Phosphorylated intermediate of the ouabain-insensitive, Na(+)-stimulated ATPase in rat kidney cortex and rainbow trout gills.

Several tissues from different animals, including the rat kidney and the freshwater rainbow trout gills, show an ouabain-insensitive, furosemide-sensitive, Na(+)-stimulated ATPase activity, which has been associated with the active control of the cell volume. This Na-ATPase is Mg(2+) dependent and it is inhibited by vanadate, which can be taken as an indication that this enzyme is a P-type ATPase. The P-type ATPases are known to form a phosphorylated intermediate during their catalytic cycle, where the phosphate binds an aspartyl residue at the enzyme's substrate site. In the current study, we partially characterized the phosphorylated intermediate of the ouabain-insensitive Na-ATPase of rat kidney cortex homogenates and that of gill microsomes from freshwater rainbow trout. While the kidney cortex homogenates, under our assay conditions, show both Na- and Na,K-ATPase activities, the gill microsomes, when assayed at pH 5.2, only show Na-ATPase activity. Both preparations showed a Mg(2+)-dependent, Na(+)-stimulated phosphorylated intermediate, which is enhanced by furosemide. Incubation of the phosphorylated enzyme with 0.6 N hydroxylamine (NH(2)OH) showed that it is acid-stable and sensitive to hydroxylamine, either when phosphorylated in the presence or absence of furosemide. Addition of ADP to the incubation medium drives the reaction cycle of the enzyme backward, diminishing its phosphorylation. Na(+) seems to stimulate both the phosphorylation and the dephosphorylation of the enzyme, at least for the Na-ATPase from gill microsomes. In a E1-E2 reaction cycle of the Na-ATPase, furosemide seems to be blocking the transition step from Na.E1 approximately P to Na.E2-P.

Chemical and biochemical parameters of cultured diatoms and bacteria from the Adriatic Sea as possible biomarkers of mucilage production.

Bacteria and diatom strains from the Adriatic Sea were investigated, under standard and altered environmental conditions, for carbohydrate production and for the presence of specific biomarkers. Algae from P-depleted cultures showed an increase in extracellular carbohydrate production, a significantly lower chlorophyll a content and unchanged total lipid levels. However, the fatty acid composition of algal cultures was severely affected by low P levels, in that, total saturated and monounsaturated fatty acids increased and total polyunsaturated fatty acids decreased. Marine heterotrophic bacteria resulted enriched by 4 to 6 orders of magnitude in mucilage samples respect to surrounding seawater, unlike other groups of bacteria such as the non-halophylic heterotrophs. The major fatty acids detected in bacteria were 16:0 and 18:1n-7; the uneven fatty acids 17:0i, 17:0 and 17:1 also constituted an important component of various strains and, as a result, the total monounsaturated fraction represented the main component of total fatty acids. All the mucilage samples analysed shared the same general fatty acid composition features with a high amount of saturated components, especially 16:0; typical marine polyunsaturated fatty acids, such as 20:5n-3 and 22:6n-3, were found at very low levels. With regard to the sterol composition, the analysed algal species and bacteria showed that different compounds prevailed in the different species, and under P-deprivation sterol distribution resulted differently affected in the various algal species. In mucilage samples an overall prevalence of cholesterol was observed and, among 4alpha-methylsterols, constantly present, dinosterol prevailed in all samples. Vibrational IR spectroscopic analyses confirmed the main results obtained with the GC analysis: a higher unsaturation degree in nutrient replete diatom cultures than in P-depleted ones, a lower amount of P-containing compounds in the latter, bacterial lipid profiles with a high amount of free carboxylic acids and/or ketones and a low unsaturation degree and, finally, mucilage samples with a very low unsaturation degree. All these results allowed some speculations on the involvement of the various microbial and phytoplankton components in mucilage genesis.

Response of rainbow trout gill Na+-ATPpase to T(3) and NaCl administration.

The effect of the administration of commercial diets supplemented with 9 mg kg(-1) 3,5,3'-triiodo-l-thyronine (T(3)) or 10% (w/w) NaCl was evaluated on the ouabain-insensitive Na+-ATPase activity in rainbow trout gill microsomes. The trial, carried out following the seasonal trend from March to mid-May, included a treatment phase in freshwater and a subsequent transfer to brackish water (22 per thousand salinity) where trout were not treated. pH dependence, apparent Km values for Mg(2+) and Na+, and Hill coefficients evaluated throughout the trial for Na+-ATPase were generally not affected by the treatments and habitat change. In comparison with the control group, in both treated groups, Na+-ATPase activity was lower during the freshwater phase and higher after brackish-water transfer. As compared with untreated trout, gill (Na++K+)-ATPase activity during the freshwater phase was stimulated by NaCl treatment and also by T(3) treatment after transfer to brackish water. The results indicate that NaCl and T(3) administration act differently on the two ATPase activities involved in Na+ regulation and suggest a prevalent role of Na+-ATPase activity in hypoosmotic conditions.

Response of rainbow trout gill (Na(+)+K (+))-ATPase and chloride cells to T 3 and NaCl administration.

With the aim of comparing the effects of oral T3 and NaCl administration on trout hypoosmoregulatory mechanisms, three groups of rainbow trout (Oncorhynchus mykiss Walbaum) held in freshwater (FW) were fed a basal diet (C), the same diet containing 8.83 ppm of 3,5,3'-triiodo-L-thyronine (T3) (T) or 10% (w/w) NaCl (N) respectively for 30 d. They were then transferred to brackish water (BW) for 22 d and fed on diet C. Gill (Na(+)+K(+))-ATPase activity and its dependence on ATP, Na(+) and pH, number of gill chloride cells (CC), serum T3 level as well as fish growth, condition factor (K) and mortality were evaluated. During the FW phase, as compared to C trout, T trout showed a two fold higher serum T3 level, had unchanged gill (Na(+)+K(+))-ATPase activity and increased CC number, whereas N trout showed higher gill (Na(+)+K(+))-ATPase activity and CC number. At the end of the experiment the enzyme activity was in the order T>N>C groups and all groups showed similar CC number. Both treatments changed the enzyme activation kinetics by ATP and Na(+). A transient increase in K value occurred in N group during the period of salt administration. In BW, T and N groups had higher and lower survival than C group respectively. Other parameters were unaffected by the treatments. This trial suggests that T3 administration promotes the development of hypoosmoregulatory mechanisms of trout but it leaves the (Na(+)+K(+))-ATPase activity unaltered till the transfer to a hyperosmotic environment.

Lipid composition and microsomal ATPase activities in gills and kidneys of warm- and cold-acclimated sea bass (Dicentrarchus labrax L.).

The response to cold of gill and kidney membrane lipid composition and microsomal (Na(+)+K(+))-ATPase, Na(+)-ATPase and Mg(2+)-ATPase activities in reared sea bass (Dicentrarchus labrax L.) was investigated. Fish acclimation was carried out according to the seasonal cycle from August to March. No cold-promoted increase in fatty acid unsaturation was shown in gill and kidney polar lipids and in total lipids of mitochondria and microsomes. In both tissues the (Na(+)+K(+))-ATPase exhibited positive compensation for cold acclimation whereas the Na(+)-ATPase displayed negative compensation. The Mg(2+)-ATPase showed no compensation in the gills and positive compensation in the kidneys. During cold acclimation the break in the Arrhenius plot of the (Na(+)+K(+))-ATPase decreased, whereas breaks of both the Na(+)-ATPase and the Mg(2+)-ATPase activities remained unchanged. The results indicate that the sea bass does not adopt membrane unsaturation as a cold-facing strategy. The cold-promoted enhancement of (Na(+)+K(+))-ATPase activity in osmoregulatory tissues may be advantageous to maintain efficient osmoregulation under thermodynamically unfavourable conditions.

Gill (Na+ +K+)-ATPase involvement and regulation during salmonid adaptation to salt water.

1. The involvement of gill (Na+ +K+)-ATPase in salmonid adaptation to salt water (SW) is discussed. 2. Gill (Na+ +K+)-ATPase increase during SW adaptation is mainly related to the increased number and complexity of chloride cells deputed to salt extrusion. 3. The temporal relationships between serum peaks of thyroid hormones, cortisol, growth hormone, prolactin and gill (Na+ +K+)-ATPase rise during salmonid smoltification, suggest a hormonal involvement in the enzyme stimulation and thus in the acquirement of SW tolerance. 4. Literature on gill (Na+ +K+)-ATPase response to hormonal treatment is reviewed. The effects produced on gill (Na+ +K+)-ATPase and chloride cells by exogenous hormones point out a complex inter-relationship between the hormones considered. The mechanisms involved in hormonal regulation of the enzyme remain a matter of debate.

Lipid composition and mitochondrial respiration in warm- and cold-adapted sea bass.

The response to cold of liver and heart membrane lipid composition and mitochondrial respiration in reared sea bass (Dicentrarchus labrax L.) was investigated. Fish acclimation was followed during the natural seasonal cycle from August to March. The data on the fatty acid composition of liver and heart polar lipids and on total lipids of liver mitochondria and microsomes did not indicate any increase in unsaturation in response to cold. The enzyme complexes of the liver and heart mitochondrial respiratory chain showed a repeated negative compensation for cold acclimation. The constancy of the break in the Arrhenius plot of liver cytochrome oxidase (EC 1.9.3.1) was consistent with the lack of homeoviscous adaptation of membrane lipids. A thermoadaptive strategy based on the reduction of sea bass metabolic activity is suggested.

Gill (Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in the gilthead bream (Sparus auratus L.).

1. Gilthead gill 10(-3) M ouabain-inhibited (Na+ + K+)-ATPase and 10(-2) M ouabain-insensitive Na+-ATPase require the optimal conditions of pH 7.0, 160 mM Na+, 20 mM K+, 5 mM MgATP and pH 4.8-5.2, 75 mM Na+, 2.5 mM Mg2+, 1.0 mM ATP, respectively. 2. The main distinctive features between the two activities are confirmed to be optimal pH, the ouabain-sensitivity and the monovalent cation requirement, Na+ plus another cationic species (K+, Rb+, Cs+, NH4+) in the (Na+ + K+)-ATPase and only one species (Na+, K+, Li+, Rb+, Cs+, NH4+ or choline+) in the Na+-ATPase. 3. The aspecific Na+-ATPase activation by monovalent cations, as well as by nucleotide triphosphates, opposed to the (Na+ + K+)-ATPase specificity for ATP and Na+, relates gilthead gill ATPases to lower organism ATPases and differentiates them from mammalian ones. 4. The discrimination between the two activities by the sensitivity to ethacrynic acid, vanadate, furosemide and Ca2+ only partially agrees with the literature. 5. Present findings are viewed on the basis of the ATPase's presumptive physiological role(s) and mutual relationship.

(Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in kidney of sea bass (Dicentrarchus labrax L.).

1. Sea bass kidney microsomal preparations contain two Mg2+ dependent ATPase activities: the ouabain-sensitive (Na+ + K+)-ATPase and an ouabain-insensitive Na+-ATPase, requiring different assay conditions. The (Na+ + K+)-ATPase under the optimal conditions of pH 7.0, 100 mM Na+, 25 mM K+, 10 mM Mg2+, 5 mM ATP exhibits an average specific activity (S.A.) of 59 mumol Pi/mg protein per hr whereas the Na+-ATPase under the conditions of pH 6.0, 40 mM Na+, 1.5 mM MgATP, 1 mM ouabain has a maximal S.A. of 13.9 mumol Pi/mg protein per hr. 2. The (Na+ + K+)-ATPase is specifically inhibited by ouabain and vanadate; the Na+-ATPase specifically by ethacrynic acid and preferentially by frusemide; both activities are similarly inhibited by Ca2+. 3. The (Na+ + K+)-ATPase is specific for ATP and Na+, whereas the Na+-ATPase hydrolyzes other substrates in the efficiency order ATP greater than GTP greater than CTP greater than UTP and can be activated also by K+, NH4+ or Li+. 4. Minor differences between the two activities lie in the affinity for Na+, Mg2+, ATP and in the thermosensitivity. 5. The comparison between the two activities and with what has been reported in the literature only partly agree with our findings. It tentatively suggests that on the one hand two separate enzymes exist which are related to Na+ transport and, on the other, a distinct modulation in vivo in different tissues.

Na+-like effect of monovalent cations in the stimulation of sea bass gill Mg2+-dependent Na+-stimulated ATPase.

The Mg2+-dependent ouabain insensitive-ATPase activity present in gill microsomal preparations from Dicentrarchus labrax is stimulated not only by Na+ but also by K=, NH4+ or Li+. These cations at 50-100 mM concentrations are similarly efficient to Na+ in stimulating the enzyme activity with similar Km values. Whatever cation stimulates the activity, the enzyme is poorly sensitive to ouabain and 100% inhibited by 1.5-2.5 mM ethacrynic acid. All activity vs cation concentration curves show a biphasic profile with activation following the Michaelis-Menten kinetics (Hill coefficient approximately 2). The absence of additivity when the enzyme is activated by binary mixtures of cations, each of which may act as competitive inhibitor of the other confirms the involvement of the same binding site for the monovalent cations.

Characterization of gill (Na+ + K+)-ATPase in the sea bass (Dicentrarchus labrax L.).

Bass gill microsomal preparations contain both a Na+, K+ and Mg2+-dependent ATPase, which is completely inhibited by 10(-3)M ouabain and 10(-2)M Ca2+, and also a ouabain insensitive ATP-ase activity in the presence of both Mg2+ and Na+. Under the optimal conditions of pH 6.5, 100 mM Na+, 20 mM K+, 5 mM ATP and 5 mM Mg2+, (Na+ + K+)-ATPase activity at 30 degrees C is 15.6 mumole Pi hr/mg protein. Bass gill (Na+ + K+)-ATPase is similar to other (Na+ + K+)-ATPases with respect to the sensitivity to ionic strength, Ca2+ and ouabain and to both Na+/K+ and Mg2+/ATP optimal ratios, while pH optimum is lower than poikilotherm data. The enzyme requires Na+, whereas K+ can be replaced efficiently by NH+4 and poorly by Li+. Both Km and Vm values decrease in the series NH+4 greater than K+ greater than Li+. The break of Arrhenius plot at 17.7 degrees C is close to the adaptation temperature. Activation energies are scarcely different from each other and both lower than those generally reported. The Km for Na+ poorly decreases as the assay temperature lowers. The comparison with literature data aims at distinguishing between distinctive and common features of bass gill (Na+ + K+)-ATPase.

Ouabain-insensitive Na+ stimulation of a microsomal Mg2+ -ATPase in gills of sea bass (Dicentrarchus labrax L.).

Bass gill microsomal preparations contain a Mg2+-dependent Na+-stimulated ATPase activity in the absence of K+, whose characteristics are compared with those of the (Na+ + K+)-ATPase of the same preparations. The activity at 30 degrees C is 11.3 mumol Pi X mg-1 protein X hr-1 under optimal conditions (5 mM MgATP, 75 mM Na+, 75 mM HEPES, pH 6.0) and exhibits a lower pH optimum than the (Na+ + K+)-ATPase. The Na+ stimulation of ATPase is only 17% inhibited by 10-3M ouabain and completely abolished by 2.5 mM ethacrinic acid which on the contrary cause, respectively, 100% and 34% inhibition of the (Na+ + K+)-ATPase. Both Na+-and (Na+ + K+)-stimulated activities can hydrolyze nucleotides other than ATP in the efficiency order ATP greater than CTP greater than UTP greater than GTP and ATP greater than CTP greater than GPT greater than UTP, respectively. In the presence of 10(-3)M ouabain millimolar concentrations of K+ ion lower the Na+ activation (90% inhibition at 40 mM K+). The Na+-ATPase is less sensitive than (Na+ + K+)-ATPase to the Ca2+ induced inhibition as the former is only 57.5% inhibited by a concentration of 1 X 10(-2)M which completely suppresses the latter. The thermosensitivity follows the order Mg2+--greater than (Na+ + K+)--greater than Na+-ATPase. A similar break of the Arrhenius plot of the three enzymes is found. Only some of these characteristics do coincide with those of a Na+-ATPase described elsewhere. A presumptive physiological role of Na+-ATPase activity in seawater adapted teleost gills is suggested.

[Na+-ATPase in the gills of bass (Dicentrarchus labrax)]. / Na+-ATPasi in branchie di spigola (Dicentrarchus labrax).

The authors evidence a Mg2+ dependent ATPase activity stimulated by Na+ in absence of K+ in bass gill microsomes. As this stimulated ATPase shows different features from "baseline" activity measured in the absence of both Na+ and K+ ions (Mg2+-ATPase) and from 1mM ouabain sensitive (Na+ + K+)-ATPase, it has been ascribed to a distinct Na+-ATPase. In the present paper the optimal conditions for bass gill Na+-ATPase assay and the temperature dependence of the enzyme are reported. Moreover the Na+-ATPase appears to be insensitive to 1mM ouabain and 100% inhibited by 2,5mM ethacrynic acid. It is suggested a parallel diffusion of Na+- and (Na+ + K+)-ATPase and a possible physiological role of Na+ATPase in osmoregulation.

[Characteristics of (Na+ + K+)-ATPase in the gills of bass]. / Caratteristiche della (Na+ + K+)-ATPasi branchiale di spigola (Dicentrarchus labrax).

A partial characterization of bass gill (Na+ + K+-ATPase is reported in the present paper. Microsomal preparation from gill homogenate showed optimal (Na+ + K+)-ATPase activity at pH 6,5 in the presence of 100 mM Na+, 20mM K+ and 5mM Mg2+. Under these conditions maximal activity was shown at 45 degrees C, even if an increased lability of the enzyme was shown at temperature greater than 30 degrees C. A complete inhibition of the enzyme occurred in the presence of 1 mM ouabain. The break in the Arrhenius plot occurred approximatively at the temperature of adaptation of these fish (18 degrees C). The energies of activation above and below the break were scarcely different from each other and lower than those reported in other Poikilotherms. Furthermore similar values of Km for Na+ were evidenced at 18 degrees C and 30 degrees C. The whole of data are discussed in comparison with other teleost gill (Na+ + K+)-ATPase reports and related to the physiological role of the enzyme in osmoregulation.

[First observations on the effect of various dietary oils on liver fatty acids in bass (Dicentrarchus labrax)]. / Prime osservazioni sull'effetto di differenti olii dietetici sugli acidi grassi del fegato di spigola (Dicentrarchus labrax).

A close relationship between dietary oils and fatty acid composition of bass liver and liver microsomes and mitochondria is reported in the present paper. Among the data the most relevant is the evidence for elongation and desaturation of dietary 18:3 n-3 and 18:2 n-6, giving as a result an increase of 22:6 n-3 and 20:4 n-6 levels respectively. The importance of such findings in carnivore marine fish is discussed and compared with literature data.

[Respiration and oxidative phosphorylation in liver mitochondria of bass (Morone labrax) and their dependence on temperature]. / Respirazione e fosforilazione ossidativa in mitocondri epatici di branzino (Morone labrax) e loro diependenza dalla temperatura.

In the present paper the respiratory function in bass liver mitochondria is studied over the temperature range 6-34 degrees C. The respiratory rate in state 3 and state 4 at the temperatures examined agrees with data from poikiloterms reported elsewhere by other investigators ICR and ADP/O values with glutamate and succinate as substrates are considered and their variations as a function of the experimental temperature are discussed. ICR shows a maximum at 20 degrees C which approximatively corresponds with the temperature of the seawater where these fishes were bred. On the contrary ADP/O ratio does not show any meaningful variation.

[Arrhenius diagrams of respiratory enzymes of liver mitochondria from bass (Morone labrax)]. / Diagrammi di Arrhenius di alcuni enzimi respiratori di mitocondri epatici di branzino (Morone labrax).

Arrhenius plot of glutamate, succinate and ascorbate+TMPD oxidation in bass liver mitochondria show a break at different temperatures. Above the break activation energies (Ea) of the three enzymes. Above the break activation energies (Ea) of the three enzymes examined are similar and comparable with literature data in poikilotermic and homeothermic animals. Below the break the Ea are again comparable with poikiloterm and homeotherm ones except for succinate-oxidase whose Ea is surprisingly higher. The data are suggested to be due to the features of the enzymes or to the microenvironmental physical state.

[Interaction of n-alkanes with respiration and oxidative phosphorylation in rabbit heart mitochondria: n-hexane]. / Interazioni di n-alcani con la respirazione e fosforilazione ossidativa di mitocondri cardiaci di coniglio: n-esano.

n-Hexane on coupled rabbit heart mitochondria induces "in vitro" uncoupling with both glutamate and succinate as substrates and the effect increases with increasing n-alkane concentration (from 0 to 160 microgram/mg mitochondrial protein) and temperature (from 15 degrees to 38 degrees C). The inner mitochondrial membrane is made permeable to proteins; moreover extrusion of some matrix enzymes and entry of exogenous NADH is produced. Furthermore at higher concentrations and temperatures NADH oxidase inhibition and increase of its thermosensitivity is shown whereas upon succinate oxidase is evidenced a biphasic effect (activation followed by inhibition). The results, qualitatively similar to those observed with detergents and solvents, suggest a fluidization of the lipid phase of the membrane.

[Interaction of n-alkanes with respiration and oxidative phosphorylation in rabbit heart mitochondria: n-nonane]. / Interazioni di n-alcani con la respirazione e fosforilazione ossidativa di mitocondri cardiaci di coniglio: n-nonano.

Studies "in vitro" on the effect of n-nonane on coupled rabbit heart mitochondria with both succinate and glutamate as substrates show that the hydrocarbon examined makes the membrane permeable to protons (uncoupling), to some matrix enzymes and to exogenous NADH. The effect increases with increasing n-alkane concentration (from 0 to 160 microgram/mg mitochondrial protein) and temperature (from 15 degrees to 38 degrees C). Furthermore at higher concentrations and temperatures NADH oxidase inhibition is observed, whereas on succinate oxidase a biphasic effect (activation at lower concentrations and inhibition at higher concentrations) is produced. However the results, qualitatively similar to those observed with n-hexane, exhibit features probably due to a longer chain and that can be ascribed to perturbations of the physical state of membrane lipids.