- Email: [email protected]
Effects of calcium and magnesium on the thermal resistance of ciliary activity in the fresh water mussel Anodonta
J. Thermal Biology, Vol. 3, pp. 153 to 157 Pergamon Press Ltd 1978. Printed in Great Britain
0306-4565/78,0701-0153S02.00 0
EFFECTS OF CALCIUM AND MAGNESIUM ON T H E T H E R M A L RESISTANCE O F CILIARY ACTIVITY IN THE F R E S H WATER MUSSEL A N O D O N T A K. E. O. SENIUS AND K. Y. H. LAGERSPETZ Department of Biomedical Sciences, University of Tampere, Box 607, SF-33101 Tampere, and Zoophysiological Laboratory, Department of Biology, University of Turku, SF-20500 Turku, Finland
(Received 12 October 1977; accepted in revised form 23 November 1977) Abstract--l. External Ca 2+ and Mg 2+ at concentrations below about 4 m M increase the thermal resistance of the activity of frontal cilia in the gills of Anodonta cygnea cellensis. 2. High external concentrations of Ca 2+ and Mg 2+, and the divalent cation ionophore A23187 in the presence of external Ca 2 + or Mg 2 +, counteract this effect. 3. The thermal resistance is increased by acetylcholine, choline, and tetramethylammonium, and decreased by ethyl alcohol. 4. Microsomal preparations from the gills show Ca 2+- or Mg2+-activated ATPase activity which is stimulated by acetylcholine, choline, and tetramethylammonium, and inhibited by ethyl alcohol. 5. It is tentatively suggested that Ca 2+- or Mg2+-activated ATPase participates in the control of membrane permeability, and also indirectly in the control of the thermal resistance of ciliary activity.
INTRODUCTION WE HAVE earlier studied the effects of temperature acclimation and of some cholinergic substances on the thermal resistance of the activity of frontal cilia in gills of the fresh water mussels Anodonta cygnea cellensis and A. anatina anatina and of the marine mussel Mytilus edulis (Lagerspetz & Dubitscher, 1966; Lagerspetz et al., 1970; Senius & Lagerspetz, 1974; Senius, 1975a, 1975b, 1977). The thermal resistance of the ciliary activity is increased in all three species by warm-acclimation of intact animals; acetylcholine (ACh), choline, and t e t r a m e t h y l a m m o n i u m (TMA) increase the thermal resistance of cilia in the two species of Anodonta, but not in Mytilus. Calcium and magnesium ions are usually considered to increase the stability of cell membranes. W h e n externally applied, they increase the thermal resistance of ciliary activity in the gills of Mytilus (Schlieper & Kowalski, 1956). O n the other hand, the entry of Ca 2 + into the cells causes arrest of the lateral cilia in the gills of Mytilus ( M u r a k a m i & Takahashi, 1975) and of the fresh water mussel Elliptio (Satir, 1975), as well as the reversal of the direction of ciliary beat in Paramecium (Eckert, 1972; N a i t o h & Kaneko, 1973). It is possible that the thermal resistance of the ciliary activity is, in part, controlled by the relative distribution of Ca 2+, and possibly Mg 2+, on both sides of the cell membrane: These considerations p r o m p t e d us to study the effects of the extra- and intracellular application of Ca 2+ and M g 2+ on the thermal resistance of ciliary activity in Anodonta, and on the m e m b r a n e - b o u n d ATPase enzymes of its ciliated gills.
MATERIALS AND METHODS
Fresh water mussels (Anodonta cyonea cellensis) were collected and maintained as previously (Lagerspetz & Dubitscber, 1966). The animals were stored at +4°C. T.B,
3/3--D
153
For the thermal resistance measurements, median gills of the mussels were excised and cut into small strips which were transferred to beakers with 50 ml of tap water kept in an accurate constant-temperature bath at 39°C. In some cases, deionized water was used as the incubation medium, or various salts or drugs were added to it. The ionophore A23187 (Eli Lilly and Co.), dissolved primarily in dimethylsulfoxide (DMSO) (20mM) and used then as colloidal water solution (final concentrations 10, 20, and 40FtM), was employed for the introduction of Ca 2÷ and Mg 2+ into the gill cells. At intervals of 10 min a strip of gill was removed for the inspection of the activity of frontal cillia by the particle transport method (Lagerspetz & Dubitscher, 1966). The time in minutes from the onset of the incubation of gill strips to the middle of the interval during which the movement of frontal cilia became unobservable, was used as the measure of the thermal resistance of ciliary activity. For microsomal preparations, the excised median gills of the mussels were homogenized in ice-cold 50 mM histidine-HCI buffer (pH 7.2) containing 1 mM EDTA. The supernatant from the first centrifugation of 10 rain at 900 g was further centrifuged for 10 min at 2,000g. and the remaining supernatant again twice for 20 min at 12,500g. The sediment from a last centrifugation of 1 h at 107,000 g was suspended by gentle stirring into 50 mM histidine-HCI buffer (pH 7.2). All centrifugations were made in cold. Electron micrographs made by courtesy of Mr. Reino Pitk~inen, Mag. Phil., Department of Biomedical Sciences, University of Tampere, showed that the preparations consisted of spherular membrane fragments. The determination of ATPase activity was carried out in tubes incubated usually for 20 min after temperature equilibration at 39°C. The standard incubation medium contained 3 mM ATP as Tris salt and 50 mM histidineHCI (pH 7.2). The salts and drugs used were added to make the total volume to 2 ml; this also contained 0.5 ml of the microsomal suspension. After incubation, inorganic phosphate liberated was determined (Atkinson et al, 1973), as well as the protein content of the microsomal suspensions (Lowry et al., 1951). The ATPase activities were calculated as micromoles of liberated inorganic phosphate per mg of protein per hour. With 4 mM of MgCI2, the hydrolysis of ATP was linear for at least 120 min. When micro-
154
K . E . O . SENIUS AND K. Y. H. LAGERSPETZ
Table 1. Activities of some enzymes in gill tissue preparations of Anodoma
Table 2. Effect of deionized water and of the divalent cation ionophore A23187 on the thermal resistance of ciliary activity in Anodonra gills
Enzyme activity Incubation medium
900 g Supernatant Alkaline phosphatase Acid phosphatase ATPase (with 4 m M Mg 2+) Microsomal preparation ATPase (with 4 mM Mg 2 +)
1.28 + 0.04 (8) 1.80 + 0.05 (8) 6.22 _+ 0.65 (3) 10.19 + 0.73 (6)
Results are expressed as micromoles of inorganic phosphate liberated per mg of protein per hour ( + S.E.). The number of preparations is given in parentheses. somal suspensions were assayed for different incubation times in the presence of 0.5mM ATP and 4 m M Mg 2÷, the hydrolysis of ATP stopped when an amount of phosphate equivalent to one phosphate group was split off. No attempt was made to purify the ATPase of the gill tissue. When compared with the starting material (the supernatant from 900 g centrifugation of the homogenate), microsomal preparations showed an average activity of Mg2÷-ATPase which was about 1.6 times that found in the 900 g supernatant (Table 1). Some alkaline phosphatase and acid phosphatase activity was also found in this material, when determined with the methods of Bessey et al. (1946) and Andersch & Szczypinski (1947), respectively. The activities of these enzymes were expressed in the same units as the ATPase activities. The statistical analysis of the results was calculated using the t-test. The deviations are expressed as standard errors of the mean. The number of exPeriments in each case refers to the number of preparations from separate animals. The enzyme activity determinations were run with duplicate or triplicate samples from each preparation. RESULTS
Thermal resistance of ciliary activity The effects of externally applied Ca 2 +, M g 2 +, and K ÷ on the thermal resistance of ciliary activity are 160
._~
[
I
I
l
I
I
I
I
4
8
120
E
~
co-
"~
40
Deionized Deionized ion'ophore Tap water Tap water ionophore Tap water ionophore (20/tM)
Thermal resistance time at 39 C in min ( + S.E.) water water + A23187 (20#M) + solvent + A23187
38 + 2 40 + 2 77 _ 3 76 + 9 57 + 3
The number of preparations at each experiment was 6. shown in Fig. 1. The increasing effect of divalent cations, Ca 2 ÷ and M g 2 + had its maximum at concentrations of about 4 raM. At higher concentrations, this effect was diminished in a concentration-dependent way. These effects were not simple osmotic ones, since K ÷ did not affect the thermal resistance except by antagonizing the effect of low C a " - concentrations. In these experiments, tap water was used as the solvent of the chloride salts and as the control medium. The local tap water contained 0 . 5 6 m M Ca 2 + and 0.07 m M M g ~ +. W h e n deionized water was used as the incubation medium, the thermal resistance time at 39°C was lower t h a n in tap water controls (Fig. 1, Table 2). While D M S O used as solvent of the i o n o p h o r e A23187 did not affect the thermal resistance of cilia by itself, the ionophore already at the low cation concentrations present in the tap water significantly decreased it (Table 21. The ionophore was without effect in deionized water. W h e n Ca 2 + or M g 2+ was added to the tap water in the incubation medium, their effect was significantly counteracted by the ionophore (Table 3). We have earlier found that A C h in concentrations of 0.55 to 11 m M increases the thermal resistance of ciliary activity and that this effect is concentrationdependent (Senius & Lagerspetz, 1974). Choline and T M A had similar effect. We now studied the dependence of this effect on Ca 2+. The addition of 5 m M ACh to deionized water increased the thermal resistance time from 38 + 2 min to 89 + 5 min (6 experiments). W h e n the small a m o u n t s of Ca 2 + probably leaking out of the tissue into the deionized water medium were enriched by 0.55 m M of C a 2 + to the level found in tap water, an addition of 5 m M A C h increased the thermal resistance time to 145 + 5 minutes (6 experiments).
ATPase activity of microsomes ~
o " [ I I 0
1
.z
Cation concentration
16
mM
Fig. 1. The effect of different concentrations of Ca 2+, Mg 2÷, and K ÷ on the thermal resistance of the activity of frontal cilia in the gills of Anodonta cygnea. All cations were dissolved as chlorides in the local tap water (0.56 mM Ca 2., 0.07 mM Mg2+). The black square at the beginning of the graphs gives the thermal resistance time in tap water; the other control value indicated by the black triangle was measured in deionized water. Each point gives the average from 3 to 8 experiments.
The concentration of Ca 2 + and Mg 2+ in the incubation medium affected the hydrolysis of A T P by the gill microsomes profoundly. Figure 2 gives results from three experiments, in which the Ca 2 + or Mg :+ concentration was varied. The maximum activity of the ATPase was attained at relatively high divalent cation concentrations (above 10 mM). Between 2 m M and 16 m M the activity was approximately doubled. W h e n added to the incubation media, 1 m M of the Ca z+ chelating substance E G T A (ethyleneglycolbis-(fl-aminoethylether)-N,N'-tetraacetic acid) caused
Calcium and magnesium in thermal resistance
155
Table 3. Effect of the divalent cation ionophore A23187 on the thermal resistance of the ciliary activity of Anodonta in the presence of Ca 2+ and Mg 2+
Cation added
Thermal resistance at 39°C in min (+ S.E.) Divalent cation ionophore A23187 Control 10/~M 20#M 40~uM
2mM Ca 2+ 2mM Mg 2+
157 + 6 167 + 3
117 + 7* --
105 + 8** 128 + 3**
112 + 7** --
The solutions were made to tap water. The ionophore solvent was present also in the control mediums. Asterisks indicate statistical significance of the difference from the controls; *: P < 0.01, **: P < 0.001. Number of preparations at each experiment was 6. 1oo r -
~"
50
0 0
I
2
3
,~
Cation
6
$
10
concentration
12
I 15
mM
Fig. 2. The effect of different concentrations of Ca: + (black circles) and Mg a+ (open circles) on the activity of ATPase in microsomal preparation made from the gills of Anodonta cyonea. Assay conditions: ATP (Tris salt) 3 mM, histidineHCI (pH 7.2) 50 mM. Results are from three experiments. The enzyme activity is expressed as a percentage of the maximum activity. a decrease of the MgZ+-ATPase activity from the control value of 10.19 + 0.73 to an average value of 9.26 + 0.66 #moles of inorganic phosphate liberated per mg protein per hour (6 experiments). Apparently the low concentrations of Ca z+ present in the microsomal preparations slightly although not significantly stimulated the ATPase activity, but Ca z+ was not necessary for the activation of the enzyme by Mg 2 +. The effects of Ca 2 + and Mg 2+ were additive at low concentrations. As the maximum activity was approached (at 16mM), a further addition of the other divalent cation did not increase the activity of
ATPase. Apparently the ATPase in question was activated either by Ca 2+ or by Mg 2+. The effects of certain substances on this Ca 2 +- or Mg2+-ATPase activity of microsomal preparations from gills were studied with 4 m M MgCI2 in the incubation medium. The results were compared with the effects of the same substances on the thermal resistance of the ciliary activity, measured as described above. As shown in Table 4, ACh, choline, and TMA, all used as chlorides, increased both the Mg 2+- or CaZ+-stimulated ATPase activity and the thermal resistance of the ciliary activity, while ethyl alcohol had an opposite effect on both variables studied. T h e stimulating effect of ACh on the ATPase activity was in so far specific, that the activities of alkaline phosphatase and acid phosphatase in 900 9 supernatants of gill tissue homogenates were unaffected by the presence of 5 m M of ACh, 8 determinations of each giving the average activities of 1.36 + 0.05 and 1.81 + 0.05 pmoles of inorganic phosphate liberated per mg of protein per hour, respectively. These values did not differ significantly from the basic activities of these enzymes given in T a b l e 1. DISCUSSION
According to the present results, Ca 2+ and Mg z+ have a double effect on the thermal resistance of ciliary activity in the gills of A~wdonta: these ions increase the thermal resistance when applied externally at concentrations below about 4 mM, and decrease
Table 4. Effects of some substances on the Mg z+ or Ca z + stimulated ATPase activity in gill tissue preparations and on the thermal resistance of the ciliary activity in the gills of Anodonta Substance added None Acetylcholine Acetyicholine Choline TMA Ethyl alcohol Ethyl alcohol
Concentr. (mM) 1 5 5 5 • 100 1000
ATPase activity 100
Thermal resistance at 39°C 100
110 5:2 (7) 127 + 2 (7) 124 + 2(5) 124+3(5) 92 _+ 1 (4) 43 + 1 (3)
180 -t- 22 (6) 267 + 12 (4) 244 + 8(4) 271 +11(6) 102 + 4 (6) 56 + 10(6)
Results are expressed as percentages of control values, typically I0.19 + 0.73 (S.E.) micromoles of inorganic phosphate liberated per mg of protein per h for the ATPase (in the presence of 4 mM MgClz) and 75 + 3 (S.E.) min at 39°C, r e~ectively. Percentage values have been calculated by comparison with paired controls Of the same series of determinations. Numbers of preparations are given in parentheses.
156
K.E.O. SENIUSAND K. Y. H. LAGERSPETZ
it when applied together with the ionophore A23187 or at high concentrations. The ionophore A23187 may not only affect the plasma membrane but also release Ca -'+ from intracellular storage sites (Babcock et al., 1976). Divalent cations in the medium apparently also interfere to some extent with its action by complexation (Pfeiffer et al., 1974; Chandler & Williams, 1977). However, as the thermal resistance of the ciliary activity is decreased by the ionophore only in the presence of divalent cations in the medium and not in deionized water (Tables 2 and 3), this effect is probably caused by the entry of divalent cations to the cells through ionophore mediation. At high external concentrations of Ca 2÷ and Mg 2÷, an increase of the intracetlular concentration of these ions could probably occur through osmosis. Increased intracellular concentrations of divalent cations are known to interfere with the microtubular mechanisms of cilia, perhaps especially those related to the bending of cilia (Satir, 1975; Tsuchiya, 1976, 1977), and may thus shorten the thermal resistance time. It is possible that the heat-induced arrest of ciliary activity is caused by the increase of membrane permeability occurring at high temperatures. Increased permeability to ions or a partial solubilization of the membrane in an ion-deficient medium leads to a leakout of ions, and hence to profound changes in the intracellular conditions. Thus ciliary arrest is known to occur in ciliated epithelium treated with the detergent Triton X-100. There is no permanent damage to the ciliary motor apparatus, which can be subsequently reactivated by Mg 2÷ and ATP (Eckert & Murakami, 1972; Tsuchiya, 1976, 1977). The cell membrane may well be the part of the cell which is most sensitive to heat and which thus determines also the thermal resistance of ciliary activity. This would explain the extremely short thermal resistance times observed in deionized water. The increasing effect of extracellular divalent cations in concentrations below about 4 mM on the thermal resistance of cilia is more difficult to explain. These concentrations are much higher than those usually found in the cytoplasm. It could be possible that extracellular Ca 2+ and Mg 2÷ permeating in small amounts into the cell stimulate the transport mechanism which expels Ca 2÷ out of the cell and keeps the intracellular Ca 2÷ concentration on a low level. The active transport of Ca 2÷ is linked to a Ca2+-stimulated Mg2*-dependent ATPase (Schatzmann, 1966; Nakamaru & Konishi, 1968; Roufogalis, 1973). This enzyme requires Mg 2÷ and is maximally stimulated by low concentrations (1-100 pmol 1-1) of Ca 2÷ (Roufogalis, 1973). Our experiments show that the microsomal preparations from A n o d o n t a gills in fact contain an ATPase enzyme. However, rather large concentrations (over 10mmol1-1) of Ca 2- or Mg 2÷ are needed for its maximal activation, and either of these ions is sufficient for this. The chelation of Ca 2÷ by EGTA causes only a slight decrease in the ATPase activity. The ATPase of A n o d o n t a gill microsomes is therefore not a Ca2÷-stimulated Mg2+-dependent ATPase, like the Ca 2÷ transport enzyme, but a Ca 2 ÷or Mg2÷-activated ATPase. The microtubular apparatus of cilia contains
dynein, a protein with ATPase activity dependent on the presence of either Ca 2 ÷ or Mg 2- (Gibbons, 1966). However, in order to solubilize dynein, the ciliary membranes should be broken, e.g. with ethyl alcohol (Watson & Hopkins, 1962; Gibbons, 1966). This was not attempted in the preparation of microsomes. On the other hand, according to electron micrographs, no cilia or their fragments were present in the microsomal preparations. Although we can not exclude the possibility of contamination by dynein, it seems therefore unlikely that the Ca z~- or Mg2+-activated ATPase found would be microtubular dynein, but is rather a membrane bound enzyme. Actomyosin-like proteins are found in membranes of many types of cells (Bowler & Duncan, 1967. 1968; Puszkin et al., 1968; Berl & Puszkin, 1970; Puszkin et al., 1972; Willingham et al.. 19741. Such proteins or membrane preparations have been shown to exhibit ATPase activity in the presence of Mg 2. or of either Ca 2+ or Mg 2+. The Mg2"-activated ATPase has been suggested to be a contractile protein controlling the passive permeability of cell membranes (Duncan, 1967). A possibility to explain the increase of the thermal resistance by externally applied Ca -'+ and Mg 2÷ is that the Ca 2+- or Mg2+-activated ATPase found in the microsomal preparations made from the gills of A n o d o n t a is a contractile protein which, when activated, decreases the passive permeability of the cell membrane. The effect of the ionophore A23187 in decreasing the thermal resistance of the cilia is very effectively counteracted by external Ca 2 + and Mg- . In experiments without the ionophore, Ca z+ and Mg 2+ increase the thermal resistance of cilia up to concentrations of about 4raM. The activity of the microsomal Ca 2+- or Mg2+-activated ATPase increases to about 75~/o of the maximal values within the same cation concentration range. The increase in the enzyme activity is smaller at high concentrations. at which this enzyme probably cannot counteract effectively enough the passive permeation of the divalent cations. This results in a decrease of the heat resistance of the cellular function studied, the ciliary activity. The hypothesis that the Ca-' +- or Mg 2÷-activated ATPase would control the passive permeability of the cell membranes obviously explains the results obtained through the experimental application of these ions. The results of our other experiments seem to confirm this hypothesis. The effects of ACh, choline, and TMA in increasing the thermal resistance of cilia, as well as the decreasing effect of ethyl alcohol on the thermal resistance, are all parallel to the changes caused by these substances at the same concentrations in the activity of the Ca 2-- or Mg2+-activated ATPase. The concentrations of the d r u ~ effective in our experiments are rather high, particularly that of ethyl alcohol (1 M = 4.6~o w/v). However, ethyl alcohol o/ concentrations of the same range (_9-- 6/0) have been found to be effective in the reversible depression of evoked compound excitatory postsynaptic potentials when applied on the ganglion of the nudibranch mollusc Tritonia (Chase, 1975). We suggest that the thermal resistance of the ciliary activity is limited by the increase in the permeability
Calcium and magnesium in thermal resistance of the cell m e m b r a n e at high temperatures, and that the Ca 2÷- or Mg2+-ATPase participates in the control of the permeability, and thus indirectly in the control of the heat resistance of ciliary activity. Acknowledgements--We wish to thank Mrs. Erika Holmbom and Miss Hanna Kurppa for competent technical assistance and Mrs. Varpu V~ilim~iki for the drawing of the figures. Eli Lilly and Co., Indianapolis, Ind., U.S.A., is acknowledged for a gift of the ionophore A23187. This work was supported by the Academy of Finland.
REFERENCES ANDEgSCH M. A. & SZCZYPINSKI A. J. (1947) Use of p-nitrophenylphosphate as the substrate in determination of serum acid phosphatase. Am. J. Clin. Pathol. 17, 571-574. ATKXNSON A., GATEN~Y A. D. & LOWE A. G. (1973) The determination of inorganic orthophosphate in biological systems. Biochim. biophys. Acta 320, 195-204. BAnCOCK D. F., Frost N. L. & LARDY H. A. (1976) Action of ionophore A23187 at the cellular level. Separation of effects at the plasma and mitochondrial membranes. J. biol. Chem. 251, 3881-3886. BERL S. & PUSZKIN S. (1970) MgZ+-Ca2+-activated adenosine triphosphatase system isolated from mammalian brain. Biochemistry 9, 2058-2067. BESSEY O. A., LOWRY O. H. & BROCK M. J. (1946) A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J. biol. Chem. 164, 321-329. BOWLER K. & DUNCAN C. J. (1967) Studies on the actomyosin-like ATPase in a membrane preparation from crayfish nerve cord. Comp. Biochem. Physiol. 20, 543-551. BOWLER K. & DUNCAN C. J. (1968) The temperature characteristics of the ATPases from a frog brain microsomal preparation. Comp. Biochem. Physiol. 24, 223-227. CHANDLER D. E. & WmHAMS J. A. (1977) Intracellular uptake and ,,-amylase and lactate dehydrogenase releasing actions of the divalent cation ionophore A23187 in dissociated pancreatic acinar cells. J. Membrane Biol. 32, 201-230. CHASE R. (1975) The suppression of excitatory synaptic responses by ethyl alcohol in the nudibranch mollusc, TritonM diomedea. Comp. Biochem. Physiol. 50C, 37-40. DUNCAN C. J. (1967) The Molecular Properties and Evolution of Excitable Cells. 253 pp. Pergamon Press, Oxford. ECKEgT R. (1972) Bioelectric control of ciliary activity. Science 176, 473-481. ECKERT R. & MURAKAMI A. (1972) Calcium dependence of ciliary activity in the oviduct of the salamander Necturus. J. Physiol., Lond. 226, 699-711. GmBONS I. R. (1966) Studies on the adenosine triphosphatase activity of 14S and 30S dynein from cilia of Tetrahymena. J. biol. Chem. 241, 5590-5596. LAGERSPETZ K. Y. H. & DUBrrSCHER I. (1966) Temperature acclimation of the ciliary activity in the gills of Anodonta. Comp. Biochem. Physiol. 17, 665-671. LAGERSPETZ K. Y. H., IMPiVAARAH. & SENIUS K. (19,70) Acetylcholine in the thermal resistance acclimation of the ciliary activity in the gills of Anodonta. Comp. yen. Pharmac. 1, 236-240.
157
LOWRY O. H., ROSEBROUGHN. J., EARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MURAKAMIA. & TAKAHASHIK. (1975) The role of calcium in the control of ciliary movement in Mytilus. II. The effects of calcium ionophores X537A and A23187 on the lateral gill cilia. J. Fac. Sci. Tokyo Univ., Sect. IV, 13, 251-256. NAITOH Y. E¢ KANEKO H. (1973) Control of ciliary activities by adenosinetriphosphate and divalent cations in Tritonextracted models of Paramecium caudatum. J. exp. Biol. 58, 657-676. NAKAMARUY. & KONISm K. (1968) Elicitation of the brain microsomal (Mg 2 + + Ca 2 ÷)-activated ATPase by digitonin treatment. Biochim. biophys. Acta 159, 206-208. PFEIFFER D. R., REED P. W. & LADY H. A. (1974) Ultraviolet and fluorescent spectral properties of the divalent cation ionophore A23187 and its metal ion complexes. Biochemistry 13, 4007--4014. PUSZKIN S., BERL S., PtJSZKIN E. & CLARKE D. D. (1968) Actomyosin-like protein isolated from mammalian brain. Science 161, 170-171. PUSZKtN S., NICKLAS W. J. & BERL S. (1972) Actomyosinlike protein in brain: subcellular distribution. J. Neurochem. 19, 1319-1333. ROUFOGALIS B. D. (1973) Properties of a (Mg 2+ + Ca2+) dependent ATPase of bovine brain cortex. Effects of detergents, freezing, cations and local anesthetics. Biochim. biophys. Acta 318, 360-370. SATIn P. (1975) Ionophore-mediated calcium entry induces mussel gill ciliary arrest. Science 190, 586-588. SCnATZMANN H. J. (1966) ATP-dependent Ca + +-extrusion from human red cells. Experientia 22, 364-365. SCHLmPEg C. & KOWALSKX R. (1956) Quantitative Beobachtungen fiber physiologische Ionenwirkungen im Brackwasser. Kieler Meeresf. 12, 154-165. SENIUS K. E. O. (1975a) Thermal resistance acclimation of ciliary activity in the gills of fresh water mussels Anodonta anatina and Anodonta cy#nea. Comp. Biochem. Physiol. 51C, 157-160. SENIUS K. E. O. (1975b) The thermal resistance and thermal resistance acclimation of ciliary activity in the Mytilus gills. Comp. Biochem. Physiol. 51A, 957-961. SENIUS K. E. O. (1977) Thermal resistance of the ciliary activity in the gills of the fresh water mussel Anodonta anatina. J. ther. Biol. 2, 233--238. SENtUS K. E. O. & L^6EgSPE'rz K. Y. H. (19741 The role of cholinergic receptors in the thermal resistance and thermal resistance acclimation of the ciliary activity in the Anodonta gills. Comp. yen. Pharmac. 5, 169-179. Tst:cntYA T. (1976) Ca-induced arrest response in Tritonextracted lateral cilia of Mytilus gill. Experientia 32, 1439-1440. TsucmvA T. (1977) Effects of calcium ions on Tritonextracted lamellibranch gill cilia: Ciliary arrest response in a model system. Comp. Biochem. Physiol. 56A, 353-361. WATSON M. R. & HOPKINS J. M. (1962) Isolated cilia from Tetrahymena pyriformis. Exptl Cell Res. 28, 280-295. WILLINGHAM M. C., OSTLUND R. E. ~ PmT^.~ I. (1974) Myosin is a component of the cell surface of cultured cells. Proc. Nat. Acad. Sci. USA 71, 4 1 ~ 4148. Key Word Index--Thermal resistance; ciliary activity; acetylcholine; choline; tetramethylammonium; ionophore A23187; calcium; magnesium; ATPase; gill tissue; Anodonta cyonea cellensis.
0306-4565/78,0701-0153S02.00 0
EFFECTS OF CALCIUM AND MAGNESIUM ON T H E T H E R M A L RESISTANCE O F CILIARY ACTIVITY IN THE F R E S H WATER MUSSEL A N O D O N T A K. E. O. SENIUS AND K. Y. H. LAGERSPETZ Department of Biomedical Sciences, University of Tampere, Box 607, SF-33101 Tampere, and Zoophysiological Laboratory, Department of Biology, University of Turku, SF-20500 Turku, Finland
(Received 12 October 1977; accepted in revised form 23 November 1977) Abstract--l. External Ca 2+ and Mg 2+ at concentrations below about 4 m M increase the thermal resistance of the activity of frontal cilia in the gills of Anodonta cygnea cellensis. 2. High external concentrations of Ca 2+ and Mg 2+, and the divalent cation ionophore A23187 in the presence of external Ca 2 + or Mg 2 +, counteract this effect. 3. The thermal resistance is increased by acetylcholine, choline, and tetramethylammonium, and decreased by ethyl alcohol. 4. Microsomal preparations from the gills show Ca 2+- or Mg2+-activated ATPase activity which is stimulated by acetylcholine, choline, and tetramethylammonium, and inhibited by ethyl alcohol. 5. It is tentatively suggested that Ca 2+- or Mg2+-activated ATPase participates in the control of membrane permeability, and also indirectly in the control of the thermal resistance of ciliary activity.
INTRODUCTION WE HAVE earlier studied the effects of temperature acclimation and of some cholinergic substances on the thermal resistance of the activity of frontal cilia in gills of the fresh water mussels Anodonta cygnea cellensis and A. anatina anatina and of the marine mussel Mytilus edulis (Lagerspetz & Dubitscher, 1966; Lagerspetz et al., 1970; Senius & Lagerspetz, 1974; Senius, 1975a, 1975b, 1977). The thermal resistance of the ciliary activity is increased in all three species by warm-acclimation of intact animals; acetylcholine (ACh), choline, and t e t r a m e t h y l a m m o n i u m (TMA) increase the thermal resistance of cilia in the two species of Anodonta, but not in Mytilus. Calcium and magnesium ions are usually considered to increase the stability of cell membranes. W h e n externally applied, they increase the thermal resistance of ciliary activity in the gills of Mytilus (Schlieper & Kowalski, 1956). O n the other hand, the entry of Ca 2 + into the cells causes arrest of the lateral cilia in the gills of Mytilus ( M u r a k a m i & Takahashi, 1975) and of the fresh water mussel Elliptio (Satir, 1975), as well as the reversal of the direction of ciliary beat in Paramecium (Eckert, 1972; N a i t o h & Kaneko, 1973). It is possible that the thermal resistance of the ciliary activity is, in part, controlled by the relative distribution of Ca 2+, and possibly Mg 2+, on both sides of the cell membrane: These considerations p r o m p t e d us to study the effects of the extra- and intracellular application of Ca 2+ and M g 2+ on the thermal resistance of ciliary activity in Anodonta, and on the m e m b r a n e - b o u n d ATPase enzymes of its ciliated gills.
MATERIALS AND METHODS
Fresh water mussels (Anodonta cyonea cellensis) were collected and maintained as previously (Lagerspetz & Dubitscber, 1966). The animals were stored at +4°C. T.B,
3/3--D
153
For the thermal resistance measurements, median gills of the mussels were excised and cut into small strips which were transferred to beakers with 50 ml of tap water kept in an accurate constant-temperature bath at 39°C. In some cases, deionized water was used as the incubation medium, or various salts or drugs were added to it. The ionophore A23187 (Eli Lilly and Co.), dissolved primarily in dimethylsulfoxide (DMSO) (20mM) and used then as colloidal water solution (final concentrations 10, 20, and 40FtM), was employed for the introduction of Ca 2÷ and Mg 2+ into the gill cells. At intervals of 10 min a strip of gill was removed for the inspection of the activity of frontal cillia by the particle transport method (Lagerspetz & Dubitscher, 1966). The time in minutes from the onset of the incubation of gill strips to the middle of the interval during which the movement of frontal cilia became unobservable, was used as the measure of the thermal resistance of ciliary activity. For microsomal preparations, the excised median gills of the mussels were homogenized in ice-cold 50 mM histidine-HCI buffer (pH 7.2) containing 1 mM EDTA. The supernatant from the first centrifugation of 10 rain at 900 g was further centrifuged for 10 min at 2,000g. and the remaining supernatant again twice for 20 min at 12,500g. The sediment from a last centrifugation of 1 h at 107,000 g was suspended by gentle stirring into 50 mM histidine-HCI buffer (pH 7.2). All centrifugations were made in cold. Electron micrographs made by courtesy of Mr. Reino Pitk~inen, Mag. Phil., Department of Biomedical Sciences, University of Tampere, showed that the preparations consisted of spherular membrane fragments. The determination of ATPase activity was carried out in tubes incubated usually for 20 min after temperature equilibration at 39°C. The standard incubation medium contained 3 mM ATP as Tris salt and 50 mM histidineHCI (pH 7.2). The salts and drugs used were added to make the total volume to 2 ml; this also contained 0.5 ml of the microsomal suspension. After incubation, inorganic phosphate liberated was determined (Atkinson et al, 1973), as well as the protein content of the microsomal suspensions (Lowry et al., 1951). The ATPase activities were calculated as micromoles of liberated inorganic phosphate per mg of protein per hour. With 4 mM of MgCI2, the hydrolysis of ATP was linear for at least 120 min. When micro-
154
K . E . O . SENIUS AND K. Y. H. LAGERSPETZ
Table 1. Activities of some enzymes in gill tissue preparations of Anodoma
Table 2. Effect of deionized water and of the divalent cation ionophore A23187 on the thermal resistance of ciliary activity in Anodonra gills
Enzyme activity Incubation medium
900 g Supernatant Alkaline phosphatase Acid phosphatase ATPase (with 4 m M Mg 2+) Microsomal preparation ATPase (with 4 mM Mg 2 +)
1.28 + 0.04 (8) 1.80 + 0.05 (8) 6.22 _+ 0.65 (3) 10.19 + 0.73 (6)
Results are expressed as micromoles of inorganic phosphate liberated per mg of protein per hour ( + S.E.). The number of preparations is given in parentheses. somal suspensions were assayed for different incubation times in the presence of 0.5mM ATP and 4 m M Mg 2÷, the hydrolysis of ATP stopped when an amount of phosphate equivalent to one phosphate group was split off. No attempt was made to purify the ATPase of the gill tissue. When compared with the starting material (the supernatant from 900 g centrifugation of the homogenate), microsomal preparations showed an average activity of Mg2÷-ATPase which was about 1.6 times that found in the 900 g supernatant (Table 1). Some alkaline phosphatase and acid phosphatase activity was also found in this material, when determined with the methods of Bessey et al. (1946) and Andersch & Szczypinski (1947), respectively. The activities of these enzymes were expressed in the same units as the ATPase activities. The statistical analysis of the results was calculated using the t-test. The deviations are expressed as standard errors of the mean. The number of exPeriments in each case refers to the number of preparations from separate animals. The enzyme activity determinations were run with duplicate or triplicate samples from each preparation. RESULTS
Thermal resistance of ciliary activity The effects of externally applied Ca 2 +, M g 2 +, and K ÷ on the thermal resistance of ciliary activity are 160
._~
[
I
I
l
I
I
I
I
4
8
120
E
~
co-
"~
40
Deionized Deionized ion'ophore Tap water Tap water ionophore Tap water ionophore (20/tM)
Thermal resistance time at 39 C in min ( + S.E.) water water + A23187 (20#M) + solvent + A23187
38 + 2 40 + 2 77 _ 3 76 + 9 57 + 3
The number of preparations at each experiment was 6. shown in Fig. 1. The increasing effect of divalent cations, Ca 2 ÷ and M g 2 + had its maximum at concentrations of about 4 raM. At higher concentrations, this effect was diminished in a concentration-dependent way. These effects were not simple osmotic ones, since K ÷ did not affect the thermal resistance except by antagonizing the effect of low C a " - concentrations. In these experiments, tap water was used as the solvent of the chloride salts and as the control medium. The local tap water contained 0 . 5 6 m M Ca 2 + and 0.07 m M M g ~ +. W h e n deionized water was used as the incubation medium, the thermal resistance time at 39°C was lower t h a n in tap water controls (Fig. 1, Table 2). While D M S O used as solvent of the i o n o p h o r e A23187 did not affect the thermal resistance of cilia by itself, the ionophore already at the low cation concentrations present in the tap water significantly decreased it (Table 21. The ionophore was without effect in deionized water. W h e n Ca 2 + or M g 2+ was added to the tap water in the incubation medium, their effect was significantly counteracted by the ionophore (Table 3). We have earlier found that A C h in concentrations of 0.55 to 11 m M increases the thermal resistance of ciliary activity and that this effect is concentrationdependent (Senius & Lagerspetz, 1974). Choline and T M A had similar effect. We now studied the dependence of this effect on Ca 2+. The addition of 5 m M ACh to deionized water increased the thermal resistance time from 38 + 2 min to 89 + 5 min (6 experiments). W h e n the small a m o u n t s of Ca 2 + probably leaking out of the tissue into the deionized water medium were enriched by 0.55 m M of C a 2 + to the level found in tap water, an addition of 5 m M A C h increased the thermal resistance time to 145 + 5 minutes (6 experiments).
ATPase activity of microsomes ~
o " [ I I 0
1
.z
Cation concentration
16
mM
Fig. 1. The effect of different concentrations of Ca 2+, Mg 2÷, and K ÷ on the thermal resistance of the activity of frontal cilia in the gills of Anodonta cygnea. All cations were dissolved as chlorides in the local tap water (0.56 mM Ca 2., 0.07 mM Mg2+). The black square at the beginning of the graphs gives the thermal resistance time in tap water; the other control value indicated by the black triangle was measured in deionized water. Each point gives the average from 3 to 8 experiments.
The concentration of Ca 2 + and Mg 2+ in the incubation medium affected the hydrolysis of A T P by the gill microsomes profoundly. Figure 2 gives results from three experiments, in which the Ca 2 + or Mg :+ concentration was varied. The maximum activity of the ATPase was attained at relatively high divalent cation concentrations (above 10 mM). Between 2 m M and 16 m M the activity was approximately doubled. W h e n added to the incubation media, 1 m M of the Ca z+ chelating substance E G T A (ethyleneglycolbis-(fl-aminoethylether)-N,N'-tetraacetic acid) caused
Calcium and magnesium in thermal resistance
155
Table 3. Effect of the divalent cation ionophore A23187 on the thermal resistance of the ciliary activity of Anodonta in the presence of Ca 2+ and Mg 2+
Cation added
Thermal resistance at 39°C in min (+ S.E.) Divalent cation ionophore A23187 Control 10/~M 20#M 40~uM
2mM Ca 2+ 2mM Mg 2+
157 + 6 167 + 3
117 + 7* --
105 + 8** 128 + 3**
112 + 7** --
The solutions were made to tap water. The ionophore solvent was present also in the control mediums. Asterisks indicate statistical significance of the difference from the controls; *: P < 0.01, **: P < 0.001. Number of preparations at each experiment was 6. 1oo r -
~"
50
0 0
I
2
3
,~
Cation
6
$
10
concentration
12
I 15
mM
Fig. 2. The effect of different concentrations of Ca: + (black circles) and Mg a+ (open circles) on the activity of ATPase in microsomal preparation made from the gills of Anodonta cyonea. Assay conditions: ATP (Tris salt) 3 mM, histidineHCI (pH 7.2) 50 mM. Results are from three experiments. The enzyme activity is expressed as a percentage of the maximum activity. a decrease of the MgZ+-ATPase activity from the control value of 10.19 + 0.73 to an average value of 9.26 + 0.66 #moles of inorganic phosphate liberated per mg protein per hour (6 experiments). Apparently the low concentrations of Ca z+ present in the microsomal preparations slightly although not significantly stimulated the ATPase activity, but Ca z+ was not necessary for the activation of the enzyme by Mg 2 +. The effects of Ca 2 + and Mg 2+ were additive at low concentrations. As the maximum activity was approached (at 16mM), a further addition of the other divalent cation did not increase the activity of
ATPase. Apparently the ATPase in question was activated either by Ca 2+ or by Mg 2+. The effects of certain substances on this Ca 2 +- or Mg2+-ATPase activity of microsomal preparations from gills were studied with 4 m M MgCI2 in the incubation medium. The results were compared with the effects of the same substances on the thermal resistance of the ciliary activity, measured as described above. As shown in Table 4, ACh, choline, and TMA, all used as chlorides, increased both the Mg 2+- or CaZ+-stimulated ATPase activity and the thermal resistance of the ciliary activity, while ethyl alcohol had an opposite effect on both variables studied. T h e stimulating effect of ACh on the ATPase activity was in so far specific, that the activities of alkaline phosphatase and acid phosphatase in 900 9 supernatants of gill tissue homogenates were unaffected by the presence of 5 m M of ACh, 8 determinations of each giving the average activities of 1.36 + 0.05 and 1.81 + 0.05 pmoles of inorganic phosphate liberated per mg of protein per hour, respectively. These values did not differ significantly from the basic activities of these enzymes given in T a b l e 1. DISCUSSION
According to the present results, Ca 2+ and Mg z+ have a double effect on the thermal resistance of ciliary activity in the gills of A~wdonta: these ions increase the thermal resistance when applied externally at concentrations below about 4 mM, and decrease
Table 4. Effects of some substances on the Mg z+ or Ca z + stimulated ATPase activity in gill tissue preparations and on the thermal resistance of the ciliary activity in the gills of Anodonta Substance added None Acetylcholine Acetyicholine Choline TMA Ethyl alcohol Ethyl alcohol
Concentr. (mM) 1 5 5 5 • 100 1000
ATPase activity 100
Thermal resistance at 39°C 100
110 5:2 (7) 127 + 2 (7) 124 + 2(5) 124+3(5) 92 _+ 1 (4) 43 + 1 (3)
180 -t- 22 (6) 267 + 12 (4) 244 + 8(4) 271 +11(6) 102 + 4 (6) 56 + 10(6)
Results are expressed as percentages of control values, typically I0.19 + 0.73 (S.E.) micromoles of inorganic phosphate liberated per mg of protein per h for the ATPase (in the presence of 4 mM MgClz) and 75 + 3 (S.E.) min at 39°C, r e~ectively. Percentage values have been calculated by comparison with paired controls Of the same series of determinations. Numbers of preparations are given in parentheses.
156
K.E.O. SENIUSAND K. Y. H. LAGERSPETZ
it when applied together with the ionophore A23187 or at high concentrations. The ionophore A23187 may not only affect the plasma membrane but also release Ca -'+ from intracellular storage sites (Babcock et al., 1976). Divalent cations in the medium apparently also interfere to some extent with its action by complexation (Pfeiffer et al., 1974; Chandler & Williams, 1977). However, as the thermal resistance of the ciliary activity is decreased by the ionophore only in the presence of divalent cations in the medium and not in deionized water (Tables 2 and 3), this effect is probably caused by the entry of divalent cations to the cells through ionophore mediation. At high external concentrations of Ca 2÷ and Mg 2÷, an increase of the intracetlular concentration of these ions could probably occur through osmosis. Increased intracellular concentrations of divalent cations are known to interfere with the microtubular mechanisms of cilia, perhaps especially those related to the bending of cilia (Satir, 1975; Tsuchiya, 1976, 1977), and may thus shorten the thermal resistance time. It is possible that the heat-induced arrest of ciliary activity is caused by the increase of membrane permeability occurring at high temperatures. Increased permeability to ions or a partial solubilization of the membrane in an ion-deficient medium leads to a leakout of ions, and hence to profound changes in the intracellular conditions. Thus ciliary arrest is known to occur in ciliated epithelium treated with the detergent Triton X-100. There is no permanent damage to the ciliary motor apparatus, which can be subsequently reactivated by Mg 2÷ and ATP (Eckert & Murakami, 1972; Tsuchiya, 1976, 1977). The cell membrane may well be the part of the cell which is most sensitive to heat and which thus determines also the thermal resistance of ciliary activity. This would explain the extremely short thermal resistance times observed in deionized water. The increasing effect of extracellular divalent cations in concentrations below about 4 mM on the thermal resistance of cilia is more difficult to explain. These concentrations are much higher than those usually found in the cytoplasm. It could be possible that extracellular Ca 2+ and Mg 2÷ permeating in small amounts into the cell stimulate the transport mechanism which expels Ca 2÷ out of the cell and keeps the intracellular Ca 2÷ concentration on a low level. The active transport of Ca 2÷ is linked to a Ca2+-stimulated Mg2*-dependent ATPase (Schatzmann, 1966; Nakamaru & Konishi, 1968; Roufogalis, 1973). This enzyme requires Mg 2÷ and is maximally stimulated by low concentrations (1-100 pmol 1-1) of Ca 2÷ (Roufogalis, 1973). Our experiments show that the microsomal preparations from A n o d o n t a gills in fact contain an ATPase enzyme. However, rather large concentrations (over 10mmol1-1) of Ca 2- or Mg 2÷ are needed for its maximal activation, and either of these ions is sufficient for this. The chelation of Ca 2÷ by EGTA causes only a slight decrease in the ATPase activity. The ATPase of A n o d o n t a gill microsomes is therefore not a Ca2÷-stimulated Mg2+-dependent ATPase, like the Ca 2÷ transport enzyme, but a Ca 2 ÷or Mg2÷-activated ATPase. The microtubular apparatus of cilia contains
dynein, a protein with ATPase activity dependent on the presence of either Ca 2 ÷ or Mg 2- (Gibbons, 1966). However, in order to solubilize dynein, the ciliary membranes should be broken, e.g. with ethyl alcohol (Watson & Hopkins, 1962; Gibbons, 1966). This was not attempted in the preparation of microsomes. On the other hand, according to electron micrographs, no cilia or their fragments were present in the microsomal preparations. Although we can not exclude the possibility of contamination by dynein, it seems therefore unlikely that the Ca z~- or Mg2+-activated ATPase found would be microtubular dynein, but is rather a membrane bound enzyme. Actomyosin-like proteins are found in membranes of many types of cells (Bowler & Duncan, 1967. 1968; Puszkin et al., 1968; Berl & Puszkin, 1970; Puszkin et al., 1972; Willingham et al.. 19741. Such proteins or membrane preparations have been shown to exhibit ATPase activity in the presence of Mg 2. or of either Ca 2+ or Mg 2+. The Mg2"-activated ATPase has been suggested to be a contractile protein controlling the passive permeability of cell membranes (Duncan, 1967). A possibility to explain the increase of the thermal resistance by externally applied Ca -'+ and Mg 2÷ is that the Ca 2+- or Mg2+-activated ATPase found in the microsomal preparations made from the gills of A n o d o n t a is a contractile protein which, when activated, decreases the passive permeability of the cell membrane. The effect of the ionophore A23187 in decreasing the thermal resistance of the cilia is very effectively counteracted by external Ca 2 + and Mg- . In experiments without the ionophore, Ca z+ and Mg 2+ increase the thermal resistance of cilia up to concentrations of about 4raM. The activity of the microsomal Ca 2+- or Mg2+-activated ATPase increases to about 75~/o of the maximal values within the same cation concentration range. The increase in the enzyme activity is smaller at high concentrations. at which this enzyme probably cannot counteract effectively enough the passive permeation of the divalent cations. This results in a decrease of the heat resistance of the cellular function studied, the ciliary activity. The hypothesis that the Ca-' +- or Mg 2÷-activated ATPase would control the passive permeability of the cell membranes obviously explains the results obtained through the experimental application of these ions. The results of our other experiments seem to confirm this hypothesis. The effects of ACh, choline, and TMA in increasing the thermal resistance of cilia, as well as the decreasing effect of ethyl alcohol on the thermal resistance, are all parallel to the changes caused by these substances at the same concentrations in the activity of the Ca 2-- or Mg2+-activated ATPase. The concentrations of the d r u ~ effective in our experiments are rather high, particularly that of ethyl alcohol (1 M = 4.6~o w/v). However, ethyl alcohol o/ concentrations of the same range (_9-- 6/0) have been found to be effective in the reversible depression of evoked compound excitatory postsynaptic potentials when applied on the ganglion of the nudibranch mollusc Tritonia (Chase, 1975). We suggest that the thermal resistance of the ciliary activity is limited by the increase in the permeability
Calcium and magnesium in thermal resistance of the cell m e m b r a n e at high temperatures, and that the Ca 2÷- or Mg2+-ATPase participates in the control of the permeability, and thus indirectly in the control of the heat resistance of ciliary activity. Acknowledgements--We wish to thank Mrs. Erika Holmbom and Miss Hanna Kurppa for competent technical assistance and Mrs. Varpu V~ilim~iki for the drawing of the figures. Eli Lilly and Co., Indianapolis, Ind., U.S.A., is acknowledged for a gift of the ionophore A23187. This work was supported by the Academy of Finland.
REFERENCES ANDEgSCH M. A. & SZCZYPINSKI A. J. (1947) Use of p-nitrophenylphosphate as the substrate in determination of serum acid phosphatase. Am. J. Clin. Pathol. 17, 571-574. ATKXNSON A., GATEN~Y A. D. & LOWE A. G. (1973) The determination of inorganic orthophosphate in biological systems. Biochim. biophys. Acta 320, 195-204. BAnCOCK D. F., Frost N. L. & LARDY H. A. (1976) Action of ionophore A23187 at the cellular level. Separation of effects at the plasma and mitochondrial membranes. J. biol. Chem. 251, 3881-3886. BERL S. & PUSZKIN S. (1970) MgZ+-Ca2+-activated adenosine triphosphatase system isolated from mammalian brain. Biochemistry 9, 2058-2067. BESSEY O. A., LOWRY O. H. & BROCK M. J. (1946) A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J. biol. Chem. 164, 321-329. BOWLER K. & DUNCAN C. J. (1967) Studies on the actomyosin-like ATPase in a membrane preparation from crayfish nerve cord. Comp. Biochem. Physiol. 20, 543-551. BOWLER K. & DUNCAN C. J. (1968) The temperature characteristics of the ATPases from a frog brain microsomal preparation. Comp. Biochem. Physiol. 24, 223-227. CHANDLER D. E. & WmHAMS J. A. (1977) Intracellular uptake and ,,-amylase and lactate dehydrogenase releasing actions of the divalent cation ionophore A23187 in dissociated pancreatic acinar cells. J. Membrane Biol. 32, 201-230. CHASE R. (1975) The suppression of excitatory synaptic responses by ethyl alcohol in the nudibranch mollusc, TritonM diomedea. Comp. Biochem. Physiol. 50C, 37-40. DUNCAN C. J. (1967) The Molecular Properties and Evolution of Excitable Cells. 253 pp. Pergamon Press, Oxford. ECKEgT R. (1972) Bioelectric control of ciliary activity. Science 176, 473-481. ECKERT R. & MURAKAMI A. (1972) Calcium dependence of ciliary activity in the oviduct of the salamander Necturus. J. Physiol., Lond. 226, 699-711. GmBONS I. R. (1966) Studies on the adenosine triphosphatase activity of 14S and 30S dynein from cilia of Tetrahymena. J. biol. Chem. 241, 5590-5596. LAGERSPETZ K. Y. H. & DUBrrSCHER I. (1966) Temperature acclimation of the ciliary activity in the gills of Anodonta. Comp. Biochem. Physiol. 17, 665-671. LAGERSPETZ K. Y. H., IMPiVAARAH. & SENIUS K. (19,70) Acetylcholine in the thermal resistance acclimation of the ciliary activity in the gills of Anodonta. Comp. yen. Pharmac. 1, 236-240.
157
LOWRY O. H., ROSEBROUGHN. J., EARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MURAKAMIA. & TAKAHASHIK. (1975) The role of calcium in the control of ciliary movement in Mytilus. II. The effects of calcium ionophores X537A and A23187 on the lateral gill cilia. J. Fac. Sci. Tokyo Univ., Sect. IV, 13, 251-256. NAITOH Y. E¢ KANEKO H. (1973) Control of ciliary activities by adenosinetriphosphate and divalent cations in Tritonextracted models of Paramecium caudatum. J. exp. Biol. 58, 657-676. NAKAMARUY. & KONISm K. (1968) Elicitation of the brain microsomal (Mg 2 + + Ca 2 ÷)-activated ATPase by digitonin treatment. Biochim. biophys. Acta 159, 206-208. PFEIFFER D. R., REED P. W. & LADY H. A. (1974) Ultraviolet and fluorescent spectral properties of the divalent cation ionophore A23187 and its metal ion complexes. Biochemistry 13, 4007--4014. PUSZKIN S., BERL S., PtJSZKIN E. & CLARKE D. D. (1968) Actomyosin-like protein isolated from mammalian brain. Science 161, 170-171. PUSZKtN S., NICKLAS W. J. & BERL S. (1972) Actomyosinlike protein in brain: subcellular distribution. J. Neurochem. 19, 1319-1333. ROUFOGALIS B. D. (1973) Properties of a (Mg 2+ + Ca2+) dependent ATPase of bovine brain cortex. Effects of detergents, freezing, cations and local anesthetics. Biochim. biophys. Acta 318, 360-370. SATIn P. (1975) Ionophore-mediated calcium entry induces mussel gill ciliary arrest. Science 190, 586-588. SCnATZMANN H. J. (1966) ATP-dependent Ca + +-extrusion from human red cells. Experientia 22, 364-365. SCHLmPEg C. & KOWALSKX R. (1956) Quantitative Beobachtungen fiber physiologische Ionenwirkungen im Brackwasser. Kieler Meeresf. 12, 154-165. SENIUS K. E. O. (1975a) Thermal resistance acclimation of ciliary activity in the gills of fresh water mussels Anodonta anatina and Anodonta cy#nea. Comp. Biochem. Physiol. 51C, 157-160. SENIUS K. E. O. (1975b) The thermal resistance and thermal resistance acclimation of ciliary activity in the Mytilus gills. Comp. Biochem. Physiol. 51A, 957-961. SENIUS K. E. O. (1977) Thermal resistance of the ciliary activity in the gills of the fresh water mussel Anodonta anatina. J. ther. Biol. 2, 233--238. SENtUS K. E. O. & L^6EgSPE'rz K. Y. H. (19741 The role of cholinergic receptors in the thermal resistance and thermal resistance acclimation of the ciliary activity in the Anodonta gills. Comp. yen. Pharmac. 5, 169-179. Tst:cntYA T. (1976) Ca-induced arrest response in Tritonextracted lateral cilia of Mytilus gill. Experientia 32, 1439-1440. TsucmvA T. (1977) Effects of calcium ions on Tritonextracted lamellibranch gill cilia: Ciliary arrest response in a model system. Comp. Biochem. Physiol. 56A, 353-361. WATSON M. R. & HOPKINS J. M. (1962) Isolated cilia from Tetrahymena pyriformis. Exptl Cell Res. 28, 280-295. WILLINGHAM M. C., OSTLUND R. E. ~ PmT^.~ I. (1974) Myosin is a component of the cell surface of cultured cells. Proc. Nat. Acad. Sci. USA 71, 4 1 ~ 4148. Key Word Index--Thermal resistance; ciliary activity; acetylcholine; choline; tetramethylammonium; ionophore A23187; calcium; magnesium; ATPase; gill tissue; Anodonta cyonea cellensis.