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ATPase stimulated by Na+ or K+ in gills of the freshwater mussel Anodonta
Comp. Biochem. Physiol., Vol. 6211, pp. 291-293
0305-0491/79/0301-0291502.00/0
© Pergamon Press Ltd 1979. Printed in Great Britain
A T P a s e S T I M U L A T E D BY N a ÷ O R K ÷ I N G I L L S OF THE FRESHWATER MUSSEL ANODONTA K. Y. H. LAOERSPETZ and K. E. O. SENIUS Zoophysiological Laboratory, Department of Biology, University of Turku, SF-20500 Turku, and Department of Biomedical Sciences, University of Tampere, Box 607, SF-33101 Tampere, Finland (Received 9 June 1978)
Abstract--1. Mierosomal preparations from the gills of the freshwater mussel Anodonta cygnea cellensis show Mg2+-dependent Na ÷- or K+-stimulated ATPase activity, which is not inhibited by ouabain. 2. Na +- or K+-ATPase activity is decreased by Ca 2+, acetylcholine, choline, and tetramethylammonium, but slightly increased by ethyl alcohol. 3. It is tentatively suggested that Na +- or K+-ATPase is involved in the mechanism of active monovalent cation uptake through the gills of freshwater mussels.
HC1 (pH 7.2). The total volume was 2 ml. After incubation, the inorganic phosphate liberated was determined by the method of Atkinson et al. (1973) and the protein content of the microsomal preparations using the method of Lowry et aL 0951) on trichloroacetic acid precipitates. The activity was subsequently calculated as/~mol of liberated inorganic phosphate/rag protein per hr. Mg2+-dependent Na+- or K+-stimulated ATPase activities were computed as the differences between enzyme activity in the presence of Na ÷, K + and Mg 2+ (medium 3) or Na + and Mg 2+ (medium 2) and in the presence of Mg 2+ alone (medium 1). ATPase reaction was linear for at least 120min at 39°C with 4raM Mg 2+ in the medium, while the additional activity found in the presence of Mg z+, Na + and/or K + was linear for at least 90 rain.
INTRODUCTION
The ciliated gills of bivalve molluscs serve both a feeding and a respiratory function. Picken (1937) and Krogh (1939) have suggested that an active uptake of ions occurs through the body surface of the freshwater mussels. It is likely that the gills by virtue of their large surface area are particularly involved in the ion uptake in these animals. The Na + transport has been associated with Na +- and K+-stimulated Mg2+-dependent ATPase activity found in the gills of fishes (Epstein et al., 1967; Pfeiler & Kirschner, 1972) and crustaceans (Quinn & Lane, 1966; Horiuchi, 1977). It has also been suggested, that Ca 2+- or Mg 2 +-activated ATPase has a role in the control of passive permeability to ions in the gills of Anodonta (Senius & Lagerspetz, 1978). It therefore seemed useful to investigate the stimulation of ATPase activity by Na + and K + in gill preparations. A short summary of some of these results has already been presented (Senius & Lagerspetz, 1976).
RESULTS
Effects o f Na +, K + and ouabain
In studies of the activity of Na +- and K+-stimu luted ATPase, the assay media typically contain, in addition to Mg 2+ at a low concentration, also about 100mM Na + and 2 0 m M K +. The effects of these cations and ouabain were investigated in the first series of experiments. The addition of 100mM Na + to an incubation medium already containing 4 r a M Mg 2+ increased the ATPase activity from 5.50 _+ 0.41/~mols inorganic phosphate liberated/rag protein per hr by around 9 3 ~ (means of experiments on 10 different microsomal preparations). The average activity of this Na+-acti vated ATPase component was thus 5.12 _ 0.38/~mol Piling protein per hr. A further addition of 20 m M K + in 16 experiments did not significantly alter the ATPase activity. The increase of the activity was in average only 0.8~o over the activity shown in the presence of 100raM Na + and 4 r a M Mg 2+ only. When the N a + + K+-ATPase inhibitor ouabain was added (12 experiments) at a concentration of 1 m M to the Na +, K + and Mg 2+ containing medium, the ATPase activity was decreased on average only by 1.5~o. This change was not statistically significant. Ouabain was also added in concentrations of 0.2 and
MATERIALS AND METHODS Freshwater mussels Anodonta cygnea cellensis with a shell length of 12-16 cm were collected from Lake Luolala near the city of Turku in southwest Finland and stored at 4°C, For microsomal preparations, one excised median gill of a mussel (about 3 g of tissue) was homogenized in 8 ml of ice-cold 50 mM histidine-HCl buffer (pH 7.2) containing 1 mM EDTA. Supernatant from an initial centrifugation of 10rain duration at 900g was further centrifuged for 10rain at 2000g, and the remaining supernatant again twice for 20 min at 12,500 g. Sediment from a final centrifugation of 1 hr duration at 107,000g was suspended by gentle stirring into 35 ml of 50mM histidine-HCl buffer (pH 7.2). All centrifugations were carried out in cold. The assay of ATPase activity was carried out in duplicate tubes containing 0.5 ml of the microsomal suspension, which were usually incubated for 20 min after temperature equilibration at 39°C. Generally three incubation media with differing cation compositions were used: ( l ) 4 r a m MgCl2, (2) 4 rnM MgCI 2 and 100 mM NaC1 and (3) 4 mM MgCl2, 100rnM NaCl and 20mM KC1. All media contained also 3 mM ATP as Tris salt and 50 mM histidineC.B.P. 62/3B---G
291
K. Y. H. LAGERSPETZ and K. E. O. SENIUS
292
°1o
°1o
lOO
lOO
50
50 Ill IIII 1111
• loo v so t, 2o
f//
I I I I
O 10
If
o
A loo o so
X 20
o
o
so
,oo
[Na
,so
+] m M
200
o
so
,oo
[K+] m M
Fig. 1. Effects of different concentrations of Na + and K + on the relative activity (%) of Na*- or K÷-stimulated ATPase in microsomal preparations from the gills of Anodonta. All incubation media contain also 4 m M Mg 2+, 3 mM Tris-ATP and 50mM histidine-HC1 (pH 7.2). Each point gives the average from three experiments. In each experiment, the highest activity found at any combination of Na + and K ÷ concentrations is represented by 100y,,, and the activity found with only Mg 2+ by 0~o.
5 m M in experiments with three microsomal preparations. Even these concentrations were inefffective. In a second set of three experiments, the depencence of the ATPase activity on the concentrations of N a ÷ and K ÷ was studied. The mean results of these three experiments are given in Fig. 1. Both N a ÷ and K ÷ were approximately equipotent activators of the ATPase, and a b o u t 80Yo of the m a x i m u m activity was reached at a total N a ÷ and/or K ÷ concentration of about 100mM. W h e n either N a ÷ or K ÷ were present at this concentration, the addition of the other alkali metal ion increased the ATPase activity maximally by a r o u n d 20Yo. This fraction of the ATPase activity therefore represented the activity specific for the N a ÷- and K÷-stimulated ATPase in these preparations. Table 1. Effects of Ca 2+ and some compounds on the activity of Na ÷- or K÷-stimulated ATPase in microsomal preparations from gills of Anodonta
Compound
ConcenRelative tration Na ÷- or K ÷(mM) ATPase activity
Ca 2÷ 2 Acetylcholine 1 Acetylcholine 5 Choline 5 Tetramethylammonium 5 Ethyl alcohol 100 Ethyl alcohol 1000
56 107 82 84 81 105 114
+ 4 + 6 + 6 + 4 + 3 +_ 2 + 6
No. of exp. 4 4 4 5 5 4 3
Results are expressed as percentages of control values (in average 4.95 + 0.30/~mol inorganic phosphate liberated/ mg protein per hr). Percentage values have been calculated by comparison with paired controls in the same series of determinations. Numbers of experiments with different preparations are given.
In three further experiments, the Na+-stimulated ATPase activity was assayed in the supernatants after centrifugation of the homogenate at 900 g. The mean activity of N a + - A T P a s e was 1.40 + 0.08/~mol Pi/mg protein per hr. The mean activity in the microsomal preparations from the same homogenates was on average 6.52 + 0.45/tmol PJmg protein per hr, i.e. about 4-fold,
Effects of Ca 2+, cholinergic agents and ethyl alcohol The effects of Ca 2+, acetylcholine, choline, tetramethylammonium, all added as chlorides, and ethyl alcohol, on the activity of N a + - A T P a s e in gill microsomes are shown in Table 1. Additions of 2 m M Ca 2 + and cholinergic substances at concentrations of 5 m M caused decreases in the Na÷-stimulated ATPase activity, while ethyl alcohol at a concentration of 1 M somewhat increased it. DISCUSSION The ATPase activity of microsomal preparations made from the gills of the fresh water mussel Anodonta was found to be dependent on Mg 2÷. The addition of Na ÷ and/or K ÷ to the incubation medium increased the ATPase activity by about 90yo. This fraction of the ATPase activity was not much affected either by additions of the other alkali metal ion, or by ouabain. There was thus a strong either N a ÷- or K+-activation of ATPase in the microsomal preparations, while under the assay conditions used there was much less ATPase activity stimulated by Na ÷ and K ÷ in combination. Na÷-activated, ouabaininsensitive ATPase has earlier been found in gill microsomes from Octopus vulgaris (Schoffeniels, 1962) and freshwater rainbow trout (Salmo gairdneri) (Pfeiler & Kirschner, 1972). The possible activation of the ATPase by K ÷ alone has not been studied
Na ÷- or K+-ATPase in the gills of Anodonta in these animals. The Na + activation of the gill ATPase is smaller in the freshwater smalimouth bass (Micropterus dolomieui), and depends on a pretreatment of the microsomes at high temperatures (Pfeiler, 1976). The Na+-ATPase activity in gill homogenates from the semiterrestrial crab Cardisoma #uanhumi was about one third of the maximal ATPase activity found in the presence of both Na + and K + (Quinn & Lane, 1966). Na+-ATPase activity could not be demonstrated in the gill preparations from the seawater adapted rainbow trout (Pfeiler & Kirschner, 1972) or from the freshwater crayfish Procambarus clarki (Horiuchi, 1977), although both these animals show Na +- and K +-stimulated ATPase activity. In summary, the ATPase systems present in the gills of aquatic animals show variations, which are not simply related to the marine or fresh water origin of the animals. However, it seems reasonable to suggest that the Na +- or K+-stimulated ATPase in the gills of Anodonta described here is involved in the mechanism of active ion uptake. The ATPase activity induced in this study by Mg 2+ can also apparently be induced by equivalent concentrations of Ca 2+. One may thus properly consider it as a Ca 2+- or Mg2+-activated ATPase (Senius & Lagerspetz, 1978). In experiments ran simultaneously with the present ones, it was found that Ca 2 + and the cholinergic substances acetylcholine, choline and tetramethylammonium significantly increased the activity of the Ca 2+- or MgZ+-activated ATPase, while ethyl alcohol decreased such activity. The effects of these substances on the Na +- or K+-stimulated ATPase activity were the reverse of these. In our previous study, we suggested that Ca 2 +- or Mg 2 +-activated ATPase could be involved in the control of the passive permeability of the cell membranes of the gill epithelium to ions and other substances (Senius & Lagerspetz, 1978). In that case, the Ca 2+or Mg2+-ATPase would probably be situated as an ecto-ATPase on the outer surface of the cell membrane, as suggested for the vertebrate brain Ca 2+or Mg2+-ATPase by Trams & Lauter 0978). The reverse effects of the substances studied on the Na +or K+-ATPase in microsomes prepared from Anodonta gills would then depend on the limitation or enhancement of the access of Na + and K + to the enzymic sites of the Na +- or K+-ATPase. The activation of the Mg 2 +- or Ca 2 +-ATPase could limit the availability of Na + and K + for Na+-ATPase by increasing the outer permeability barrier and/or by in-
293
creasing the microviscosity of the membrane. Further studies are needed to elucidate the relations between the different types of ATPase activity found in the gills of bivalve molluscs and ion transport.
Acknowledgements--The authors wish to thank Mrs Erika Holmbom for her skilful assistance, Mrs Sinikka Hillgren for the drawing of the figure, and the Academy of Finland for financial support.
REFERENCES
ATKINSON A., GATENBYA. D. & LOWE A. G. (1973) The determination of inorganic orthophosphate in biological systems. Biochim. biophys. Acta 320, 195-204. EPSTEIN F. H., KATZ A. I. & PICKFORD G. E. (1967) Sodium- and potassium-activated adenosine triphosphatase of gills: role in adaptation of teleosts to salt water. Science, N.Y. 156, 1245-1247. HORIUCHI S. (1977) Characterization of gill Na, K-ATPase in the freshwater crayfish, Procambarus clarki (Girard). Comp. Biochem. Physiol. 56B, 135-138. KROGH A. (1939) Osmotic Regulation in Aquatic Animals. Cambridge University Press, Cambridge. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagents. J. biol. Chem. 193, 265-275. PFEILER E. (1976) Gill ATPase activities in the smallmouth bass (Micropterus dolomieui). Comp. Biochem. Physiol. 53B, 119-121. PFEILER E. & KIRSCHNER L. B. (1972) Studies on gill ATPase of rainbow trout (Sahno gairdneri). Biochim. biophys. Acta 282, 301-310. PIC~EN L. E. R. (1937) The mechanism of urine formation in invertebrates: II. The excretory mechanism in certain Mollusca. J. exp. Biol. 14, 20-37. QUINN D. J. & LANE C. E. (1966) Ionic regulation and Na,K-stimulated ATPase activity in the land crab, Cardisoma guanhumi. Comp. Biochem. Physiol. 19, 533-543. SCHOFFENIELSE. (1962) Isolation of a sodium-dependent ATPase from the gills of Octopus vulgaris L. Life Sci. 1, 437-440. SENIUS K. E. O. & LAGERSPETZK. Y. H. (1976) Functions of microsomal ATPases in the ciliated gills of fresh-water mussels. Acta physiol, scand., Suppl. 440, 73. SENIUS K. E. O. & LAGERSPETZK. Y. H. (1978) Effects of calcium and magnesium on the thermal resistance of ciliary activity in the fresh water mussel Anodonta. J. Thermal Biol. 3, 153-157. TRAMS E. G. & LAUTERC. J. (1978) A comparative study of brain Ca 2 +-ATPases. Comp. Biochem. Physiol. 59B, 191-194.
0305-0491/79/0301-0291502.00/0
© Pergamon Press Ltd 1979. Printed in Great Britain
A T P a s e S T I M U L A T E D BY N a ÷ O R K ÷ I N G I L L S OF THE FRESHWATER MUSSEL ANODONTA K. Y. H. LAOERSPETZ and K. E. O. SENIUS Zoophysiological Laboratory, Department of Biology, University of Turku, SF-20500 Turku, and Department of Biomedical Sciences, University of Tampere, Box 607, SF-33101 Tampere, Finland (Received 9 June 1978)
Abstract--1. Mierosomal preparations from the gills of the freshwater mussel Anodonta cygnea cellensis show Mg2+-dependent Na ÷- or K+-stimulated ATPase activity, which is not inhibited by ouabain. 2. Na +- or K+-ATPase activity is decreased by Ca 2+, acetylcholine, choline, and tetramethylammonium, but slightly increased by ethyl alcohol. 3. It is tentatively suggested that Na +- or K+-ATPase is involved in the mechanism of active monovalent cation uptake through the gills of freshwater mussels.
HC1 (pH 7.2). The total volume was 2 ml. After incubation, the inorganic phosphate liberated was determined by the method of Atkinson et al. (1973) and the protein content of the microsomal preparations using the method of Lowry et aL 0951) on trichloroacetic acid precipitates. The activity was subsequently calculated as/~mol of liberated inorganic phosphate/rag protein per hr. Mg2+-dependent Na+- or K+-stimulated ATPase activities were computed as the differences between enzyme activity in the presence of Na ÷, K + and Mg 2+ (medium 3) or Na + and Mg 2+ (medium 2) and in the presence of Mg 2+ alone (medium 1). ATPase reaction was linear for at least 120min at 39°C with 4raM Mg 2+ in the medium, while the additional activity found in the presence of Mg z+, Na + and/or K + was linear for at least 90 rain.
INTRODUCTION
The ciliated gills of bivalve molluscs serve both a feeding and a respiratory function. Picken (1937) and Krogh (1939) have suggested that an active uptake of ions occurs through the body surface of the freshwater mussels. It is likely that the gills by virtue of their large surface area are particularly involved in the ion uptake in these animals. The Na + transport has been associated with Na +- and K+-stimulated Mg2+-dependent ATPase activity found in the gills of fishes (Epstein et al., 1967; Pfeiler & Kirschner, 1972) and crustaceans (Quinn & Lane, 1966; Horiuchi, 1977). It has also been suggested, that Ca 2+- or Mg 2 +-activated ATPase has a role in the control of passive permeability to ions in the gills of Anodonta (Senius & Lagerspetz, 1978). It therefore seemed useful to investigate the stimulation of ATPase activity by Na + and K + in gill preparations. A short summary of some of these results has already been presented (Senius & Lagerspetz, 1976).
RESULTS
Effects o f Na +, K + and ouabain
In studies of the activity of Na +- and K+-stimu luted ATPase, the assay media typically contain, in addition to Mg 2+ at a low concentration, also about 100mM Na + and 2 0 m M K +. The effects of these cations and ouabain were investigated in the first series of experiments. The addition of 100mM Na + to an incubation medium already containing 4 r a M Mg 2+ increased the ATPase activity from 5.50 _+ 0.41/~mols inorganic phosphate liberated/rag protein per hr by around 9 3 ~ (means of experiments on 10 different microsomal preparations). The average activity of this Na+-acti vated ATPase component was thus 5.12 _ 0.38/~mol Piling protein per hr. A further addition of 20 m M K + in 16 experiments did not significantly alter the ATPase activity. The increase of the activity was in average only 0.8~o over the activity shown in the presence of 100raM Na + and 4 r a M Mg 2+ only. When the N a + + K+-ATPase inhibitor ouabain was added (12 experiments) at a concentration of 1 m M to the Na +, K + and Mg 2+ containing medium, the ATPase activity was decreased on average only by 1.5~o. This change was not statistically significant. Ouabain was also added in concentrations of 0.2 and
MATERIALS AND METHODS Freshwater mussels Anodonta cygnea cellensis with a shell length of 12-16 cm were collected from Lake Luolala near the city of Turku in southwest Finland and stored at 4°C, For microsomal preparations, one excised median gill of a mussel (about 3 g of tissue) was homogenized in 8 ml of ice-cold 50 mM histidine-HCl buffer (pH 7.2) containing 1 mM EDTA. Supernatant from an initial centrifugation of 10rain duration at 900g was further centrifuged for 10rain at 2000g, and the remaining supernatant again twice for 20 min at 12,500 g. Sediment from a final centrifugation of 1 hr duration at 107,000g was suspended by gentle stirring into 35 ml of 50mM histidine-HCl buffer (pH 7.2). All centrifugations were carried out in cold. The assay of ATPase activity was carried out in duplicate tubes containing 0.5 ml of the microsomal suspension, which were usually incubated for 20 min after temperature equilibration at 39°C. Generally three incubation media with differing cation compositions were used: ( l ) 4 r a m MgCl2, (2) 4 rnM MgCI 2 and 100 mM NaC1 and (3) 4 mM MgCl2, 100rnM NaCl and 20mM KC1. All media contained also 3 mM ATP as Tris salt and 50 mM histidineC.B.P. 62/3B---G
291
K. Y. H. LAGERSPETZ and K. E. O. SENIUS
292
°1o
°1o
lOO
lOO
50
50 Ill IIII 1111
• loo v so t, 2o
f//
I I I I
O 10
If
o
A loo o so
X 20
o
o
so
,oo
[Na
,so
+] m M
200
o
so
,oo
[K+] m M
Fig. 1. Effects of different concentrations of Na + and K + on the relative activity (%) of Na*- or K÷-stimulated ATPase in microsomal preparations from the gills of Anodonta. All incubation media contain also 4 m M Mg 2+, 3 mM Tris-ATP and 50mM histidine-HC1 (pH 7.2). Each point gives the average from three experiments. In each experiment, the highest activity found at any combination of Na + and K ÷ concentrations is represented by 100y,,, and the activity found with only Mg 2+ by 0~o.
5 m M in experiments with three microsomal preparations. Even these concentrations were inefffective. In a second set of three experiments, the depencence of the ATPase activity on the concentrations of N a ÷ and K ÷ was studied. The mean results of these three experiments are given in Fig. 1. Both N a ÷ and K ÷ were approximately equipotent activators of the ATPase, and a b o u t 80Yo of the m a x i m u m activity was reached at a total N a ÷ and/or K ÷ concentration of about 100mM. W h e n either N a ÷ or K ÷ were present at this concentration, the addition of the other alkali metal ion increased the ATPase activity maximally by a r o u n d 20Yo. This fraction of the ATPase activity therefore represented the activity specific for the N a ÷- and K÷-stimulated ATPase in these preparations. Table 1. Effects of Ca 2+ and some compounds on the activity of Na ÷- or K÷-stimulated ATPase in microsomal preparations from gills of Anodonta
Compound
ConcenRelative tration Na ÷- or K ÷(mM) ATPase activity
Ca 2÷ 2 Acetylcholine 1 Acetylcholine 5 Choline 5 Tetramethylammonium 5 Ethyl alcohol 100 Ethyl alcohol 1000
56 107 82 84 81 105 114
+ 4 + 6 + 6 + 4 + 3 +_ 2 + 6
No. of exp. 4 4 4 5 5 4 3
Results are expressed as percentages of control values (in average 4.95 + 0.30/~mol inorganic phosphate liberated/ mg protein per hr). Percentage values have been calculated by comparison with paired controls in the same series of determinations. Numbers of experiments with different preparations are given.
In three further experiments, the Na+-stimulated ATPase activity was assayed in the supernatants after centrifugation of the homogenate at 900 g. The mean activity of N a + - A T P a s e was 1.40 + 0.08/~mol Pi/mg protein per hr. The mean activity in the microsomal preparations from the same homogenates was on average 6.52 + 0.45/tmol PJmg protein per hr, i.e. about 4-fold,
Effects of Ca 2+, cholinergic agents and ethyl alcohol The effects of Ca 2+, acetylcholine, choline, tetramethylammonium, all added as chlorides, and ethyl alcohol, on the activity of N a + - A T P a s e in gill microsomes are shown in Table 1. Additions of 2 m M Ca 2 + and cholinergic substances at concentrations of 5 m M caused decreases in the Na÷-stimulated ATPase activity, while ethyl alcohol at a concentration of 1 M somewhat increased it. DISCUSSION The ATPase activity of microsomal preparations made from the gills of the fresh water mussel Anodonta was found to be dependent on Mg 2÷. The addition of Na ÷ and/or K ÷ to the incubation medium increased the ATPase activity by about 90yo. This fraction of the ATPase activity was not much affected either by additions of the other alkali metal ion, or by ouabain. There was thus a strong either N a ÷- or K+-activation of ATPase in the microsomal preparations, while under the assay conditions used there was much less ATPase activity stimulated by Na ÷ and K ÷ in combination. Na÷-activated, ouabaininsensitive ATPase has earlier been found in gill microsomes from Octopus vulgaris (Schoffeniels, 1962) and freshwater rainbow trout (Salmo gairdneri) (Pfeiler & Kirschner, 1972). The possible activation of the ATPase by K ÷ alone has not been studied
Na ÷- or K+-ATPase in the gills of Anodonta in these animals. The Na + activation of the gill ATPase is smaller in the freshwater smalimouth bass (Micropterus dolomieui), and depends on a pretreatment of the microsomes at high temperatures (Pfeiler, 1976). The Na+-ATPase activity in gill homogenates from the semiterrestrial crab Cardisoma #uanhumi was about one third of the maximal ATPase activity found in the presence of both Na + and K + (Quinn & Lane, 1966). Na+-ATPase activity could not be demonstrated in the gill preparations from the seawater adapted rainbow trout (Pfeiler & Kirschner, 1972) or from the freshwater crayfish Procambarus clarki (Horiuchi, 1977), although both these animals show Na +- and K +-stimulated ATPase activity. In summary, the ATPase systems present in the gills of aquatic animals show variations, which are not simply related to the marine or fresh water origin of the animals. However, it seems reasonable to suggest that the Na +- or K+-stimulated ATPase in the gills of Anodonta described here is involved in the mechanism of active ion uptake. The ATPase activity induced in this study by Mg 2+ can also apparently be induced by equivalent concentrations of Ca 2+. One may thus properly consider it as a Ca 2+- or Mg2+-activated ATPase (Senius & Lagerspetz, 1978). In experiments ran simultaneously with the present ones, it was found that Ca 2 + and the cholinergic substances acetylcholine, choline and tetramethylammonium significantly increased the activity of the Ca 2+- or MgZ+-activated ATPase, while ethyl alcohol decreased such activity. The effects of these substances on the Na +- or K+-stimulated ATPase activity were the reverse of these. In our previous study, we suggested that Ca 2 +- or Mg 2 +-activated ATPase could be involved in the control of the passive permeability of the cell membranes of the gill epithelium to ions and other substances (Senius & Lagerspetz, 1978). In that case, the Ca 2+or Mg2+-ATPase would probably be situated as an ecto-ATPase on the outer surface of the cell membrane, as suggested for the vertebrate brain Ca 2+or Mg2+-ATPase by Trams & Lauter 0978). The reverse effects of the substances studied on the Na +or K+-ATPase in microsomes prepared from Anodonta gills would then depend on the limitation or enhancement of the access of Na + and K + to the enzymic sites of the Na +- or K+-ATPase. The activation of the Mg 2 +- or Ca 2 +-ATPase could limit the availability of Na + and K + for Na+-ATPase by increasing the outer permeability barrier and/or by in-
293
creasing the microviscosity of the membrane. Further studies are needed to elucidate the relations between the different types of ATPase activity found in the gills of bivalve molluscs and ion transport.
Acknowledgements--The authors wish to thank Mrs Erika Holmbom for her skilful assistance, Mrs Sinikka Hillgren for the drawing of the figure, and the Academy of Finland for financial support.
REFERENCES
ATKINSON A., GATENBYA. D. & LOWE A. G. (1973) The determination of inorganic orthophosphate in biological systems. Biochim. biophys. Acta 320, 195-204. EPSTEIN F. H., KATZ A. I. & PICKFORD G. E. (1967) Sodium- and potassium-activated adenosine triphosphatase of gills: role in adaptation of teleosts to salt water. Science, N.Y. 156, 1245-1247. HORIUCHI S. (1977) Characterization of gill Na, K-ATPase in the freshwater crayfish, Procambarus clarki (Girard). Comp. Biochem. Physiol. 56B, 135-138. KROGH A. (1939) Osmotic Regulation in Aquatic Animals. Cambridge University Press, Cambridge. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagents. J. biol. Chem. 193, 265-275. PFEILER E. (1976) Gill ATPase activities in the smallmouth bass (Micropterus dolomieui). Comp. Biochem. Physiol. 53B, 119-121. PFEILER E. & KIRSCHNER L. B. (1972) Studies on gill ATPase of rainbow trout (Sahno gairdneri). Biochim. biophys. Acta 282, 301-310. PIC~EN L. E. R. (1937) The mechanism of urine formation in invertebrates: II. The excretory mechanism in certain Mollusca. J. exp. Biol. 14, 20-37. QUINN D. J. & LANE C. E. (1966) Ionic regulation and Na,K-stimulated ATPase activity in the land crab, Cardisoma guanhumi. Comp. Biochem. Physiol. 19, 533-543. SCHOFFENIELSE. (1962) Isolation of a sodium-dependent ATPase from the gills of Octopus vulgaris L. Life Sci. 1, 437-440. SENIUS K. E. O. & LAGERSPETZK. Y. H. (1976) Functions of microsomal ATPases in the ciliated gills of fresh-water mussels. Acta physiol, scand., Suppl. 440, 73. SENIUS K. E. O. & LAGERSPETZK. Y. H. (1978) Effects of calcium and magnesium on the thermal resistance of ciliary activity in the fresh water mussel Anodonta. J. Thermal Biol. 3, 153-157. TRAMS E. G. & LAUTERC. J. (1978) A comparative study of brain Ca 2 +-ATPases. Comp. Biochem. Physiol. 59B, 191-194.