Alessandra Pagliarani

Salinity-dependence of the properties of gill (Na++K+-ATPase in rainbow trout Oncorhynchus mykiss

Abstract 1. 1. The expected higher gill (Na + +K + )-ATPase activity in rainbow trout adapted to brackish water (BW) with respect to fresh water (FW) is accompanied by some changes in the enzyme kinetics while the enzyme sensitivity to ouabain is unaffected 2. 2. Maximal activation is attained under the optimal conditions of 4 mM ATP, 7.5 mM Mg 2+ , 50 mM Na + , 2.5 mM K + , pH 7.0 in FW, and 3 mM ATP, 10 mM Mg 2+ , 100 mM Na + , 10 mM K + , pH 7.5 in BW. 3. 3. The change of the enzyme activation kinetics by Mg 2+ , ATP, Na + and K + from simple saturation in FW to cooperativity in BW and other habitat-dependent variations including the pH alkaline shift in BW are hypothetically related to an adaptive significance to the different environmental salinity. 4. 4. Gill total lipids and phospholipids are 30% lower in BW than in FW while their ratio is constant; some differences in gill total lipid fatty acid composition between FW and BW do not significantly affect the unsaturation parameters.

Toxicity of organotin compounds: Shared and unshared biochemical targets and mechanisms in animal cells

Most biochemical effects of organotin compounds leading to toxicity are astonishingly similar in different animal species. In vitro tests, designed to explore organotin action modes at cell level by minimizing interfering factors, point out akin responses to these man-made environmental pollutants from prokaryotes to mammals. On the other hand, a broad susceptibility range to organotin toxicants of animal cells and variegated action mechanisms of these compounds have been reported both in vitro and in vivo studies. Endocrine and lipid homeostasis perturbations span from mollusks to mammals, in which organotins mainly favor fat accumulation. Lipid changes were also found in Bacteria. Organotin are immunotoxic both in invertebrates and humans. Mitochondria and membrane functions seem to be a preferred target of these lipophilic pollutants. The inhibition of key membrane-bound enzyme complexes such as Na,K-and F0F1-ATPases, accompanied by perturbation of hydromineral balance, membrane potential and bioenergetics, has been widely reported. Highly conserved mechanisms could be involved in organotin binding to nuclear receptors, membrane components and intracellular proteins as well as in promoting DNA damage, all widely shared action modes of these toxicants. Accordingly, the different responsiveness/refractoriness to organotins, here overviewed, may mirror the biochemical-physiological selectivity of biomembranes, signalling pathways and intracellular protein components.

Ouabain-insensitive Na+ stimulation of a microsomal Mg+-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.

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+)-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.

Tributyltin (TBT) and mitochondrial respiration in mussel digestive gland

The toxicity of organotins and especially tri-n-butyltin (TBT) on mitochondria is well known. However as far as we are aware, effects on mitochondrial respiration are unexplored in mollusks. In this work mitochondria isolated from the digestive gland of Mytilus galloprovincialis and susceptive to the classical respiratory chain inhibitors, were assayed in the presence of micromolar TBT concentrations to investigate mitochondrial respiratory activities. Intact and freeze-thawed mitochondria were used. TBT significantly inhibited oxygen consumption in the presence of glutamate/malate or succinate as substrates. Conversely cytochrome c oxidase activity (complex IV), assayed both polarographically and spectrophotometrically, was unaffected. The addition of 1,4-dithioerythritol (DTE) decreased the TBT-driven inhibition of complexes I and III. The TBT capability of covalent binding to thiol groups of mitochondrial proteins in a dose-dependent manner was confirmed by the aid of Ellmans reagent. Data strongly suggests that TBT may prevent the electron transfer from complexes I and III to downhill respiratory chain complexes by binding to critical SH residues.

Tributyltin (TBT) inhibition of oligomycin-sensitive Mg-ATPase activity in mussel mitochondria

Tributyltin (TBT), one of the most toxic lipophilic aquatic pollutants, can be efficiently incorporated from sea water and sediments by filter-feeding molluscs. As far as we are aware TBT effects on the mitochondrial oligomycin-sensitive Mg-ATPase, the enzymatic core of energy production and a known target of TBT toxicity in mammals, have not been yet investigated in molluscs; thus the hydrolytic capability of the mitochondrial complex in the presence of micromolar concentrations of TBT was assayed in the mussel Mytilus galloprovincialis. Gill and mantle ATPase activities were progressively depressed by increasing TBT doses up to a maximal inhibition (82% in the gills and 74% in the mantle) at 0.62 microM TBT. Non-cooperative inhibition kinetics (n(H) approximately -1) and a non-competitive mechanism with respect to ATP substrate were pointed out. The mitochondrial Mg-ATPase susceptivity to TBT in the marine mussel was consistent with the formation of a TBT-Mg-ATPase complex, apparently more stable in the gills than in the mantle. The complex shape of the dose-response curve and the partial release of Mg-ATPase inhibition within the 0.6-34.4 microM TBT range suggest multiple interactions of TBT with the enzyme complex putatively related to its molecular mechanism of toxicity.

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 ATPases presumptive physiological role(s) and mutual relationship.

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.

Mussel microsomal Na+-Mg2+-ATPase sensitivity to waterborne mercury, zinc and ammonia

Abstract 1. 1. In Mytilus galloprovincialis Lam. both the Na + + K + )- and the Na + -ATPases show a tissue-and dose-dependent response to mercury (0.1 and 0.25 mg · L −1 Hg 2+ ), zinc (0.5 and 1.0 mg · L −1 Zn 2+ ) and ammonia (0.4 and 1.0 mg · L −1 ammonia-N). 2. 2. The two ATPases were generally more susceptive to the toxicants in the gills than in the mantle; in the case of heavy metals this finding complies with the higher metal incorporation in the gills with respect to the mantle. 3. 3. In the gills the two ATPase inhibition by mercury was enhanced by the co-presence of environmental 0.4 mg · L −1 ammonia-N; zinc removed the ( Na + + K + )-ATPase inhibition by 0.1 mg · L −1 Hg 2+ as well as the enzyme activation promoted by 0.4 mg · L −1 ammonia-N; both ATPases were inhibited by the 1.0 mg · L −1 ammonia-N treatment. 4. 4. Mantle ATPases were apparently refractory to mercury but not to zinc: the ( Na + + K + )-ATPase was similarly stimulated by the exposure to 1.0 mg · L −1 Zn 2+ and to the mixture Zn 2+ + Hg 2+ and unaffected by 0.5 mg · L −1 Zn 2+ alone; the Na + -ATPase was depressed by the exposures to 1.0 mg · L −1 ammonia-N and to the mixture Zn 2+ + ammonia-N and conversely stimulated by Hg 2+ + ammonia-N, whereas all other treatments were uneffective. 5. 5. In vitro approaches pointed out a susceptivity to the toxicants only for the (Na + + K + )-ATPase. The enzyme response in vitro often differed from that in vivo .