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Aluminium, phosphatase activities and shell-repair. Studies in the snail Helix pomatia L.
Vol. 96C,
Camp. Biochem. Physiol.
NO. 2, pp. 357-360,
0306-4492/90$3.00+ 0.00 0 1990Pergamon Press plc
1990
Printed in Great Britain
ALUMINIUM, PHOSPHATASE ACTIVITIES AND SHELL-REPAIR. STUDIES IN THE SNAIL HELIX POMA TIA L. MICHAEL REINE~KGG Department of Zoophysiology, Uppsala University, Box 560, S-751 22 Uppsala, Sweden (Telephone: 46 18 182500) (Received
27 February
1990)
Abstract-l. Aluminum, injected as AlCl, in the snail Helix pomatiu L., affected the activities of both acid and alkaline phosphatase in homogenates prepared from the mantle. 2. In snails with intact as well as damaged shells the activity of acid phosphatase was increased by Al, while that of alkaline phosphatase was decreased. 3. In the shell-damaged snails the activity of the acid phosphatase was approximately 15% lower and the activity of the alkaline phosphatase roughly 20% higher than that of the intact snails receiving the same Al-dose. 4. As the changes in enzyme activity associated with shell-repair are unaffected by Al, it is concluded that reduced weights of the shell-repair membranes and effects on the phosphatases, although both a result of the Al-injections, are independent of each other
INTRODUCTION phosphatase is an enzyme believed to be involved in the repair of an injury to the molluscan shell, a process shown by Reineskog (1990) to be inhibited by aluminium (Al). The activity of the enzyme, which is suggested to be involved in the production of the organic matrix of the repaired shell, in the removal of crystallization inhibitors, or in the deposition of calcium (Durning, 1957; Kado, 1960; Ganagarajah and Saleuddin, 1972; Timmermanns, 1973), is increased in the mantle, the tissue responsible for shell-repair, following shell damage (Wagge, 1951; Saleuddin, 1967; Timmermanns, 1973). Inhibitors of the enzyme have also been demonstrated to reduce the calcium deposition in the shell (Kado, 1960). Alkaline phosphatase is widely accepted to be involved also in calcification in vertebrates. This process is also affected by Al and one of several possible causes of the Al-associated vitamin D-resistant dialysis osteomalacia, a human syndrome characterized by an excess of unmineralized bone (Ellis et al., 1979; Goodman, 1986; Andress et al., 1987), has been proposed to be an inhibition, by Al, of alkaline phosphatase (Ganrot, 1986). The activity of the enzyme, which is present in particularly high concentrations in the mineralizing zone of the skeletal bones (Gomori, 1943; Salomon, 1974; Dunham et al., 1983), is, in contrast to the increase usually observed in vitamin D deficiency, normal or even low in the Al-associated osteomalacia (Lieberherr et al., 1982; Ganrot, 1986). Furthermore, alkaline phosphatase has been shown to be affected by Al also in vitro (Bamberger et al., 1968; Lieberherr et al., 1982). The reduced activity of acid phosphatase observed in the molluscan mantle during shell regeneration indicates a role in the repair process, perhaps in the extracellular processing of the organic matrix (Ganagarajah and Saleuddin, 1972; Chan and Alkaline
Saleuddin, 1974) for this enzyme too. As in vitro studies have shown the acid phosphatase of vertebrate bone to be affected by Al (Lieberherr et al., 1982), the molluscan enzyme, too, might be inhibited by this metal. The aim of the present study was to experimentally investigate the in vivo effects of Al on the activities of acid and alkaline phosphatase in the mantle of the snail Helix pomatia L. during shell-repair, and to correlate the possible effects on these enzymes with the Al-induced reduction of the weights of the repair membranes as observed by Reineskog (1990). Also, in vitro effects of Al on the phosphatase activities were to be studied. To investigate if possible effects of Al are limited to the mantle only, the enzyme activities in the kidneys and digestive glands, too, were to be determined. MATERIALS AND
METHODS
Snails, Helix pomatio L., were collected and kept in the laboratory as described by Reineskog (1990). The animals, all adults with a mean weight of approximately 25 g, were divided in 5 groups (A-E). On day 1 of the experiment shell-damage was inflicted according to Reineskog (1990) on 10 of the 18 snails of group A, in 8 of group B (16 animals), 10 of group C (18 animals), 6 of group D (12 animals) and 10 of group E (18 animals). The weights of the resulting shell-repair membranes, removed on day 2 (these membranes being discarded), on day 3, and, following repeated damage on day 8, on day 9, were then recorded. On days 4, 5 and 6 the snails were injected, once daily, with lOpl/g body weight of the following substances: group A (controls) with 200 mM NaCl, group B with 150 mM HCl, and groups C, D and E with AlCL of the concentrations 10. 50 and l&3 mM, respectively. All-solutions except 100 mM AlCl, and 150 mM HCl were adiusted. with HCl. to DH 3.2. the pH of the most concentraied Al-solution. ‘On’day 9: following the removal of the membranes, the snails were killed by decapitation. The shell was removed and the part of the mantle approximately underlying the area of
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MICHAELREINESKOG
shell-damage (or the corresponding part in the snails without injury) was immediately cut out and homogenized in 3 ml ice-cold 50mM Tris-H,SO,, pH 7.80. The homogenates were diluted with Tris-H, SO, to a volume of approximately 5 ml and centrifuged at 1OOOgfor 10 min; the pellets being discarded. Following determination of protein according to Lowry ef al. (1951), the homogenates were frozen at - 20°C. Kidney and digestive gland homogenates were prepared in the same way from tissues of 5 shelldamaged snails from each of groups A and E. The activities of both acid and alkaline phosphatase were determined as described by Wheeler and Harrison (1982), using p-nitrophenylphosphate as a substrate, with the following modifications: the acid assays were performed at pH 5.5 in a 55 mM Na-succinate buffer and the alkaline ones at pH 10.5 in 55 mM Na-glycinate. The assays were
run for 20 min at 20°C using homogenates not more than 2 weeks old. For the in vitrostudies Al was added to the assay buffers in a volume of 100 ~1 to give different final concentrations of between 1 nM and 1 mM, the concentrations being increased in steps of 10 times. The pH of each Al-containing buffer was checked and, if necessary, adjusted to the control value with NaOH. Homogenates from 10 snails of group A, 5 with intact and 5 with damaged shells, were used for the in vitro studies of both acid and alkaline phosphatase. All chemicals used were of analytical grade or, as AlCl, (Fisher Scientific Co., Fair Lawn, NJ, Lot No. 711755), of certified grade. The Student’s r-test (P < 0.05 being considered significant) was employed for statistical evaluation of the experimental data. RESULTS In vivo experiments
The activity of the acid phosphatase in the mantle was, in both intact and shell-damaged snails, found to be significantly increased by the Al-treatments (Fig. 1). However, the enzyme activities of the damaged animals were regardless of the type of treatment, including injections of NaCl and HCl, on a level approximately 15% below that of the correspondingly-treated undamaged snails. The
enzyme activities in the digestive glands and kidneys of the shell-damaged and AlCl,-injected snails of group E were not different from those recorded in the shell-damaged animals of group A (NaCl); the mean activities of these groups being 380 + 58 and 33 f 3.7 pkat/kg protein in the digestive glands and kidneys, respectively. The alkaline phosphatase activities in the mantles of both intact and damaged snails were, in contrast to those of acid phosphatase, significantly decreased by the Al-injections (Fig. 2). Also in contrast to the acid phosphatase, the activity of the alkaline phosphatase was increased by shell-damage; roughly 20% for AlCl,- as well as NaCl- and HCl-injected snails. The kidneys of the shell-damaged snails of group E showed enzyme activities (100 + 9.0 p kat/kg protein) significantly lower than those of the shelldamaged animals of group A (170 +_8.3 pkat/kg protein). No such difference could be detected concerning the digestive glands; the activities of both groups being approximately 160 p kat/kg protein. There were no significant differences between the mean membrane weights of the treatment groups on day 3, before the injections. However, on day 9 the membrane weights of groups A (5.84 f 0.984mg) and B (5.74 + 0.847 mg) were significantly increased, whereas those of the Al-injected groups C, D and E (3.95kO.566, 4.10f1.07 and 2.78+1.15mg, respectively) were significantly decreased, all compared with the mean weight (4.80 f 0.989 mg) of day 3. The activity of the acid phosphatase and the weights of the shell-repair membranes are both related to the Al-dose, as well as to each other. The alkaline phosphatase activity is also related to both membrane weight and Al-dose, and also to the activity of the acid phosphatase (Table 1). In vitro experiments The activities of both acid and alkaline phosphatase were, in intact as well as in damaged snails, unaffected by all the Al-concentrations tested.
+
200 T
0'
1
0 10
[AlClnl
100
50
(mM)
0
I 0 10
I
I
50
[AlClnl
100
(mM)
Fia. 1. Acid (a) and alkaline (b) nhosnhatase activities (mean f SD) in mantle homogenates from snails (H\/ix porn& L.) injected with‘nond (200mM NaCl instead), 10, 50 or IOOmM AlCl, (circles). Also shown are the activities in animals receiving 150 mM HCl (trianales). Activities recorded in snails with damaged shells (closed symbols) were signilficantly different (Student’s t-test, P < 0.05) compared with those of correspondingly injected intact snails (open symbols) for all treatments except 10 mM AlCl, (acid phosphatase) and 100 mM AlCl, (alkaline phosphatase). Significant differences within each type of shell treatment, from the controls (NaCl) and from both the controls and the snails receiving 10 mM AlCl,, are indicated by * and **, respectively.
Al and shell repair in snail
359
Table 1. Correlation coefficients (r) and the probability of dependence (P) between Al-dose, mantle activities of acid and alkaline phosphatase, and weights of the shell-repair membranes Variahlm
Al-dose and membrane weights Al-dose and acid phosphatase Al-dose and alkaline phosphatase Acid phosphatase and membrane weights Alkaline phosphatase and membrane weights Acid and alkaline phosphatase
DISCUSSION
The activities of both acid and alkaline phosphatase in the mantles were affected, in a dose-dependent manner, by Al. These changes are, since the enzyme activity in HCl-injected snails did not differ from that in the controls, apparently not the result of the simultaneously introduced protons. As none of the substances injected, including Al, altered the relationship between similarly injected intact and damaged snails, the damage-induced changes in enzyme activities are unaffected by these injections. In the mantle Al thus affects a part of the enzyme activity, for acid as well as for alkaline phosphatase, that is not directly associated with shell-repair. The increased activity of the alkaline phosphatase in the mantles recorded 24 hr after shell-damage is in agreement with earlier observations (Wagge, 1951; Saleuddin, 1969; Ganagarajah and Saleuddin, 1972). Saleuddin (1969) suggested that this increase actually is a result of an increased activity in the digestive gland. The enzyme is transported from the gland to the mantle, increasing the activity in the latter tissue more than that in the former. The activity of the alkaline phosphatase in the kidneys of the shelldamaged snails was found to be reduced by Al, while the activity in the digestive glands was unaffected by the same treatment. This implies, if the suggestion of Saleuddin (see above) is taken into consideration, that the enzyme activity in the kidney, as well as that in the mantle, might be decreased by Al, while that in the digestive gland itself and that exported to the mantle during shell-repair, is unaffected. As discussed by Reineskog (1990), using the equations presented by Martin (1986), the highest possible concentrations of both free AI’+ and of total soluble Al is present in the haemolymph of all the Al-injected snails. The low concentration of free A13+, calculated to be only 20 pM in the hemolymph of a 25 g snail injected with 10 mM AlCl,, in combination with the lack of Al-effects in the in vitro experiments of the present study, in which all solutions had the highest possible free A13+-concentration, suggest that Al has no direct effect on either alkaline or acid phosphatase. This lack of in vitro effects stands in contrast to previous reports. Lieberherr et al. (1982), using cultured rat calvaria, found the alkaline phosphatase activity to be affected by Al in the same concentration range as that used in the present study. Moreover, Bamberger et al. (1968), using alkaline phosphatase of bovine intestinal origin, found 1 mM Al to reduce the activity with approximately 20%. Thus, phosphatase from different sources, or assays of different types, may give different results.
r
P
-0.662 0.724 0.538 -0.634 -0.767 -0.638 0.663 - 0.424 -0.583
Intact shell Damaged shell Intact shell Damaged shell
Intact shell Damaged shell
The presence of a metal-detoxifying system consisting of metal-binding mineralized phosphate-containing granules in the digestive gland (Simkiss and Mason, 1983), but not in the other tissues, may possibly explain the observed differences between the tissues regarding the alkaline phosphatase activity. Aluminium in the digestive gland is incorporated into the granules as AlPO, and eventually excreted with the faeces. In the mantle and kidney no such phosphate-containing granules exist; Al, shown by OndreiEka et al. (1966) to disturb the phosphate metabolism, might thus interact with the substrate(s) of alkaline phosphatase. Reduced amounts of the substrate(s) may then result in an Al-dose-dependent down-regulation of the production of alkaline phosphatase. The actual substrate(s) of the enzyme is not known, several compounds being equally well hydrolyzed by the digestive gland enzyme (Dumitru et al., 1983). The possibility that shell-damage induces the production of isoenzymes with different characteristics, regarding substrates and Al-interactions, in the different tissues, also exists. However, the isoenzymes found by Saleuddin (1969) in Anodonru, were equally present in both mantles, kidneys and digestive glands. In the digestive gland of Helix only one form seems to be present (Principato et al., 1982; Dumitru et al., 1983); the situation in the other tissues being unknown. The decreased activity of the acid phosphatase in the mantle recorded 24 hr after shell-damage is in agreement with previous observations (Ganagarajah and Saleuddin, 1972; Chan and Saleuddin, 1974). The enzyme, proposed to be involved in the extracellular processing of the organic matrix, may be released in increased amounts following shell-damage; the apparent activity in the mantle tissue thus being reduced. The endocytosis of particulate Al (AlPOd or Al associated with protein) may explain the Al-doserelated increase in acid phosphatase activity recorded in the mantle. The endocytosis as such is associated with an increased enzyme activity attributable to the formation of secondary lysosomes (Ryder and Bowen, 1977). The storage of Al as AlPO, may then increase the demand for phosphate ions furthermore. This probably requires an increased activity of the acid phosphatase, especially as the availability of substrate may be reduced as a consequence of a disturbed phosphorus metabolism. Such an increase, recorded in the mantles of both intact and shelldamaged snails, could explain the differences related to the Al-dose. The way the investigated parameters are correlated to each other (Table 1) implies that it still remains to be settled if the decreased membrane weights
MICHAELREINESKOG
360
resulting from the Al-injections are a consequence of altered phosphatase activities, or if the decreased membrane weights and the altered enzyme activities, though both a result of the Al-injections, are independent of each other. The latter seems more plausible since the changes in enzyme activity related to shellrepair are unaffected by aluminium. Acknowledgements-This work was financially supported by grants -from the Swedish Council for Planning and Coordination of Research to Professor Jan-Erik KihlstrBm. REFERENCES Andress D. L., Maloney N. A., Coburn J. W., Endres D. B. and Sherrard D. J. (1987) Osteomalacia and aplastic bone disease in aluminum-related osteodystrophy. J. clin. Endocr. Merab. 65, 11-16. Bamberger C. E., Botbol J. and Cabrini R. L. (1968) Inhibition of alkaline phosphatase by beryllium and aluminum. Archs Biochem. Biophys. 123, 195-200. Chan J. F. Y. and Saleuddin A. S. M. (1974) Acid phosphatase in the mantle of the shell-regenerating snail Helisoma duryi duryi. Calcif. Tissue Res. 15, 213-220. Dumitru I. F., IordHchescu D. and Constantin D. (1983) Evidenciation and purification of one alkaline phosphatase from the hepatopancreas of Helixpomatia Rev. roum. Biochim. 20, 21-27.
Dunham J., Catterall A., Bitensky L. and Chayen J. (1983) Metabolic changes in the callus during fracture healing in the rat. C&if. Tiss. Inr. 35, 56-61. Duming W. C. (1957) Repair of a defect in the shell of the snail Helix aspersa. J. Bone Joinf Surg. 39A, 377-393. Ellis H. A., McCarthy J. H. and Herrington J. (1979) Bone aluminium in haemodialysed patients and in rats injected with aluminium chloride: relationship to impaired bone mineralisation. J. clin. Path. 32, 8321844. _ Ganaearaiah M. and Saleuddin A. S. M. (1972) Electron hi&chemistry of the outer mantle epithelium in Helix pomatia during shell regeneration. Proc. M&c. Sot. Lond. 40, 71-77.
Ganrot P. 0. (1986) Metabolism and possible health effects of aluminum. Environ. Hlth Persp.-65, 363441. Gomori G. (1943) Calcification and phosphatase. Am. J. Pathol. 19, 197-205.
Goodman W. G. (1986) Experimental aluminum-induced bone disease: studies in uivo. Kidney Znt. 29, (Suppl. 18), 32.-36.
Kado Y. (1960) Studies on shell formation in Mollusca. J. Sci. Hiroshima Univ. Ser BI 19, 163-210. Lieberherr M., Grosse B., Cournot-Witmer G., Thil C. L. and Balsan S. (1982) In oitro effects of aluminum on bone phosphatases: a possible interaction with bPTH and vitamin D, metabolites. Calcif. Tissue Znt. 34, 280-284. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-215. Martin R. B. (1986) The chemistry of aluminum as related to biology and medicine Clin. khem. 32, 1797-1806. OndreiEka R.. Ginter E. and Kortus J. (1966) Chronic toxicity of aluminium in rats and mice and its’effects on phosphorus metabolism. Br. J. ind. Med. 23, 305-312. Princioato G. B.. Aisa M. C.. Biaaioni M. and Giovannini E. (1982) Partial purification agd characterization of an alkaline phosphatase in Helix nemoralis and in Octopus vulgaris. Camp. Biochem. Physiol. 72B, 325-329.
Reineskog M. (1989) Aluminium chloride injected in the snail Helix pomatia L. decreases the weights and calcium contents of the shell-repair membranes. Comp. Biochem. Physiol. 94C, 695-701.
Ryder T. A. and Bowen I. D. (1977) Endocytosis and aspects of autophagy in the foot epithelium of the slug Agriolimax reticulatus. Cell Tissue Res. 181, 129-142. Saleuddin A. S. M. (1967) The histochemistry of the mantle during the early stage of shell repair. Proc. Malac, Sot. Lond. 37, 371-379.
Saleuddin A. S. M. (1969) Isoenzymes of alkaline phosphatase in Anodonta grandis (Bivalvia: Unionidae) during shell regeneration. Malacologia 9, 501-508. Salomon C. D. (1974) A fine structural study on the extracellular activity of alkaline phosphatase and its role in calcification. Calcif. Tissue Res. 15,201-212. Simkiss K. and Mason A. Z. (1983) Metal ions: metabolic and toxic effects. In The Mollusca, Vol 2: Environmental Biochemistry and Physiology (Edited by Wilbur K. M. and Hochachka P. W.), pp. 101-164. Academic Press, New York. Timmermans L. P. M. (1973) Mantle activity following shell injury in the pond snail Lymnea stagnalis L. Malacologia 14, 53-61.
Wagge L. E. (1951) The activity of amoebocytes and of alkaline phosphatases during the regeneration of the shell in the snail, Helix aspersa. Q. J. microsc. Sci. 92,307-321. Wheeler A. P. and Harrison E. W. (1982) Subcellular localization and characterization of HCO; -ATPase from the mantle of the fresh-water clam Anodonta cataracta. Comp. Biochem. Physiol. 71B, 629-636.
Camp. Biochem. Physiol.
NO. 2, pp. 357-360,
0306-4492/90$3.00+ 0.00 0 1990Pergamon Press plc
1990
Printed in Great Britain
ALUMINIUM, PHOSPHATASE ACTIVITIES AND SHELL-REPAIR. STUDIES IN THE SNAIL HELIX POMA TIA L. MICHAEL REINE~KGG Department of Zoophysiology, Uppsala University, Box 560, S-751 22 Uppsala, Sweden (Telephone: 46 18 182500) (Received
27 February
1990)
Abstract-l. Aluminum, injected as AlCl, in the snail Helix pomatiu L., affected the activities of both acid and alkaline phosphatase in homogenates prepared from the mantle. 2. In snails with intact as well as damaged shells the activity of acid phosphatase was increased by Al, while that of alkaline phosphatase was decreased. 3. In the shell-damaged snails the activity of the acid phosphatase was approximately 15% lower and the activity of the alkaline phosphatase roughly 20% higher than that of the intact snails receiving the same Al-dose. 4. As the changes in enzyme activity associated with shell-repair are unaffected by Al, it is concluded that reduced weights of the shell-repair membranes and effects on the phosphatases, although both a result of the Al-injections, are independent of each other
INTRODUCTION phosphatase is an enzyme believed to be involved in the repair of an injury to the molluscan shell, a process shown by Reineskog (1990) to be inhibited by aluminium (Al). The activity of the enzyme, which is suggested to be involved in the production of the organic matrix of the repaired shell, in the removal of crystallization inhibitors, or in the deposition of calcium (Durning, 1957; Kado, 1960; Ganagarajah and Saleuddin, 1972; Timmermanns, 1973), is increased in the mantle, the tissue responsible for shell-repair, following shell damage (Wagge, 1951; Saleuddin, 1967; Timmermanns, 1973). Inhibitors of the enzyme have also been demonstrated to reduce the calcium deposition in the shell (Kado, 1960). Alkaline phosphatase is widely accepted to be involved also in calcification in vertebrates. This process is also affected by Al and one of several possible causes of the Al-associated vitamin D-resistant dialysis osteomalacia, a human syndrome characterized by an excess of unmineralized bone (Ellis et al., 1979; Goodman, 1986; Andress et al., 1987), has been proposed to be an inhibition, by Al, of alkaline phosphatase (Ganrot, 1986). The activity of the enzyme, which is present in particularly high concentrations in the mineralizing zone of the skeletal bones (Gomori, 1943; Salomon, 1974; Dunham et al., 1983), is, in contrast to the increase usually observed in vitamin D deficiency, normal or even low in the Al-associated osteomalacia (Lieberherr et al., 1982; Ganrot, 1986). Furthermore, alkaline phosphatase has been shown to be affected by Al also in vitro (Bamberger et al., 1968; Lieberherr et al., 1982). The reduced activity of acid phosphatase observed in the molluscan mantle during shell regeneration indicates a role in the repair process, perhaps in the extracellular processing of the organic matrix (Ganagarajah and Saleuddin, 1972; Chan and Alkaline
Saleuddin, 1974) for this enzyme too. As in vitro studies have shown the acid phosphatase of vertebrate bone to be affected by Al (Lieberherr et al., 1982), the molluscan enzyme, too, might be inhibited by this metal. The aim of the present study was to experimentally investigate the in vivo effects of Al on the activities of acid and alkaline phosphatase in the mantle of the snail Helix pomatia L. during shell-repair, and to correlate the possible effects on these enzymes with the Al-induced reduction of the weights of the repair membranes as observed by Reineskog (1990). Also, in vitro effects of Al on the phosphatase activities were to be studied. To investigate if possible effects of Al are limited to the mantle only, the enzyme activities in the kidneys and digestive glands, too, were to be determined. MATERIALS AND
METHODS
Snails, Helix pomatio L., were collected and kept in the laboratory as described by Reineskog (1990). The animals, all adults with a mean weight of approximately 25 g, were divided in 5 groups (A-E). On day 1 of the experiment shell-damage was inflicted according to Reineskog (1990) on 10 of the 18 snails of group A, in 8 of group B (16 animals), 10 of group C (18 animals), 6 of group D (12 animals) and 10 of group E (18 animals). The weights of the resulting shell-repair membranes, removed on day 2 (these membranes being discarded), on day 3, and, following repeated damage on day 8, on day 9, were then recorded. On days 4, 5 and 6 the snails were injected, once daily, with lOpl/g body weight of the following substances: group A (controls) with 200 mM NaCl, group B with 150 mM HCl, and groups C, D and E with AlCL of the concentrations 10. 50 and l&3 mM, respectively. All-solutions except 100 mM AlCl, and 150 mM HCl were adiusted. with HCl. to DH 3.2. the pH of the most concentraied Al-solution. ‘On’day 9: following the removal of the membranes, the snails were killed by decapitation. The shell was removed and the part of the mantle approximately underlying the area of
357
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MICHAELREINESKOG
shell-damage (or the corresponding part in the snails without injury) was immediately cut out and homogenized in 3 ml ice-cold 50mM Tris-H,SO,, pH 7.80. The homogenates were diluted with Tris-H, SO, to a volume of approximately 5 ml and centrifuged at 1OOOgfor 10 min; the pellets being discarded. Following determination of protein according to Lowry ef al. (1951), the homogenates were frozen at - 20°C. Kidney and digestive gland homogenates were prepared in the same way from tissues of 5 shelldamaged snails from each of groups A and E. The activities of both acid and alkaline phosphatase were determined as described by Wheeler and Harrison (1982), using p-nitrophenylphosphate as a substrate, with the following modifications: the acid assays were performed at pH 5.5 in a 55 mM Na-succinate buffer and the alkaline ones at pH 10.5 in 55 mM Na-glycinate. The assays were
run for 20 min at 20°C using homogenates not more than 2 weeks old. For the in vitrostudies Al was added to the assay buffers in a volume of 100 ~1 to give different final concentrations of between 1 nM and 1 mM, the concentrations being increased in steps of 10 times. The pH of each Al-containing buffer was checked and, if necessary, adjusted to the control value with NaOH. Homogenates from 10 snails of group A, 5 with intact and 5 with damaged shells, were used for the in vitro studies of both acid and alkaline phosphatase. All chemicals used were of analytical grade or, as AlCl, (Fisher Scientific Co., Fair Lawn, NJ, Lot No. 711755), of certified grade. The Student’s r-test (P < 0.05 being considered significant) was employed for statistical evaluation of the experimental data. RESULTS In vivo experiments
The activity of the acid phosphatase in the mantle was, in both intact and shell-damaged snails, found to be significantly increased by the Al-treatments (Fig. 1). However, the enzyme activities of the damaged animals were regardless of the type of treatment, including injections of NaCl and HCl, on a level approximately 15% below that of the correspondingly-treated undamaged snails. The
enzyme activities in the digestive glands and kidneys of the shell-damaged and AlCl,-injected snails of group E were not different from those recorded in the shell-damaged animals of group A (NaCl); the mean activities of these groups being 380 + 58 and 33 f 3.7 pkat/kg protein in the digestive glands and kidneys, respectively. The alkaline phosphatase activities in the mantles of both intact and damaged snails were, in contrast to those of acid phosphatase, significantly decreased by the Al-injections (Fig. 2). Also in contrast to the acid phosphatase, the activity of the alkaline phosphatase was increased by shell-damage; roughly 20% for AlCl,- as well as NaCl- and HCl-injected snails. The kidneys of the shell-damaged snails of group E showed enzyme activities (100 + 9.0 p kat/kg protein) significantly lower than those of the shelldamaged animals of group A (170 +_8.3 pkat/kg protein). No such difference could be detected concerning the digestive glands; the activities of both groups being approximately 160 p kat/kg protein. There were no significant differences between the mean membrane weights of the treatment groups on day 3, before the injections. However, on day 9 the membrane weights of groups A (5.84 f 0.984mg) and B (5.74 + 0.847 mg) were significantly increased, whereas those of the Al-injected groups C, D and E (3.95kO.566, 4.10f1.07 and 2.78+1.15mg, respectively) were significantly decreased, all compared with the mean weight (4.80 f 0.989 mg) of day 3. The activity of the acid phosphatase and the weights of the shell-repair membranes are both related to the Al-dose, as well as to each other. The alkaline phosphatase activity is also related to both membrane weight and Al-dose, and also to the activity of the acid phosphatase (Table 1). In vitro experiments The activities of both acid and alkaline phosphatase were, in intact as well as in damaged snails, unaffected by all the Al-concentrations tested.
+
200 T
0'
1
0 10
[AlClnl
100
50
(mM)
0
I 0 10
I
I
50
[AlClnl
100
(mM)
Fia. 1. Acid (a) and alkaline (b) nhosnhatase activities (mean f SD) in mantle homogenates from snails (H\/ix porn& L.) injected with‘nond (200mM NaCl instead), 10, 50 or IOOmM AlCl, (circles). Also shown are the activities in animals receiving 150 mM HCl (trianales). Activities recorded in snails with damaged shells (closed symbols) were signilficantly different (Student’s t-test, P < 0.05) compared with those of correspondingly injected intact snails (open symbols) for all treatments except 10 mM AlCl, (acid phosphatase) and 100 mM AlCl, (alkaline phosphatase). Significant differences within each type of shell treatment, from the controls (NaCl) and from both the controls and the snails receiving 10 mM AlCl,, are indicated by * and **, respectively.
Al and shell repair in snail
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Table 1. Correlation coefficients (r) and the probability of dependence (P) between Al-dose, mantle activities of acid and alkaline phosphatase, and weights of the shell-repair membranes Variahlm
Al-dose and membrane weights Al-dose and acid phosphatase Al-dose and alkaline phosphatase Acid phosphatase and membrane weights Alkaline phosphatase and membrane weights Acid and alkaline phosphatase
DISCUSSION
The activities of both acid and alkaline phosphatase in the mantles were affected, in a dose-dependent manner, by Al. These changes are, since the enzyme activity in HCl-injected snails did not differ from that in the controls, apparently not the result of the simultaneously introduced protons. As none of the substances injected, including Al, altered the relationship between similarly injected intact and damaged snails, the damage-induced changes in enzyme activities are unaffected by these injections. In the mantle Al thus affects a part of the enzyme activity, for acid as well as for alkaline phosphatase, that is not directly associated with shell-repair. The increased activity of the alkaline phosphatase in the mantles recorded 24 hr after shell-damage is in agreement with earlier observations (Wagge, 1951; Saleuddin, 1969; Ganagarajah and Saleuddin, 1972). Saleuddin (1969) suggested that this increase actually is a result of an increased activity in the digestive gland. The enzyme is transported from the gland to the mantle, increasing the activity in the latter tissue more than that in the former. The activity of the alkaline phosphatase in the kidneys of the shelldamaged snails was found to be reduced by Al, while the activity in the digestive glands was unaffected by the same treatment. This implies, if the suggestion of Saleuddin (see above) is taken into consideration, that the enzyme activity in the kidney, as well as that in the mantle, might be decreased by Al, while that in the digestive gland itself and that exported to the mantle during shell-repair, is unaffected. As discussed by Reineskog (1990), using the equations presented by Martin (1986), the highest possible concentrations of both free AI’+ and of total soluble Al is present in the haemolymph of all the Al-injected snails. The low concentration of free A13+, calculated to be only 20 pM in the hemolymph of a 25 g snail injected with 10 mM AlCl,, in combination with the lack of Al-effects in the in vitro experiments of the present study, in which all solutions had the highest possible free A13+-concentration, suggest that Al has no direct effect on either alkaline or acid phosphatase. This lack of in vitro effects stands in contrast to previous reports. Lieberherr et al. (1982), using cultured rat calvaria, found the alkaline phosphatase activity to be affected by Al in the same concentration range as that used in the present study. Moreover, Bamberger et al. (1968), using alkaline phosphatase of bovine intestinal origin, found 1 mM Al to reduce the activity with approximately 20%. Thus, phosphatase from different sources, or assays of different types, may give different results.
r
P
-0.662 0.724 0.538 -0.634 -0.767 -0.638 0.663 - 0.424 -0.583
Intact shell Damaged shell Intact shell Damaged shell
Intact shell Damaged shell
The presence of a metal-detoxifying system consisting of metal-binding mineralized phosphate-containing granules in the digestive gland (Simkiss and Mason, 1983), but not in the other tissues, may possibly explain the observed differences between the tissues regarding the alkaline phosphatase activity. Aluminium in the digestive gland is incorporated into the granules as AlPO, and eventually excreted with the faeces. In the mantle and kidney no such phosphate-containing granules exist; Al, shown by OndreiEka et al. (1966) to disturb the phosphate metabolism, might thus interact with the substrate(s) of alkaline phosphatase. Reduced amounts of the substrate(s) may then result in an Al-dose-dependent down-regulation of the production of alkaline phosphatase. The actual substrate(s) of the enzyme is not known, several compounds being equally well hydrolyzed by the digestive gland enzyme (Dumitru et al., 1983). The possibility that shell-damage induces the production of isoenzymes with different characteristics, regarding substrates and Al-interactions, in the different tissues, also exists. However, the isoenzymes found by Saleuddin (1969) in Anodonru, were equally present in both mantles, kidneys and digestive glands. In the digestive gland of Helix only one form seems to be present (Principato et al., 1982; Dumitru et al., 1983); the situation in the other tissues being unknown. The decreased activity of the acid phosphatase in the mantle recorded 24 hr after shell-damage is in agreement with previous observations (Ganagarajah and Saleuddin, 1972; Chan and Saleuddin, 1974). The enzyme, proposed to be involved in the extracellular processing of the organic matrix, may be released in increased amounts following shell-damage; the apparent activity in the mantle tissue thus being reduced. The endocytosis of particulate Al (AlPOd or Al associated with protein) may explain the Al-doserelated increase in acid phosphatase activity recorded in the mantle. The endocytosis as such is associated with an increased enzyme activity attributable to the formation of secondary lysosomes (Ryder and Bowen, 1977). The storage of Al as AlPO, may then increase the demand for phosphate ions furthermore. This probably requires an increased activity of the acid phosphatase, especially as the availability of substrate may be reduced as a consequence of a disturbed phosphorus metabolism. Such an increase, recorded in the mantles of both intact and shelldamaged snails, could explain the differences related to the Al-dose. The way the investigated parameters are correlated to each other (Table 1) implies that it still remains to be settled if the decreased membrane weights
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resulting from the Al-injections are a consequence of altered phosphatase activities, or if the decreased membrane weights and the altered enzyme activities, though both a result of the Al-injections, are independent of each other. The latter seems more plausible since the changes in enzyme activity related to shellrepair are unaffected by aluminium. Acknowledgements-This work was financially supported by grants -from the Swedish Council for Planning and Coordination of Research to Professor Jan-Erik KihlstrBm. REFERENCES Andress D. L., Maloney N. A., Coburn J. W., Endres D. B. and Sherrard D. J. (1987) Osteomalacia and aplastic bone disease in aluminum-related osteodystrophy. J. clin. Endocr. Merab. 65, 11-16. Bamberger C. E., Botbol J. and Cabrini R. L. (1968) Inhibition of alkaline phosphatase by beryllium and aluminum. Archs Biochem. Biophys. 123, 195-200. Chan J. F. Y. and Saleuddin A. S. M. (1974) Acid phosphatase in the mantle of the shell-regenerating snail Helisoma duryi duryi. Calcif. Tissue Res. 15, 213-220. Dumitru I. F., IordHchescu D. and Constantin D. (1983) Evidenciation and purification of one alkaline phosphatase from the hepatopancreas of Helixpomatia Rev. roum. Biochim. 20, 21-27.
Dunham J., Catterall A., Bitensky L. and Chayen J. (1983) Metabolic changes in the callus during fracture healing in the rat. C&if. Tiss. Inr. 35, 56-61. Duming W. C. (1957) Repair of a defect in the shell of the snail Helix aspersa. J. Bone Joinf Surg. 39A, 377-393. Ellis H. A., McCarthy J. H. and Herrington J. (1979) Bone aluminium in haemodialysed patients and in rats injected with aluminium chloride: relationship to impaired bone mineralisation. J. clin. Path. 32, 8321844. _ Ganaearaiah M. and Saleuddin A. S. M. (1972) Electron hi&chemistry of the outer mantle epithelium in Helix pomatia during shell regeneration. Proc. M&c. Sot. Lond. 40, 71-77.
Ganrot P. 0. (1986) Metabolism and possible health effects of aluminum. Environ. Hlth Persp.-65, 363441. Gomori G. (1943) Calcification and phosphatase. Am. J. Pathol. 19, 197-205.
Goodman W. G. (1986) Experimental aluminum-induced bone disease: studies in uivo. Kidney Znt. 29, (Suppl. 18), 32.-36.
Kado Y. (1960) Studies on shell formation in Mollusca. J. Sci. Hiroshima Univ. Ser BI 19, 163-210. Lieberherr M., Grosse B., Cournot-Witmer G., Thil C. L. and Balsan S. (1982) In oitro effects of aluminum on bone phosphatases: a possible interaction with bPTH and vitamin D, metabolites. Calcif. Tissue Znt. 34, 280-284. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-215. Martin R. B. (1986) The chemistry of aluminum as related to biology and medicine Clin. khem. 32, 1797-1806. OndreiEka R.. Ginter E. and Kortus J. (1966) Chronic toxicity of aluminium in rats and mice and its’effects on phosphorus metabolism. Br. J. ind. Med. 23, 305-312. Princioato G. B.. Aisa M. C.. Biaaioni M. and Giovannini E. (1982) Partial purification agd characterization of an alkaline phosphatase in Helix nemoralis and in Octopus vulgaris. Camp. Biochem. Physiol. 72B, 325-329.
Reineskog M. (1989) Aluminium chloride injected in the snail Helix pomatia L. decreases the weights and calcium contents of the shell-repair membranes. Comp. Biochem. Physiol. 94C, 695-701.
Ryder T. A. and Bowen I. D. (1977) Endocytosis and aspects of autophagy in the foot epithelium of the slug Agriolimax reticulatus. Cell Tissue Res. 181, 129-142. Saleuddin A. S. M. (1967) The histochemistry of the mantle during the early stage of shell repair. Proc. Malac, Sot. Lond. 37, 371-379.
Saleuddin A. S. M. (1969) Isoenzymes of alkaline phosphatase in Anodonta grandis (Bivalvia: Unionidae) during shell regeneration. Malacologia 9, 501-508. Salomon C. D. (1974) A fine structural study on the extracellular activity of alkaline phosphatase and its role in calcification. Calcif. Tissue Res. 15,201-212. Simkiss K. and Mason A. Z. (1983) Metal ions: metabolic and toxic effects. In The Mollusca, Vol 2: Environmental Biochemistry and Physiology (Edited by Wilbur K. M. and Hochachka P. W.), pp. 101-164. Academic Press, New York. Timmermans L. P. M. (1973) Mantle activity following shell injury in the pond snail Lymnea stagnalis L. Malacologia 14, 53-61.
Wagge L. E. (1951) The activity of amoebocytes and of alkaline phosphatases during the regeneration of the shell in the snail, Helix aspersa. Q. J. microsc. Sci. 92,307-321. Wheeler A. P. and Harrison E. W. (1982) Subcellular localization and characterization of HCO; -ATPase from the mantle of the fresh-water clam Anodonta cataracta. Comp. Biochem. Physiol. 71B, 629-636.