Monovalent cation activated ouabain-insensitive ATPase in the gills of freshwater mussel Anodonta cygnea

Comp. Biochem. Physiol. Vol. 103B,No. 4, pp. 903-908, 1992 Printed in Great Britain

0305-0491/92$5.00+ 0.00 © 1992PergamonPress Ltd

MONOVALENT CATION ACTIVATED OUABAIN-INSENSITIVE ATPase IN THE GILLS OF FRESHWATER MUSSEL ANODONTA CYGNEA K. Y. H. LAGERSPETZ,*~N. B. PIVOVAROVAf'~and K. E. O. SENIUSt ~'Laboratory of Animal Physiology, Department of Biology, University of Turku, SF-20500 Turku, Finland (Tel. 358-21-63351; Fax 358-21-633-6590); and :~Instituteof Evolutionary Physiology and Biochemistry, Russian Academy of Sciences,194223 St Petersburg,Russia (Received 22 May 1992; accepted 26 June 1992)

Abstract--1. The ouabain-insensitive Na+-ATpese of gill microsomes of the freshwater mussel Anodonta cygnea is activated similarly also by K +, Li+, TI+, Rb +, Cs + and NH + . 2. Na+-ATPase also hydrolyses UTP and ADP, but with lower activity. 3. The Na+-ATPase activity is maximal at pH 6.5. 4. Na+-ATPase activity is inhibited by furosemide, but by ethacrynlc acid and vanadate only at high concentrations. 5. The temperature dependence of Na+-ATPase activity of Anodonta gill microsomes is linear between 5 and 50°C without the discontinuities usually observed in the function of this enzyme from other sources. 6. Cd2+, but not Ca2+, may substitute Mg2+ as the necessary cofactor. 7. No significant ouabain-sensitive (Na++ K+)-ATPase activity has been detected in Anodonta gill microsomes, but this and the relative unspecificityof Na+-ATPase do not at present allow for evolutionary conclusions.

INTRODUCTION The (Na + +K+)-ATPase is a membrane-bound, Mg2+-dependent enzyme, activated in the presence of both Na + and K +, and inhibited by ouahain. ( N a + + K + ) - A T P a s e has been identified with the transmembrane Na+/K + exchange mechanism (Skou, 1965), and it has been considered an enzyme ubiquitous in animal cells, and especially important for the functions of excitable cells. In addition, many animal tissues contain an ouabaln-insensitive, Mg2+dependent, membrane-bound ATPase, for the activation of which Na + alone is sufficient (Na+-ATPase; Proverbio et al., 1975; Moretti et al., 1991; Proverbio et al., 1991). Such an enzyme is found in the gills of many aquatic animals (Schoeffeniels, 1962; Lagerspetz and Senius, 1979; Borgatti et al., 1985; Howland and Fans, 1985; Ventrella et al., 1987, 1990; Pagliarani et al., 1988; Proverbio et al., 1990, 1991; Moretti et al., 1991). In some cases it has been shown to be activated also by other monovalent cations, and to be relatively sensitive to the inhibition by ethacrynic acid. The occurrence and the relative activities of (Na + + K +)-ATPase and Na+-ATPase especially in the kidneys and other excretory organs, and in the gills of various animals have been compared by Pagliarani et al. (1988), Moretti et al. (1991) and Proverbio et al. (1991). This has led to the hypothesis that the single monovalent cation activated ATPase (Na+-ATPase above) could have been replaced *To whom correspondence should be addreued. Abbrevlatlons--HEPES, N-2-hydroxyethyl-piperazine-N'2-ethanesulphonic acid; Tris, Tris(hydroxymethyl)amino-methane.

during the evolution gradually by the more specific (Na + + K +)-ATPase (Pagliarani et aL, 1988). The functions of Na+-ATPase in ion and water regulation are not known. Proverbio et al. (1988, 1991; Moretti et al., 1991) have shown that it may participate in the active regulation of cell volume by extruding monovalent cations together with CI- and water from the cell. We have earlier reported the occurrence of Na +ATPase activity in microsomal membranes prepared from the gills of the freshwater mussel Anodonta cygnea (Lagerspetz and Senins, 1979). The Na +ATPase was found to be ouabaln-insensitive, and it could be activated also by K + alone. We suggested a physiological role for that enzyme in the ion uptake of this animal. No significant ouahain-sensitive (Na + +K+)-ATPase activity was found in our preparations. We have now further characterized the Na +ATPase of Anodonta gills, and compared our results with data about monovalent cation activated ouabain-insensitive ATPases from other sources. The Anodonta gill enzyme seems to be relatively unspecific in its monovalent cation activation, in its sensitivity to inhibitors, and also in its dependence on divalent cations. MATERIALSAND METHODS The mussels (Anodonta cygnea; shell length 10-14 cm) were collected at several occasions from lakes in SW Finland and kept in aerated dechlorinated tap water at 8-10°C without feeding. We used their median gills, which do not contain developing giochidia. For ATPase assay, microsomes were prepared from median gill homogenates as described earlier (Senins and

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mM Fig. 1 (A, B). The relative activation of ATPase by different monovalent cations in gill microsomes. The activity values are given as percentages of the highest Na+-stimulation of ATPase activity observed in the same series of experiments. The control (basic Mg2+-ATPase) assay medium contained 4 mM MgCI2, 3 mM Tris--ATP and 50 mM HEPES (pH 7.2), and the experimental media the same plus the monovalent cation at the appropriate concentration. The differences between experimental and control activities are used as the measure of ATPase activation by monovalent cations. Means from 3-4 experiments. Lagerspetz, 1978; Pivovarova et al., 1992). The incubation medium contained, unless otherwise stated, 50 mM HEPES, 3 mM ATP as Tris salt, 4 mM MgC12, and the required monovalent cation chloride concentration. In experiments with thallium, TINO3 was used. The incubation temperature was usually 39°C, the incubation time 20min, and the reaction was started by adding the microsome suspension. The activity of the monovalent cation ATPase was determined (1) as the difference between the rates of ATP hydrolysis in the presence of the monovalent cation salt + 4 mM MgCI2 and of 4 mM MgC12 only, or (2) as the difference between the rates of ATP hydrolysis in the presence of 100 mM NaCI + 20 mM KC1 + I mM ouabain + 4 mM MgC12 and of 4 mM MgCI2 only. The ATPase activities were expressed as/~mol of liberated inorganic phosphate (P0 per mg of protein per hr. The inorganic phosphate liberated was determined by the method of Atkinson et aL (1973) and the protein content of the microsomai suspensions using the method of Lowry et aL (1951). When determining the temperature dependence of the monovalent cation induced ouabain-insensitive ATPase ac. tivity, a serial thermostate bath was used. This allowed the incubation of several sets of three tubes (blank, Mg 2+,

Mg 2+ + N a + +K+-ouabain) simultaneously at different temperatures. The actual temperature in the incubated tubes was measured with a thermocouple. The chemicals were purchased from Merck (Darmstadt), except HEPES, Tris, Tris-UTP, Tris-ADP, and the prospective enzyme inhibitors, which were from Sigma (St Louis). Student's t-test was used to assessed the statistical significance of the differences in the means, which are given with +SEM.

RESULTS

Figures IA and 1B show that a Mg:+-dependent ATPase was activated similarly by all monovalent cations tested (Na +, K +, Li +, T1 +, Rb +, Cs + and NH2- ), maximally at concentrations of about 50 raM, and a 50% activation was found at 10-20 raM. The ATPase activation by T1 + could not be studied at concentrations higher than 25 m M because of the precipitation of Tl-salts. The maximal ATPase activity found with Li + was about 20% lower than with other monovalent cations.

M o n o v a l e n t c a t i o n A T P a s e in mussels Table 1. Effect of some substrates on the ATPase activities in gill microsomes Substrate ATP UTP ATP ADP

Mg 2+

Table 2. Effects of enzyme inhibitors on the Na+-ATPase activity in gill microsomes

Pi produced ~mol/mg/prot/h) Na + + K + Na + + K + + ouabain

10.3+1.6 7.8 + 0.8 76% 9.4 + 0.6 3.4+0.5 36%

7.0 :[: 0.8 2.6 + 0.7 37% 5.7 + 0.4 1.9_+0.4 33%

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6.7+1.0 2.8 + 0.6 42% 5.6 + 0.3 2.1 _+0.6 37%

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All incubation media contained 4 mM MgCI2 and 3 mM of Tris-salt of the respective suhstrate in 50 mM histidine-HCl (pH 7.2). (Na + +K+)-ATPase media contained in addition 100raM NaCI and 20 raM KCI, the third medium also 1 mM ouabain. (Na + + K +)-ATpase activity is the difference between Mg 2+ + N a + + K+-induced and Mg2+-induced activity. Percentages of activity with the two other substrates are calculated from the activity with ATP as the substrate found in the same experiment. Means from 3 (ATP-UTP) and 4 (ATP-ADP) experiments.

The substrate specificity of monovalent cation stimulated nucleotidase and nucleosidase activity was studied using uridine triphosphate (UTP) and ADP as the substrates (Table 1). UTP as the substrate gave, in the presence of ouabain, a mean enzyme activity 42% of that found with ATP, and ADP 37% of that found with ATP. The results given in Table 1 also confirm that there is no significant ouabain-sensitive (Na++K+)-stimulated ATPase activity in the microsomal fraction. Figure 2 shows the pH dependence of the ouabaininsensitive Na+-activated ATPase in comparison with the basic Mge+-ATPase activity from the same experiments. Na+-ATPase activity is at its maximum at pH 6.5, and thus at a pH lower than pH 8 and above at which Mg 2+-ATPase is maximally activated. Table 2 shows the effects of some enzyme inhibitors on the activity of Na+-ATPase. Furosemide at a concentration of 2 mM inhibits about 66% of the Na+-ATPase activity. Ethacrynic acid at 5 mM and sodium orthovanadate at 1 mM (data not shown) do not affect the Na+-ATPase activity significantly.

The incubation media contained for basic Mg2+-ATPase 4raM MgCI2, 3 mM Tris-ATP and 50 mM HEPES (pH 7.2), and for Na+-ATPase in addition 100 mM NaCI. Na+-ATPase activity is the difference of ATP hydrolysis in these two media. The inhibitors have been added to both types of media, and their effects on Na+-ATPase are given. Means from 5-7 experiments.

These two substances even at concentrations as high as 10 mM do not cause complete inhibition of the enzyme activity (Table 2). Results of the experiments, in which the temperature dependence of ouabain-insensitive Na+-ATPase activity was studied, are given in Fig. 3. Between 5 and 50°C the Arrhenius plot is linear, and the apparent activation energy E, is about 13.5 kcal tool -~. We have also depicted the results obtained with the 10 gill microsome preparations from different animals separately, but no clear discontinuity points were found in these individual Arrhenius plots. DISCUSSION

Two types of Mg2+-dependent ATPase activity can be clearly detected in microsomal membrane preparations made from gills of Anodonta: the Mg 2+ATPase, which is also activated by C a 2+ (Senius and Lagerspetz, 1978) and possibly by Cd 2+ (Pivovarova et ai., 1992), and the Na+-ATPase, which is activated also by K + (Lagerspetz and Senius, 1979) and by several other monovalent cations, as shown in the present study (Fig. 1). These two ATPase activities are not affected by ouabain at the usually effective

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Temperot.ure (1000/T°K) Fig. 3. Arrhenius plot of the temperature dependence of Na+-ATPase activity in gill microsomes. The graph is based on experiments with preparations from 10 different animals. Each preparation has been assayed at 10 different temperatures (some points overlap). The regression line is that given by the method of least means squares. Na+-ATPase activity has been determined as the difference between ATP hydrolysis with 4raM Mg2+ + 100mM Na + + 20raM K + + 1 mM ouabain, and with 4mM Mg2+ only. The medium contained also 3 mM of Tris-ATP and 50 mM of histidine-HCl buffer adjusted to pH 7.2. concentrations. A Mg2+-dependent, ouabain-insensitive Na+-activated ATPase (Na+-ATPase) is found in the gills of many aquatic animals (see the references in the Introduction). The third common type of membrane-bound ATPase activity, ouabain-inhibited ( N a + + K + ) ATPase, is lacking in membrane preparations of Anodonta gills (Lagerspetz and Senius, 1979). This is confirmed in the present study (Table 1). The gills of Anodonta are in this respect similar to Mytilus gills (Howland and Faus, 1985) and to the tissues of the planarian Dugesia gonocephala (Venturini et al., 1981). However, as the gills and other tissues of another bivalve mollusc, the brackish-water clam Rangia cuneata show ouabain-sensitive (Na + + K + )ATPase activity (Saintsing and Towle, 1978), it appears premature to draw any evolutionary conclusions. Taking into account the about fourfold enrichment of Na+-ATPase activity from homogenates to microsomes (Lagerspetz and Senius, 1979), the level of specific enzyme activity (about 100 nmol Pi/mg protein/min; Table 1) is of the same order as that found by Moretti et al. (1991) in tissues from various animals. The enzyme activity at 20°C (Fig. 3) corresponds to that found in the gill cell membranes of the marine mussel Mytilus edulis (Howland and Faus, 1985) at that temperature. In addition to the lack of ( N a ÷ + K+)-ATPase activity, the main finding in this study concerns the relative unspecificity of the Na+-ATPase of Anodonta gills. The specificity of Na + as the activating cation has been studied in some cases. In the membrane preparations from the gills of Anodonta, K + was found to activate ATPase at about the same concentrations as Na +, maximally at about 100ram (Lagerspetz and Senius, 1979). We therefore called it Na +- or K+-ATPase. In membranes from sea mussel Mytilus edulis gills, Mg-dependent monovalent cation activation of ATPase was found with Na +, K +, Li +,

Rb + and NH~, and to a lesser extent with Cs + (Howland and Faus, 1985). In the sea bass (Dicentrarchus labrax) gill cell membranes K +, Li + and NH~ activated ATPase similarly as Na + (Ventrella et al., 1987). In addition to these, also Rb +, Cs +, and choline + activated ATPase in the microsomal membranes from the gills of the squid Doriteuthis plei (Proverbio et al., 1988) and of the gilthead bream Sparus auratus (Ventrella et al., 1990). In the gill homogenates from a crustacean, the shrimp Macrobrachium amazonicum, Na + activation of ATPase was higher than with other monovalent cations in the order K + > Rb + > Li +, while NH + and Cs + had no stimulating effect (Proverbio et al., 1990). All monovalent cations mentioned above, and TI +, have now been tested for their Na+-ATPase-like action in the gill microsomes of Anodonta. All these cations activate the so-called Na+ATP-ase to the same extent at 39°C, as well as T1+ at the concentrations used. The activating effect of Li + is smaller. Choline has a slight inhibitory effect (Lagerspetz and Senius, 1979, Table 1). Therefore, the Na+-ATPase of Anodonta gill cells seems to be a single monovalent cation activated ATPase. The substrate specificity of Na+-ATPase has been previously studied in the gill and kidney microsomes of two fish species and in the gill microsomes of two molluscs. In the sea bass (Dicentrarchus labrax) gill microsomes the activity was equal with CTP and ATP as the substrates, and 60% and 27% of that with UTP and GTP, respectively (Borgatti et al., 1985). The Na+-ATPase-type of hydrolysis of other nucleotides than ATP was less efficient (24-62%) in the kidney microsomes of the sea bass (Pagliarani et ai., 1988). In the gilthead bream (Sparus auratus) the Na +ATPase activity of gill microsomes was with CTP, GTP, and UTP 52-61% of that found with ATP as the substrate. The Na+-ATPase activity of kidney microsomes of the giltbead bream showed higher

Monovalent cation ATPase in mussels substrate specificity as CTP and UTP were not hydrolysed detectably (but GTP at a rate of 79% of that of ATP) (Trombetti et al., 1990). The fish [gill enzyme seems to be a less substrate specific nucleotidase (with some specificity for ATP) than the fish kidney enzyme. In microsomes prepared from the gills of the marine mussel Mytilus edulis, Howiand and Faus (1985) found the activity of Na+-ATPase to be similar with ATP and CTP as substrates, and about 80 and 67% of that with ITP (inosine triphosphate) and GTP, respectively. The Na+-ATPase activity in the gill microsomes of the squid Doritheutis plei is even less substrate specific, as the all five nucleotides mentioned above are hydrolysed at not significantly different rates, and ADP at a rate of about 40% of those (Proverbio et al., 1988). The Na+-ATPase of Anodonta gills is also relatively unspecific in regard to its substrate, as UTP and ADP are hydrolyzed with an enzyme activity of 42, respectively 37% of that observed in parallel experiments with ATP. This substrate unspecificity is typical for Na+-ATPase when compared with (Na + + K + )-ATPase. In previous studies, furosemide and ethacrynic acid have been shown to inhibit Na+-ATPase activity at 1-2 millimolar concentrations. The Anodonta enzyme is as sensitive to furosemide, but less sensitive to ethacrynic acid. It resembles Mytilus edu//s gill Na +ATPase (Howland and Faus, 1985) in its low sensitivity to vanadate, as compared with the gill and kidney microsomal enzymes of the gilthead bream (Ventrella et al., 1990; Trombetti et aL, 1990). Since the pioneering work of Skou (1957, 1965) Mg 2+ has been considered an obligatory cofactor for the (Na + +K+)-ATPase activation, and in later studies necessary also for Na+-ATPase activity. We have recently found that although Mg 2+ cannot be substituted by Ca 2+, Cd 2+ at concentrations from 0.2 to 1.5mM acts as a substitute for Mg 2+ in the activation of Na+-ATPase (Pivovarova et al., 1992, Table 1). This is an additional evidence of the unspecificity of the Anodonta gill enzyme. The activity of Mg2+-ATPase has its maximal values at the alkaline pH in gilthead bream gills (Ventrella et al., 1990) and in the gills of Anodonta (Fig. 2). In contrast to this, the maximal activity of Na+-ATPase is found at subneutral pH values in the gilthead bream gills and kidneys and the rainbow trout gills (Ventrella et al., 1990: Trombetti et al., 1990; Ventrella et al., 1992) and in the Anodonta gills. This enzyme could perhaps be involved in monovalent metal cation/proton exchange at gill cell membranes. The apparent activation energies (E,) of N a +A T P a s e have been determined by Borgatti et al. (1985), Pagliarani et al. (1988), Trombetti et al. (1990) and by Ventrella et al. (1990). They found a discontinuity in the Arrhenius plots at temperatures of 16.5-26.2°C. In our preparations, we did not find any break points in Arrhenius plots between 5 and 50°C. This may be due to differences in methods and needs further study. That applies also on the generally observed high temperature resistance of Na +ATPase, and to its less known kinetics at lower, ecological temperatures.

907

Acknowledgements--The authors wish to think Sirpa Lehti, Sinikka Hil~eu and Ari T'fiska for their help and the A~d,~ny of Finland and Turku University Foundation for support, especially for the visitor's grant to N. B. Pivovarova. REFERENCES Atkinson A., Cratenby A. D. and Lowe A. G. (1973) The determination of inorganic orthophmphete in biological systems. Biochim. Biophys. Acta 230, 195-204. Borgatti A. R., Trigari G., Pagiiarani, A. and Venttella V. (1985) Ouabain-insensitive Na + stimulation of a microsomal Mg2+-ATPase in [gills of sea bass (Dicentrarchus iabrax). Comp. Biochem. Physiol. 81A, 127-135. Howland J. L. and Faus I. (1985) Cation-sensitive ATPase from gills of the salt water mussel, Mytilus edulis. Comp. Biochem. Physiol. 81E, 551-553. Lagerspetz K. Y. H. and Senius K. E. O. (1979) ATPase stimulated by Na + or K + in gills of the freshwater mussel Anodonta. Comp. Biochem. Physiol. 67~ 291-293. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagents. J. biol. Chem. 193, 265-275. Moretti R., Martin M., Proverbio T., proverbio F. and Matin R. (1991) Ouabain-insensitive Na-ATPase activity in homogenates from different aninlal tissues. Comp. Biochem. Physiol. 98E, 623-626. Pagiiarani A., Ventrella V., Trombetti F., Trignri G. and Borgatti A. R. (1988) (Na + + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in kidney of sea bass (Dicentrarchus labrax L.). Comp. Biochem. Physiol. 90E, 41-52. Pagilarani A., Ventrella V., Ballestrazzi R., Trombetti F., Pirini M. and Trigari G. (1991) Salinity-dependence of the properties of gill (Na + + K+)-ATPase in rainbow trout (Oncorhynchus mykiss ). Comp. Biochem. Physiol. 1003, 229-236. Pivovarova N. B., Lagerspetz K. Y. H. and Sknlsk~ I. A. (1992) Effect of cadmium on ciliary and ATPase activity in the gillsof freshwater mussel Anodonta cygnea. Comp. Biochem. Physiol. C (in press). proverbio F., Condrescu-Guldi M. and Whittembury G. (1975) Ouabain-insensitive Na + stimulation ofa Mg2+-dependent ATPase in kidney tissue. Biochim. Biophys. Acta 394, 281-292. proverbio F., Duque J. A., proverbio T. and Matin R. (1988) Cell volume sensitive Na +-ATPase in rat kidney cortex cell membranes. Biochim. Biophys. Acta 941, 107-110. Proverbio F., Matin R. and Proverbio T. (1991) The ouabain-insensitive sodium pump. Comp. Biochem. Physiol. 99A, 279-283. Proverbio T., Matin R. and Proverbio F. (1988) Ouabaininsensitive, Na+-stimulated ATPase activity in squid gill microsomes. Comp. Biochem. Physiol. 90]$, 341-345. Proverbio T., Zanders I. P., Matin, R., Rodrignez J. M. and Proverbio F. (1990) Effects of Na + and/or K + on the Mg2+-dependent ATPe~ activities in shrimp (Macrobrachiom anmzonicum ) ~11 homogenates. Comp. Biochem. Physiol. 97B, 383-390. Saintsing D. G. and Towle D. W. (1978) Ha + + K+-ATPase in the osmoregnlating clam Rangio cuneata. J. exp. Zool. 435-442. Schoeffeniels E. (1962) Isolation of a sodium-dependent ATPase from the gills of Octopus vulgaris L. Life Sci. 1, 437--440. Senins K. E. O. and Lagerspetz K. Y. H. (1978) Effects of calcium and magnesium on the thermal resistance of ciliary activity in the fresh water mussel Anodonta. Y. therm. BioL 3, 153-157. Skou J. C. (1957) The influence of some cations on an adenosine triphosphatase from peripheral nerves. Biochim. Biophys. Acta 7,3, 394--401.

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Skou J. C. (1965) Enzymatic basis for active transport ofNa + and K + across cell membrane. Physiol. Rev. 45, 596-617. Trombetti F., Ventrella V., Pagiiarani A., Trigari G. and Borgatti A. R. (1990) Mg2+.dependent (Na + + K+)- and Na+-ATPases in the kidneys of the gilthead bream (Sparus auratus L.). Comp. Biochem. Physiol. 97B, 343-354. VentreUa V., Pagiiarani A., Trigari G., Trombetti F. and Borgatti A. R. (1987) Na+-like effect of monovalent cations in the stimulation of sea bass gill Mg2+-dependent Na+-stimulated ATPase. Comp. Biochem. Physiol. 88B, 691-695. Ventrella V., Trombetti F., Pagliarani A., Trigari G. and Borgatti A. R. (1990) Gill (Na + + K + )- and Na+-stimu-

lated Mg2+.dependent ATPase activities in the gilthead bream (Sparus auratus L.). Comp. Biochem. Physiol. 95B, 95-105. Ventrella V., Trombetti F., Pagiiarani A., Trigari G., Pirini M. and Borsatti A. R. (1992) Salinity dependence of the ouabain-insensitiveMg2+.dependent Na+-ATPase in gills of rainbow trout (Oncorhyncus mykiss Walbaum) adapted to fresh- and brackish water. Comp. Biochem. Physiol. 101B, 1-7. Venturini G., PaUadini G., Margotta V., Medolago-Albani, L., Carolei A. and Hernandez M. C. (1981) Ouabain insensitive ATPase in planaria. Comp. Biochem. Physiol. 70B, 775-778.