimated crabs was markedly increased relative to that in SW. Increased Na⁺ + K⁺-ATPase activity under hyposmotic stress may be modulated at least partially by the changed proportion of fatty acids in the purified membranes of posterior gills. Long-term acclimation of shore crabs to DSW resulted in a 2.6-fold increase in cytosolic metallothionein (MT) content in posterior gills over those in SW crabs. Assuming an antioxidant role of MT associated with intracellular zinc partitioning, the observed MT induction in posterior gills may be considered an adaptive response of C. aestuarii to hyposmotic stress.     

The effect of acidity on gill variations in the aquatic air-breathing fish,Trichogaster lalius

Climate change affects organisms that inhabit not only in aerial but also in aquatic environments by making water more hypoxic and acidic. In the past, we evaluated morphological and functional variations in the gills of 12 species of aquatic air-breathing fishes. The aim of the present study is to examine the degree of gill modification in the aquatic air-breathing fish, Trichogaster lalius, in response to acidic stress. This provides a link between the ecological and physiological studies. We evaluated the changes in morphology and function of the gills, labyrinth organ, and kidney when the fish were subjected to acidic water and deionized water (DW). In the first experiment, fish were sampled at 1, 2, 4, and 7 days after acidic treatment. Apparent morphological modification was observed on day 4 and recovery was noted on day 7. Protein expression and enzyme activity of vacuolar-type H⁺-ATPase (VHA) and the protein expression of the proliferating cell nuclear antigen (PCNA) of the 1st and 4th gill arches both increased in the 4-day and 7-day acidic groups while the enzyme activity of Na⁺/K⁺-ATPase (NKA) decreased. In the second experiment, fish were tested for changes in the 1st and 4th gill arches and kidney after exposure to DW and acidic water for 4 days. The gill structure of the fish in the DW was not different from that of the control group (fresh water). The protein expression and enzyme activity of the VHA of the 1st and 4th gill arches increased in both the DW and acidic groups for 4 days. We found a decrease in the protein expression of NKA in the kidney and in the enzyme activity of NKA in the 1st and 4th gill arches in the DW and acidic groups. From these results, we suggest that T. lalius exhibited significantly different ionic regulation and acid-base regulatory abilities in the DW and acidic groups in the 1st and 4th gill arches and kidney. The responses of the gills in T. lalius were different from those fish that show apparent morphological variations between the 1st and 4th gill arches.     

An endogenous factor which interacts with synaptosomal membrane Na+, K+-ATPase activation by K+

In previous papers, the isolation of brain soluble fractions able to modify neuronal Na⁺, K⁺-ATPase activity has been described. One of those fractions-peak I-stimulates membrane Na⁺, K⁺-ATPase while another-peak II-inhibits this enzyme activity, and has other ouabain-like properties. In the present study, synaptosomal membrane Na⁺, K⁺-ATPase was analyzed under several experimental conditions, using ATP orp-nitrophenylphosphate (p-NPP) as substrate, in the absence and presence of cerebral cortex peak II. Peak II inhibited K⁺-p-NPPase activity in a concentration dependent manner. Double reciprocal plots indicated that peak II uncompetitively inhibits K⁺-p-NPPase activity regarding substrate, Mg²⁺ and K⁺ concentration. Peak II failed to block the known K⁺-p-NPPase stimulation caused by ATP plus Na⁺. At various K⁺ concentrations, percentage K⁺-p-NPPase inhibition by peak II was similar regardless of the ATP plus Na⁺ presence, indicating lack of correlation with enzyme phosphorylation. Na⁺, K⁺-ATPase activity was decreased by peak II depending on K⁺ concentration. It is postulated that the inhibitory factor(s) present in peak II interfere(s) with enzyme activation by K⁺.     

Growth hormone and cortisol treatment stimulate seawater tolerance in both anadromous and landlocked Arctic charr

In a comparative experiment the effect of cortisol and growth hormone (GH) on the hypo-osmoregulatory ability of a landlocked and an anadromous strain of Arctic charr (Salvelinus alpinus) was investigated. Cortisol and GH were implanted either alone or in combination, and the fish were exposed to a 24 h seawater challenge test (SWT) on days 14 and 28 after implantation. Hypo-osmoregulatory ability, measured as plasma osmolality and chloride concentration after the SWTs, was better in the anadromous than in the landlocked strain, irrespective of treatment. However, cortisol provided a strong stimulation of hypo-osmoregualtory ability in both strains, and this stimulation seemed to be potentiated by GH in an additive manner. Improved hypo-osmoregulatory ability in GH + cortisol treated anadromous Arctic charr was accompanied by increased gill Na⁺, K⁺-ATPase activity and Na⁺–K⁺–2Cl⁻ cotransporter protein abundance, but no changes in gill Na⁺,K⁺-ATPase α1a and α1b mRNA levels. For landlocked charr the improved hypo-osmoregulatory ability in GH +cortisol treated fish was accompanied only with an increase in gill Na⁺–K⁺–2Cl⁻ cotransporter protein abundance. Hormone treatment caused an improvement of hypo-osmoregulatory ability that was of approximately the same magnitude in the landlocked as in the anadromous Arctic charr. This suggests that the lack of spontaneous development of hypo-osmoregulatory ability often seen in landlocked populations of Arctic charr may depend, at least partly, on a lack of the hormonal activation seen in anadromous populations.     

Expression of the Catalytic Subunit of Ouabain-Sensitive H+,K+-ATPase in Rat Skin Epidermis

A comparative localization of Na⁺,K⁺-ATPase and ouabain-sensitive H⁺,K⁺-ATPase in rat skin was performed using in situ RNA hybridization and immunohistochemistry. Na⁺,K⁺-ATPase was predominantly detected in the basal layer of the epithelium, whereas the ouabain-sensitive H⁺,K⁺-ATPase, in the granular and prickle cell layers. The genes of these ATPases are thus expressed in epithelial cells at different stages of their development. The hypothesis was advanced that the ouabain-sensitive H⁺,K⁺-ATPase is involved in maintaining the skin pH value. The probes specific to the mRNAs of the full-size -subunit of the ouabain-sensitive H⁺,K⁺-ATPase and its truncated form were used to establish a similar distribution of both mRNA variants in skin.     

In search of synaptosomal Na+, K+-ATPase regulators

The arrival of the nerve impulse to the nerve endings leads to a series of events involving the entry of sodium and the exit of potassium. Restoration of ionic equilibria of sodium and potassium through the membrane is carried out by the sodium/potassium pump, that is the enzyme Na⁺,K⁺-ATPase. This is a particle-bound enzyme that concentrates in the nerve ending or synaptosomal membranes. The activity of Na⁺,K⁺-ATPase is essential for the maintenance of numerous reactions, as demonstrated in the isolated synaptosomes. This lends interest to the knowledge of the possible regulatory mechanisms of Na⁺,K⁺-ATPase activity in the synaptic region. The aim of this review is to summarize the results obtained in the author's laboratory, that refer to the effect of neurotransmitters and endogenous substances on Na⁺,K⁺-ATPase activity. Mention is also made of results in the field obtained in other laboratories. Evidence showing that brain Na⁺,K⁺-ATPase activity may be modified by certain neurotransmitters and insulin have been presented. The type of change produced by noradrenaline, dopamine, and serotonin on synaptosomal membrane Na⁺,K⁺-ATPase was found to depend on the presence or absence of a soluble brain fraction. The soluble brain fraction itself was able to stimulate or inhibit the enzyme, an effect that was dependent in turn on the time elapsed between preparation and use of the fraction. The filtration of soluble brain fraction through Sephadex G-50 allowed the separation of two active subfractions: peaks I and II. Peak I increased Na⁺,K⁺- and Mg²⁺-ATPases, and peak II inhibited Na⁺,K⁺-ATPase. Other membrane enzymes such as acetylcholinesterase and 5′-nucleotidase were unchanged by peaks I or II. In normotensive anesthetized rats, water and sodium excretion were not modified by peak I but were increased by peak II, thus resembling ouabain effects.³H-ouabain binding was unchanged by peak I but decreased by peak II in some areas of the CNS assayed by quantitative autoradiography and in synaptosomal membranes assayed by a filtration technique. The effects of peak I and II on Na⁺,K⁺-ATPase were reversed by catecholamines. The extent of Na⁺,K⁺-ATPase inhibition by peak II was dependent on K⁺ concentration, thus suggesting an interference with the K⁺ site of the enzyme. Peak II was able to induce the release of neurotransmitter stored in the synaptic vesicles in a way similar to ouabain. Taking into account that peak II inhibits only Na⁺,K⁺-ATPase, increases diuresis and natriuresis, blocks high affinity³H-ouabain binding, and induces neurotransmitter release, it is suggested that it contains an ouabain-like substance.     

Na+, K+-ATPase Isozyme Diversity; Comparative Biochemistry and Physiological Implications of Novel Functional Interactions

Na⁺, K⁺-ATPase is ubiquitously expressed in the plasma membrane ofall animal cells where it serves as the principal regulator of intracellularion homeostasis. Na⁺, K⁺-ATPase is responsible for generating andmaintaining transmembrane ionic gradients that are of vital importance forcellular function and subservient activities such as volume regulation, pHmaintenance, and generation of action potentials and secondary activetransport. The diversity of Na⁺, K⁺-ATPase subunit isoforms andtheir complex spatial and temporal patterns of cellular expression suggestthat Na⁺, K⁺-ATPase isozymes perform specialized physiologicalfunctions. Recent studies have shown that the subunit isoformspossess considerably different kinetic properties and modes of regulationand the subunit isoforms modulate the activity, expression and plasmamembrane targeting of Na⁺, K⁺-ATPase isozymes. This review focuseson recent developments in Na⁺, K⁺-ATPase research, and in particular reportsof expression of isoforms in various tissues and experiments aimed atelucidating the intrinsic structural features of isoforms important forNa⁺, K⁺-ATPase function.     

Kinetics of K+-p-Nitrophenyl Phosphatase Stimulation by a Brain Soluble Fraction

We have already described the separation of two brain soluble fractions by Sephadex G-50, one of which stimulates (peak I) and the other inhibits (peak II) Na⁺, K⁺-ATPase and K⁺-p-nitrophenylphosphatase (K⁺-p-NPPase) activities. Here we examine the features of synaptosomal membrane p-NPPase activity in the presence and absence of brain peak I. It was observed that stimulation of Mg²⁺, K⁺-p-NPPase activity by peak I was concentration dependent, The ability of peak I to stimulate p-NPPase activity was lost by heat treatment followed by brief centrifugation. Pure serum albumin also stimulated enzyme activity. K⁺-p-NPPase stimulation by peak I proved dependent on K⁺ concentration but independent of Mg²⁺ and substrate p-nitrophenylphosphate concentrations. Since our determinations were performed in a non-phosphorylating condition reflecting the Na⁺, K⁺-ATPase Na⁺ site, it is suggested that peak I may stimulate the Na⁺-dependent enzyme phosphorylation known to take place from the internal cytoplasmic side.     

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.     

A comparative study between a brain Na+,K(+)-ATPase inhibitor (endobain E) and ascorbic acid

In the search of Na⁺,K⁺-ATPase modulators, we have reported the isolation by gel filtration and HPLC of a brain fraction, termed endobain E, which highly inhibits Na⁺,K⁺-ATPase activity. In the present study we compared some properties of endobain E with those of ascorbic acid. Kinetic experiments assaying synaptosomal membrane K⁺-p-nitrophenylphosphatase (K⁺-p-NPPase) activity in the presence of endobain E or ascorbic acid showed that in neither case did enzyme inhibition prove competitive in nature versus K⁺ or p-NPP concentration. At pH 5.0, endobain E and ascorbic acid maximal UV absorbance was 266 and 258 nm, respectively; alkalinization to pH 14.0 led to absorption drop and shift for endobain E but to absorbance disappearance for ascorbic acid. After cysteine treatment, endobain E absorbance decreased, whereas that of ascorbic acid remained unaltered; iodine treatment led to absorbance drop and shift for endobain E but to absorbance disappearance for ascorbic acid. HPLC analysis of endobain E disclosed the presence of two components: one eluting with retention time and UV spectrum indistinguishable from those of ascorbic acid and a second, as yet unidentified, both exerting Na⁺,K⁺-ATPase inhibition.     

The Na<Superscript>+</Superscript>-translocating ATPase in the plasma membrane of the marine microalga<Emphasis Type="Italic"> Tetraselmis viridis</Emphasis> catalyzes Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange

Our previous investigations have established that Na⁺ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na⁺-pump, Na⁺-ATPase, is accompanied by H⁺ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na⁺-ATPase of T. viridis operates as an Na⁺/H⁺ exchanger is tested in the present work. The study of Na⁺ and H⁺ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO₃^– caused (i) an increase in ATP-driven Na⁺ uptake by the vesicles, (ii) an increase in (Na⁺+ATP)-dependent vesicle lumen alkalization resulting from H⁺ efflux out of the vesicles and (iii) dissipation of electrical potential, , generated across the vesicle membrane by the Na⁺-ATPase. The (Na⁺+ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K⁺ abolished at the vesicle membrane. The fact that the Na⁺-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane is consistent with a primary role of the Na⁺-ATPase in driving H⁺ outside the vesicles. The findings allowed us to conclude that the Na⁺-ATPase of T. viridis directly performs an exchange of Na⁺ for H⁺. Since the Na⁺-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa⁺/nH⁺, where m>n.Abbreviations BTP Bis-Tris-Propane, 1,3-bis[tris(hydroxymethyl)methylamino]-propane - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DTT Dithiothreitol - NCDC 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate - PMSF Phenylmethylsulfonyl fluoride - PM Plasma membrane     

Evidence That Antioxidants Prevent the Inhibition of Na+,K+-ATPase Activity Induced by Octanoic Acid in Rat Cerebral Cortex in Vitro

The objective of the present study was to investigate the in vitro effects of octanoic acid, which accumulates in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency and in Reye syndrome, on key enzyme activities of energy metabolism in the cerebral cortex of young rats. The activities of the respiratory chain complexes I–IV, creatine kinase, and Na⁺, K⁺-ATPase were evaluated. Octanoic acid did not alter the electron transport chain and creatine kinase activities, but, in contrast, significantly inhibited Na⁺, K⁺-ATPase activity both in synaptic plasma membranes and in homogenates prepared from cerebral cortex. Furthermore, decanoic acid, which is also increased in MCAD deficiency, and oleic acid strongly reduced Na⁺, K⁺-ATPase activity, whereas palmitic acid had no effect. We also examined the effects of incubating glutathione and trolox (-tocopherol) alone or with octanoic acid on Na⁺, K⁺-ATPase activity. Tested compounds did not affect Na⁺, K⁺-ATPase activity by itself, but prevented the inhibitory effect of octanoic acid. These results suggest that inhibition of Na⁺, K⁺-ATPase activity by octanoic acid is possibly mediated by oxidation of essential groups of the enzyme. Considering that Na⁺, K⁺-ATPase is critical for normal brain function, it is feasible that the significant inhibition of this enzyme activity by octanoate and also by decanoate may be related to the neurological dysfunction found in patients affected by MCAD deficiency and Reye syndrome.