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The role of carbonic anhydrase in facilitating the transport of CO2 in the tooth of Lytechinus variegatus (Echinodermata: Echinoidea)
Camp. Biochem. Physiol. Vol. VA, No. 2, pp. 327-331, 1987
0300-9629/87$3.00+ 0.00 0 1987Pergamon Journals Ltd
Printed in Great Britain
THE ROLE OF CARBONIC ANHYDRASE IN FACILITATING THE TRANSPORT OF CO, IN THE TOOTH OF LY7’ECHINUS VARlEGATUS (ECHINODERMATA: ECHINOIDEA) CHANG-PO CHEN* and JOHN M. LAWRENCE Department of Biology, University of South Florida, Tampa, Florida 33620, USA (Received 29 July 1986)
Abstract-l. CO, is the form in which dissolved inorganic carbon enters the tooth from the external medium. 2. Carbonic anhydrase facilitates the transport of CO, from the external medium to the soft tissue and from the soft tissue to the calcareous part of the tooth.
INTRODUCTION
through the membrane and intercellular space to promote calcification. The purpose of this study was to evaluate this hypothesis.
Carbonic anhydrase (CA) catalyzes the reversible hydration of CO,, and is involved in the formation of carbonate in many biological systems (Degens,
1976; Tashian and Hewett-Emett, 1984). The rate of calcification is reduced by inhibitors of CA in regenerating spines of sea urchins (Heatfield, 1970), in the shell of barnacles (Costlow, 1959; Yule et al., 1982), in the cuticle of crabs (Giraud, 1981), in the shell of molluscs (reviewed in Wilbur and Saleuddin, 1983) and in the skeleton of corals (Goreau, 1959). The teeth of sea urchins grow continuously, 1-1.5 mm weekly in Parancentrotus hidus (Mtirkel, 1969) and renew in about 75 days in Strongylocentrotus purpuratus (Holland, 1965). The teeth of S. purpurutus grow continuously, even when individuals are starved (Fransler, 1983). The tooth of the sea urchin is enclosed in a single layer of epithelium. In the growing tip (plumula) of the tooth there are two types of odontoblasts: syncytial and free. In the plumula calcification occurs intracellularly by syncytial odontoblasts. The calcareous part of the tooth is surrounded by two membranes (an outer plasma membrane and an inner vacuolar membrane). Some organic particles occur on the surface of the vacuolar membrane, but no trace of organic matter is found within the calcareous part. Some fibroblasts and collagen fibers are located in the soft part of the tooth as well (Kniprath, 1974; Chen and Lawrence, 1986a). In the plumula of the tooth of the sea urchin Lytechinus uuriegutus, CA is localized (1) intracellularly in the epithelium cells, odontoblasts and fibroblasts, (2) extracellularly near fibroblasts and collagen fibers, and (3) adjacent on or in the plasma membrane and the vacuolar membrane of the calcareous deposits (Chen and Lawrence, 1986b). The morphological characteristics of the plumula and the localization of CA in the plumula suggest that CA functions in facilitating the movement of CO* *Present address: Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan 11529, R.O.C.
MATERIALS AND METHODS Lyrechinusuariegutus(Lamarck) were collected from Sar-
asota, Florida and maintained in the laboratory by feeding them with turtle grass Thalassiatestudinum,ad libitum.The teeth of the sea urchins were removed from the Aristotle’s lantern. The chewing tip and the adjacent hard shaft were wrapped with a soft-wax. The wax served as a buoy during incubation and also as an insulator to prevent physical isotopic exchange (Barnes and Crossland, 1982). The tooth-wax complexes were pre-incubated in the dissolved inorganic carbon-free (DIGfree) medium, then incubated in 3 ml of labeled medium. The DIC-free artificial sea-water was prepared following Horner and Smith (1982) by dissolving Instant OceanrM salts with distilled water up to 34 or 68% salinity, adding concentrated HCl to acidify the solution to a pH of 2.3, bubbling the solution with CO,-free air for 3 hr and finally adjusting the pH with IONNaOH to various pH’s. The labeled medium was prepared by mixing i4C-NaHCO, (New England Nuclear, 10 PCi, 100 fig of NaHCO, in 1 ml distilled water, pH 9.6) with the DIC-free medium. The randomized complete blocks design was used to avoid the difference among individuals (Sokal and Rohlf, 1981). To study the time-course of W-entry. the teeth were incubated in DIC-free medium (pH 7.6) for 40 min and then in the labeled medium with 2 uCi. 0.079 mM NaHCO, for periods ranging from 2 min to 6 hr. To study the s&ies of DIC entry, the teeth were incubated in nonlabeled medium in five different pH solutions (7.0-8.4) for 30min and then in labeled medium (2pCi) for 1.5 hr. The percentage molar fraction of dissolved CO, (including free H,CO,) at various pH’s was taken from Saruhashi’s table (Riley and Skirrow, 1965). To study the effect of CA inhibition, the teeth were incubated in DIC-free medium (PH 7.6) for 30 min. then in labeled medium ~DH 7.6) at five differen; bicarbonate concentrations (0.0?&0.198 mM) without or with acetazolamide (A specific inhibitor of CA, 6.7 x 1Om5M) for 1 hr. After incubation, the tooth-wax complexes were rinsed with tap water for 1 min, then the soft part of the tooth (including the plumula and adjacent soft shaft) was dissolved in I ml of 1 N NaOH at 60°C for I .5hr. The cal-
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Fig. 1. Time course of the entry of 14Cin the soft tissue and the incorporation into the calcareous part of the tooth of the sea urchin Lyrechinus uariegatus (mean + 1 SD). The number of sea urchins used is given in the figure. The negative regression of the “‘C-activity in the soft tissue from 2 to 6 hr is significant (P < 0.05).
+
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Fig. 2. Effect of pH of the medium on the entry of 14Cin the soft tissue and the incorporation into the calcareous part of the tooth of the sea urchin Lytechinus uariegatus (mean + 1 SD, N = 4 for each pH).
subsequent experiments mental conditions, limited to less than 2 hr duration. careous part and soft tissue were separated by centrifuging with a clinical centrifuge for Smin. The supernatant is referred to as the Soft-rissuefraction. The pellets were rinsed with 1 ml of 1 N NaOH solution. After centrifuging, the second supernatant was discarded and the calcareous part was dissolved in 1 ml of ethylenediamine tetraacetic acid (EDTA, 12%. pH9.6) at 60°C for 12-14 hr. This calcareous-part-EDTA solution is referred to as the “calcareous-part fraction”. The protein level of the soft tissue was measured by the Bradford method (Spector, 1978). A 0.2-m! sample of the soft-tissue fraction was acidified with 0.4 ml 1 N HC!, bubbled with air for 10 min and 2 ml of !.25-fold concentrated Bradford reagents added. Bovine serum albumin was used as the protein standard. The “C-activity was counted with a Beckman LS 1OOC liquid scintillation counter. The liquid scintillation fluids of the incubation medium, the soft-tissue fraction and the calcareous-part fraction were prepared as 0.1 ml of incubation medium with 3 ml HandifluorTM (Mallinckrodt), 0.1 ml of the soft-tissue fraction plus 0.5 ml Trizma HC! (0.2 M) and 3 ml of Handifluor and 0.2 ml of the calcareouspart fraction plus 0.4ml distilled water and 6ml of Handifluor, respectively. The counting efficiency was about 70%. After the correction of background counts, the 14C activity was expressed as cpm/pg protein or cpm/pg protein per hr.
were
2. The species of DIC entry When the concentration of DIC was low in the medium, the 14C-activity of the soft tissue decreased proportionally from a high of 125 cpm/pg protein per hr at pH 7.0 to a low of 65 cpm/pg protein per hr at pH 8.4 (Fig. 2). In contrast, the 14C-activity of the calcareous part did not vary significantly with pH. A direct linear relationship was evident between the “C-activity of soft tissue and the percentage molar fraction of dissolved CO2 of the medium (Fig. 3), indicating that CO, is the major C-species which enters the sea-urchin tooth from the external medium. 3. Effect of acetazolamide inhibition of CA When the concentration of external NaHCO, increased from 0.016 to 0.198 mM, the 14C-activity of
RESULTS 1.
Time course of DIC uptake
The time-course of the entry of 14Cin the soft tissue and the incorporation of 14Cinto the calcareous part of the tooth revealed three phases: (1) a quick and linear increase during the first hour, (2) a continuous but slow increase up to the maximal activity during the second hour and (3) a significant decrease in activity in the soft tissue but a maintained activity in the calcareous part (Fig. 1). Because these results indicate that 14Cis incorporated into the tooth only during the first 2 hr of incubation under the experi-
0
I
1
% molar fraction
I
1
,
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Fig. 3. The relationship between the average “C-activity of the soft tissue of the tooth of the sea urchin Lytechinus variegatus and the percentage molar fraction of dissolved CO, (including free H&O,) of the medium. The straight line is fitted by least squares method (r = 0.9964).
CA
in CO, transport
of sea-urchin
tooth
329
DISCUSSION
1. Isotopic exchange us calczjication 0 acetazolamide
I
NaHC031 mM
Fig. 4. Effect of 14C-NaHCO, concentration and acetazolamide on the entry of “‘C in the soft tissue and the incorporation into the calcareous part of the tooth of the sea urchin Lytec&nLts variegutus(mean f 1 SD). The number of sea urchins is given in the figure for the acetatolamidetreated groups, and is six for the control groups. For the soft tissue, the straight line (Y = 6.21 + 1043.9765 X) is fitted by least squares method (r = 0.9995), the curve is fitted by the double reciprocal plot (r = 0.8506, V,,, = 145, = 0.049). For the calcareous part, the regression K e,‘d”Lt%n is Y = 0.56 + 136.8024 X fr = 0.98261 for the adetazolamide treated group and Y = 2.66 + 187.7212 X (r =0.9180) for the control group. These two regression lines are significantly different in both the adjusted means and slopes (P K 0.05).
The use of a radioactive tracer in demonstrating calcification has to be considered cautiously in interpreting isotopic exchange phenomena. A typical time-course of 45Ca-uptake in calcareous algae is composed of two phases: a rapid exchange between the radioisotope and the calcium pool and then a slow net deposition (Bohm, 1978). However, the incorporation of both 45Ca and “C-DIC from the medium into CaCO, spicules of sea-urchin larvae has only one phase, a straight-line response (Sikes et al., 1981). One reason for the lack of an obvious isotopic exchange in sea-urchin spicules is that calcification occurs intracellularly, since the living tissues can decrease considerably the amount of isotopic exchange (Barnes and Crossland, 1977). In the seaurchin tooth, calcification occurs intra~llularly. In addition, the chewing tip of the tooth was not exposed to the labeled medium in the present study. Therefore, isotopic exchange with the calcareous part of sea-urchin tooth would be low. The time-course results suggest that calcification occurred in vitro only during the first 2 hr. However, the time-course of 14C incorporation into the teeth could indicate saturation. This would suggest that these teeth are not mineralizing and that isotopic-exchange is occurring during the first 2 hr and then becomes saturated. 2. The transport of CO, between external medium and
the soft tissue of the tooth treated with acetazolamide increased proportionally from 25 to 214cpmjpg protein per hr (Fig. 4). In contrast, the i4C-activity of the
untreated tooth increased hyperbolically from 40 to 163 cpm/pg protein per hr. At low concentrations of external bicarbonate (less than 0.05 mM), “C-activity of the soft tissue was higher in the absence of acetazolamide. These results indicate that (1) CO2 diffuses passively when CA is inhibited and (2) CA facilitates the transport of CO, from external medium to the soft tissue when the concentration of DIC was extremely low in external medium. The incorporation of 14Cinto the calcareous part increased in proportion to the concentration of external bicarbonate, both in the groups treated and untreated with acetazolamide. The acetazolamidetreated group had a lower rate of incorporation (average 40%) than the untreated group (Fig. 4). The 14C-activity found in the calcareous part of the tooth is plotted against that in the soft tissue in Fig. 5. At the same level of 14C-activity in the soft tissue, the incorporation of 14Cinto the calcareous part was higher in the untreated group than the CA-inhibited group, indicating that CA promotes the transport of DIC between the soft tissue and the calcareous part (Fig. 5). When CA was not inhibited, the incorporation of 14C into the calcareous part increased proportionaIly to the increase of the 14C-activity of the soft tissue, indicating that the incorporation of 14Coccurs at an unsaturated CA-mediated transport. When CA was inhibited, the incorporation of 14Cinto the calcareous part became hyperbolic to the increase of the 14C-activity of the soft tissue, revealing that the incorporation of 14Cdoes not occur only by a physical process such as diffusion.
the soft tissue In sea-urchin teeth, CO, crosses the external plumula membrane, entering into the soft tissue from the external medium. Within the plumula CO, reacts with
H,O to form H&O,, or reacts with OH- to form HCO,. The former reaction is important at pH < 8, and the latter dominates at pH > 10. In the absence of CA and at pH < 8, the diffusion of CO2 and the uncatalyzed hydration~ehydration are rate-limiting in the biological transport process. In the presence of CA the hydration-dehydration of CO, is not ratelimiting (Gutknecht et al., 1977). Some aquatic plants can take up exogeous HCO; for photosynthetic
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240
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Fig. 5. The relationship between the “C activity of the soft tissue and that of the cakareous part of the tooth of sea urchin Lytechinus variegates. ‘The straight line (Y = -0.30 + 0.2070 X) is fitted by least squares method (r = 0.8382). The curve (Y = -3.91 + 0.2340 X - 0.0004 X2) is fitted significantly (r = 0.9639).
330
CHANG-POCHEN and JOHN M. LAWRENCE
assimilation (Raven, 1970; Lycas, 1983). In coccolithophorids, the carbon source for coccolith formation is HCO; in the medium rather than CO, or CO:-, produced internally or provided externally. In this case, a CA has only a minor role in the supply
of DIC (Sikes and Wheeler, 1983). In the present study, CA facilitates the entry of CO* in extremely low concentrations. The concentrations of DIC used in the experimental medium are lower than that of the perivisceral fluid, 2.2 mM in most of echinoids (Shick, 1983). This seems to suggest that CA is not too important in the transport of CO, between the coelomic fluid and the teeth. However, the teeth are not surrounded by perivisceral fluid (Hyman, 1955). Each tooth is enclosed by a little dental sac, which is connected to the peripharyngeal cavity. The peripharyngeal cavity is a minor coelomic compartment completely or partly closed off from the perivisceral coelomic cavity. In addition, there are gills at the edge of the peristome. The gill lumen opens into the peripharyngeal coelomic cavity. Thus, the concentration of CO2 in the dental sacs may be different from the perivisceral coelomic fluid. Thus, the importance of CA in the transport of DIC from the coelomic fluid to the tooth is still not clear. 3. The transport of CO? from calcareous part of the tooth
the soft tissue to the
In the plumula of the sea urchin Lytechinus uarieCA is localized adjacent to or in the vacuolar membrane of the calcareous part (Chen and Lawrence, 1986b). Between the soft tissue and the calcareous part, CA is unsaturated, suggesting a very low concentration of COz. This low level of CO, may be due to an alkaline microenvironment. The biological meaning of this is that precipitation of CaCO, occurs in an alkaline environment. For example, in the developing teeth of young mice, the cellular layers have pH values of 7.1 and the pH values in enamel. predentine and dentine range from 7.3 to 8.5 (Lyman and Waddell, 1977). In addition, the pH of the shell fluid compartment is maintained above pH 8 and at 0.330.5 pH units above the blood during mineralization of the blue crab following molting (Cameron and Wood, 1985). The results of the present study suggests that an external alkaline environment favors the incorporation of 14C into the calcareous part of the seaurchin tooth. At the same pH, the incorporation of 14Cinto the calcareous part increased proportionally with activity of 14Cin the soft tissue. Thus the ratio between the 14C-activity of the calcareous part to that of the soft tissue is constant. However, the ratios obtained from the teeth treated with different pH’s increase from 0.15 at pH 7.0 to 0.25 at pH 8.4, indicating that relatively more i4C is being incorporated into the calcareous part in an alkaline environment. The pH adjacent to the vacuolar membrane of the sea-urchin tooth is not known. However, the pH at the choroid plexus, which secretes the cerebrospinal fluid (consisting of HCO,) is thought to be about 9 (Maren and Vogh, 1980; Vogh et al., 1985). In this cerebrospinal fluid system, inhibition of CA reduces secretion by 50%. These authors, based on experimental data and theoretical calculation, concluded gatus,
that if the pH were above 10, the uncatalyzed reaction of OH- and COZ would be so rapid that catalysis would be unnecessary for generation of HCO; and inhibitors of CA would have no effect on secretion. If the pH were 7-8, the uncatalyzed rate would be nearly zero, and there would be no residual rate after inhibition of CA. There are some similarities between the cerebrospinal fluid secretion and the sea-urchin calcification: CA is required and the inhibition of CA reduces calcification by 4&60% (Heatfield, 1970; the present study), suggesting that the pH adjacent to the mineralizing may be 8-9. One possible explanation for the maintenance of an alkaline environment adjacent to the vacuolar membrane involves the characteristics of the membrane-bound CA. Wistrand (1984) suggested that the membrane-bound CA forms an ion-channel, which has negative charges on one end to separate the reactants Hi and HCO; to opposite directions. The accumulation of HCO; inside the vacuolar membrane makes the microenvironment alkaline (Atkinson and Camien, 1982). The reaction is HCO, + H,O-+H$O, + OH-. The relatively alkaline pH of sea-water is a consequence of this type of reaction (Skirrow, 1965). Acknowledgements-We thank Dr Clinton J. Dawes for the “‘C-NaHCO,, and Mr Thomas G. Ferguson for the use of the liquid scintillation counter. Chen thanks both the Academia Sinica and the National Science Council of the Republic of China for financial support.
REFERENCES Atkinson D. E. and Camien M. N. (1982) The role of urea synthesis in the removal of metabolic bicarbonate and the regulation of blood pH. Curr. Top. Cell. Regul. 21, 261-302. Barnes D. J. and Crossland C. J. (1977) Coral calcification: sources of error in radioisotopic techniques. Mar. Bid. 42, 119-129.
Barnes D. J. and Crossland C. J. (1982) Variability in the calcification rate of Acropora acuminata measured with radioisotopes. Coral Reefs 1, 53-57. Bohm L. (1978) Application of the 45Ca tracer method for determination of calcification rates in calcareous algae: effect of calcium exchange and differential saturation of algal calcium pools. Mar. Bid. 47, 9914. Cameron J. N. and Wood C. M. (1985) Apparent H+ excretion and CO, dynamics accompanying carapace mineralization in the blue crab (Callinectes sapidus) following moulting. J. exp. Biol. 114, 181-196. Chen C. P. and Lawrence J. M. (1986a) the ultrastructure of the plumula of the tooth of Lytechinus variegatus (Echinodermata: Echinoidea). Acta Zool. 67(l), 3341. Chen C. P. and Lawrence J. M. (1986b) Localization of carbonic anhydrase in the plumula of the tooth of Lytechnius uarieaatus (Echinodermata: Echinoidea). Acta Zool. 67(l), 27-32. Costlow J. D., Jr. (1959) Effect of carbonic anhydrase inhibitors on shell development and growth of Balanus improvisus Darwin. Physiol. Zool. 32, 177-184. Deeens E. T. (1976) Molecular mechanisms of carbonate, ihosphate and sihca deposition in the living cell. Top. Curr. Chem. 64, l-112. Fransler S. C. (1983) Phenotypic plasticity of skeletal elements in the purple sea urchin, Strongylocenrrotus purpuratus. MSc. Thesis, San Diego State University, San Diego, California.
CA in CO, transport of sea-urchin tooth Giraud M. M. (1981) Carbonic anhydrase activity in the integument of the crab Curcinus meanas during the intermolt cycle. Comp. Biochem. Physiol. 69A, 381-387. Goreau T. F. (1959) The physiology of skeleton formation in corals. I. A method for measuring the rate of calcium deposition by corals under different conditions. Biof. Bull. Mar. biol. Lab., Woods Hole 116, 59-75. Gutknecht J., Bisson M. A. and Tosteson F. C. (1977) Diffusion of carbon dioxide through lipid bilayer membranes: effects of carbonic anhydrase bicarbonate and unstirred layers. J. Gen. Physiol. 69, 779.-794. Heatfield B. M. (1970) Calcification in echinoderms: effects of temperture and Diamox on incorporation of calcium4.5 in vitro by regenerating spines of ~rrongylocentrofus purpuratus. Biol. Bull. Mar. biol. Lab., Wood.s Hole 139, 151-163. Holland N. D. (1965) An autoradiographic investigation of tooth renewal in the purple sea urchin (Strongylocentrotus purpuratus). J. exp. Zool. 158, 275-282.
Horner S. M. J. and Smith D. F. (1982) Measurement of total inorganic carbon in seawater by a substoichiometric assay using NaHJ4C0,. Litnnol. Oceanogr. 27, 9788983. Hyman L. H. (1955) The invertebrates: Echinodermata. McGraw-Hill, New York. Kniprath E. (1974) Ultrastructure and growth of the sea urchin tooth. Calcif. Tissue Res. 14, 2i l-228. Lucas W. J. (1983) Photosvnthetic assimilation of exogeneous H&J, by aquatic-plants. A. Rev. P. Physiol. 34, 71-104.
Lyman G. E. and Waddell W, J. (1977) pH gradients in the developing teeth of young mice from autoradiography of i4C DMO. Am. J. Physiol. 232(4), F363-F367. Maren T. H. and Vogh B. P. (1980) Formation and ionic composition of cerebrospinal fluid: the role of carbonic anhydrase. In Biophysics and Physiology of Carbon Dioxide. (Edited by Bauer C., Gross G. and Bartels H.), pp. 426435. Springer, Berlin. Mirkel K. (1969) Morphology of sea-urchin teeth. II. The keeled teeth of Echinacea (Echinodermata, Echinoidea). Z. Morph. dkol. Tiere 66, I-50.
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Raven J. A. (1970) Exogenous inorganic carbon sources in plant photosynthesis. Biol. Rev. 45, 167-221. Riley J. P. and Skirrow G. (Eds.) (1965) Chemical Oceanography, Vol. 1. Academic Press, London. Shick J. M. (1983) Respiratory gas exchange in echinoderms. Echinoderm Stud. 1, 67-l 10. Sikes C. S., Okazaki K. and Fink R. D. (1981) Respiratory COz and the supply of inorganic carbon for calcification of sea urchin embryos. Camp. Biochem. Physiol. 70A, 285-292.
Sikes C. S. and Wheeler A. P. (1983) A systematic approach to some fundamental questions of carbonate calcification. In B~om~neralization and Bio/ogieai metaf Accumulation. (Edited by Westbroek P. and De Jong E. W.), pp. 2855289. Reidel, Holland. Skirrow G. (1965) The dissolved gases-carbon dioxide. In Chemical Oceanography. (Edited by Riley J. P. and Skirrow G.), PP. 227-321. Academic Press. New York. Sokal R. R. and Rohlf F. J. (1981) Biometry: The Principles and Practice of Statistics in Biological Research, 2nd edn. W. H. Freeman & Co., San Francisco. Spector T. (1978) Refinement of the Coomassie blue method of protein quantitation: A simple and linear spectrophotometric assay for 0.5 to 5Opg of protein. Analyt. Biochem. 86, 142-146.
Tashian R. E. and Hewett-Emett (Eds.) (1984) Biology and Chemistry of the CA, Ann. N. Y. Acad. Sci. 429. Vogh B. P., Godman D. R. and Maren T. H. (1985) Aluminum and gallium arrest formation of ~rebrospinal fluid by the mechanism of OH- depletion. J. Pharmac. exp. Ther. 233, 715721. Wilbur K. M. and Saleuddin A. S. M. (1983) Shell formation. In The Mollusca. Vol. 411). fEdited bv Saleuddin A. S. M. and Wilbur K. M.),‘pp.‘235-287: Academic Press, New York. Wistrand P. J. (1984) Properties of membrane-bound carbonic anhydrase. Ann. N. Y. Acad. Sci. 429, 195-206. Yule A. B., Crisp D. J. and Cotton I. H. (1982) The action of acetazolamide on calcification in juvenile Balanus balanoides. Mar. Biol. Lett. 3, 273-288.
0300-9629/87$3.00+ 0.00 0 1987Pergamon Journals Ltd
Printed in Great Britain
THE ROLE OF CARBONIC ANHYDRASE IN FACILITATING THE TRANSPORT OF CO, IN THE TOOTH OF LY7’ECHINUS VARlEGATUS (ECHINODERMATA: ECHINOIDEA) CHANG-PO CHEN* and JOHN M. LAWRENCE Department of Biology, University of South Florida, Tampa, Florida 33620, USA (Received 29 July 1986)
Abstract-l. CO, is the form in which dissolved inorganic carbon enters the tooth from the external medium. 2. Carbonic anhydrase facilitates the transport of CO, from the external medium to the soft tissue and from the soft tissue to the calcareous part of the tooth.
INTRODUCTION
through the membrane and intercellular space to promote calcification. The purpose of this study was to evaluate this hypothesis.
Carbonic anhydrase (CA) catalyzes the reversible hydration of CO,, and is involved in the formation of carbonate in many biological systems (Degens,
1976; Tashian and Hewett-Emett, 1984). The rate of calcification is reduced by inhibitors of CA in regenerating spines of sea urchins (Heatfield, 1970), in the shell of barnacles (Costlow, 1959; Yule et al., 1982), in the cuticle of crabs (Giraud, 1981), in the shell of molluscs (reviewed in Wilbur and Saleuddin, 1983) and in the skeleton of corals (Goreau, 1959). The teeth of sea urchins grow continuously, 1-1.5 mm weekly in Parancentrotus hidus (Mtirkel, 1969) and renew in about 75 days in Strongylocentrotus purpuratus (Holland, 1965). The teeth of S. purpurutus grow continuously, even when individuals are starved (Fransler, 1983). The tooth of the sea urchin is enclosed in a single layer of epithelium. In the growing tip (plumula) of the tooth there are two types of odontoblasts: syncytial and free. In the plumula calcification occurs intracellularly by syncytial odontoblasts. The calcareous part of the tooth is surrounded by two membranes (an outer plasma membrane and an inner vacuolar membrane). Some organic particles occur on the surface of the vacuolar membrane, but no trace of organic matter is found within the calcareous part. Some fibroblasts and collagen fibers are located in the soft part of the tooth as well (Kniprath, 1974; Chen and Lawrence, 1986a). In the plumula of the tooth of the sea urchin Lytechinus uuriegutus, CA is localized (1) intracellularly in the epithelium cells, odontoblasts and fibroblasts, (2) extracellularly near fibroblasts and collagen fibers, and (3) adjacent on or in the plasma membrane and the vacuolar membrane of the calcareous deposits (Chen and Lawrence, 1986b). The morphological characteristics of the plumula and the localization of CA in the plumula suggest that CA functions in facilitating the movement of CO* *Present address: Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan 11529, R.O.C.
MATERIALS AND METHODS Lyrechinusuariegutus(Lamarck) were collected from Sar-
asota, Florida and maintained in the laboratory by feeding them with turtle grass Thalassiatestudinum,ad libitum.The teeth of the sea urchins were removed from the Aristotle’s lantern. The chewing tip and the adjacent hard shaft were wrapped with a soft-wax. The wax served as a buoy during incubation and also as an insulator to prevent physical isotopic exchange (Barnes and Crossland, 1982). The tooth-wax complexes were pre-incubated in the dissolved inorganic carbon-free (DIGfree) medium, then incubated in 3 ml of labeled medium. The DIC-free artificial sea-water was prepared following Horner and Smith (1982) by dissolving Instant OceanrM salts with distilled water up to 34 or 68% salinity, adding concentrated HCl to acidify the solution to a pH of 2.3, bubbling the solution with CO,-free air for 3 hr and finally adjusting the pH with IONNaOH to various pH’s. The labeled medium was prepared by mixing i4C-NaHCO, (New England Nuclear, 10 PCi, 100 fig of NaHCO, in 1 ml distilled water, pH 9.6) with the DIC-free medium. The randomized complete blocks design was used to avoid the difference among individuals (Sokal and Rohlf, 1981). To study the time-course of W-entry. the teeth were incubated in DIC-free medium (pH 7.6) for 40 min and then in the labeled medium with 2 uCi. 0.079 mM NaHCO, for periods ranging from 2 min to 6 hr. To study the s&ies of DIC entry, the teeth were incubated in nonlabeled medium in five different pH solutions (7.0-8.4) for 30min and then in labeled medium (2pCi) for 1.5 hr. The percentage molar fraction of dissolved CO, (including free H,CO,) at various pH’s was taken from Saruhashi’s table (Riley and Skirrow, 1965). To study the effect of CA inhibition, the teeth were incubated in DIC-free medium (PH 7.6) for 30 min. then in labeled medium ~DH 7.6) at five differen; bicarbonate concentrations (0.0?&0.198 mM) without or with acetazolamide (A specific inhibitor of CA, 6.7 x 1Om5M) for 1 hr. After incubation, the tooth-wax complexes were rinsed with tap water for 1 min, then the soft part of the tooth (including the plumula and adjacent soft shaft) was dissolved in I ml of 1 N NaOH at 60°C for I .5hr. The cal-
327 c B.P.8712.4-H
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and
JOHN M. LAWRENCE
130.
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I\ \
ao70.
6o
T
soft tissue
. calcareous part
‘1 i \
20/‘--f-..t-_I_ 7.0 7.3 Incubation
7.6
0.0
8.4
PH
time h
Fig. 1. Time course of the entry of 14Cin the soft tissue and the incorporation into the calcareous part of the tooth of the sea urchin Lyrechinus uariegatus (mean + 1 SD). The number of sea urchins used is given in the figure. The negative regression of the “‘C-activity in the soft tissue from 2 to 6 hr is significant (P < 0.05).
+
I
10
Fig. 2. Effect of pH of the medium on the entry of 14Cin the soft tissue and the incorporation into the calcareous part of the tooth of the sea urchin Lytechinus uariegatus (mean + 1 SD, N = 4 for each pH).
subsequent experiments mental conditions, limited to less than 2 hr duration. careous part and soft tissue were separated by centrifuging with a clinical centrifuge for Smin. The supernatant is referred to as the Soft-rissuefraction. The pellets were rinsed with 1 ml of 1 N NaOH solution. After centrifuging, the second supernatant was discarded and the calcareous part was dissolved in 1 ml of ethylenediamine tetraacetic acid (EDTA, 12%. pH9.6) at 60°C for 12-14 hr. This calcareous-part-EDTA solution is referred to as the “calcareous-part fraction”. The protein level of the soft tissue was measured by the Bradford method (Spector, 1978). A 0.2-m! sample of the soft-tissue fraction was acidified with 0.4 ml 1 N HC!, bubbled with air for 10 min and 2 ml of !.25-fold concentrated Bradford reagents added. Bovine serum albumin was used as the protein standard. The “C-activity was counted with a Beckman LS 1OOC liquid scintillation counter. The liquid scintillation fluids of the incubation medium, the soft-tissue fraction and the calcareous-part fraction were prepared as 0.1 ml of incubation medium with 3 ml HandifluorTM (Mallinckrodt), 0.1 ml of the soft-tissue fraction plus 0.5 ml Trizma HC! (0.2 M) and 3 ml of Handifluor and 0.2 ml of the calcareouspart fraction plus 0.4ml distilled water and 6ml of Handifluor, respectively. The counting efficiency was about 70%. After the correction of background counts, the 14C activity was expressed as cpm/pg protein or cpm/pg protein per hr.
were
2. The species of DIC entry When the concentration of DIC was low in the medium, the 14C-activity of the soft tissue decreased proportionally from a high of 125 cpm/pg protein per hr at pH 7.0 to a low of 65 cpm/pg protein per hr at pH 8.4 (Fig. 2). In contrast, the 14C-activity of the calcareous part did not vary significantly with pH. A direct linear relationship was evident between the “C-activity of soft tissue and the percentage molar fraction of dissolved CO2 of the medium (Fig. 3), indicating that CO, is the major C-species which enters the sea-urchin tooth from the external medium. 3. Effect of acetazolamide inhibition of CA When the concentration of external NaHCO, increased from 0.016 to 0.198 mM, the 14C-activity of
RESULTS 1.
Time course of DIC uptake
The time-course of the entry of 14Cin the soft tissue and the incorporation of 14Cinto the calcareous part of the tooth revealed three phases: (1) a quick and linear increase during the first hour, (2) a continuous but slow increase up to the maximal activity during the second hour and (3) a significant decrease in activity in the soft tissue but a maintained activity in the calcareous part (Fig. 1). Because these results indicate that 14Cis incorporated into the tooth only during the first 2 hr of incubation under the experi-
0
I
1
% molar fraction
I
1
,
2 3 4% of dissolved CO2
Fig. 3. The relationship between the average “C-activity of the soft tissue of the tooth of the sea urchin Lytechinus variegatus and the percentage molar fraction of dissolved CO, (including free H&O,) of the medium. The straight line is fitted by least squares method (r = 0.9964).
CA
in CO, transport
of sea-urchin
tooth
329
DISCUSSION
1. Isotopic exchange us calczjication 0 acetazolamide
I
NaHC031 mM
Fig. 4. Effect of 14C-NaHCO, concentration and acetazolamide on the entry of “‘C in the soft tissue and the incorporation into the calcareous part of the tooth of the sea urchin Lytec&nLts variegutus(mean f 1 SD). The number of sea urchins is given in the figure for the acetatolamidetreated groups, and is six for the control groups. For the soft tissue, the straight line (Y = 6.21 + 1043.9765 X) is fitted by least squares method (r = 0.9995), the curve is fitted by the double reciprocal plot (r = 0.8506, V,,, = 145, = 0.049). For the calcareous part, the regression K e,‘d”Lt%n is Y = 0.56 + 136.8024 X fr = 0.98261 for the adetazolamide treated group and Y = 2.66 + 187.7212 X (r =0.9180) for the control group. These two regression lines are significantly different in both the adjusted means and slopes (P K 0.05).
The use of a radioactive tracer in demonstrating calcification has to be considered cautiously in interpreting isotopic exchange phenomena. A typical time-course of 45Ca-uptake in calcareous algae is composed of two phases: a rapid exchange between the radioisotope and the calcium pool and then a slow net deposition (Bohm, 1978). However, the incorporation of both 45Ca and “C-DIC from the medium into CaCO, spicules of sea-urchin larvae has only one phase, a straight-line response (Sikes et al., 1981). One reason for the lack of an obvious isotopic exchange in sea-urchin spicules is that calcification occurs intracellularly, since the living tissues can decrease considerably the amount of isotopic exchange (Barnes and Crossland, 1977). In the seaurchin tooth, calcification occurs intra~llularly. In addition, the chewing tip of the tooth was not exposed to the labeled medium in the present study. Therefore, isotopic exchange with the calcareous part of sea-urchin tooth would be low. The time-course results suggest that calcification occurred in vitro only during the first 2 hr. However, the time-course of 14C incorporation into the teeth could indicate saturation. This would suggest that these teeth are not mineralizing and that isotopic-exchange is occurring during the first 2 hr and then becomes saturated. 2. The transport of CO, between external medium and
the soft tissue of the tooth treated with acetazolamide increased proportionally from 25 to 214cpmjpg protein per hr (Fig. 4). In contrast, the i4C-activity of the
untreated tooth increased hyperbolically from 40 to 163 cpm/pg protein per hr. At low concentrations of external bicarbonate (less than 0.05 mM), “C-activity of the soft tissue was higher in the absence of acetazolamide. These results indicate that (1) CO2 diffuses passively when CA is inhibited and (2) CA facilitates the transport of CO, from external medium to the soft tissue when the concentration of DIC was extremely low in external medium. The incorporation of 14Cinto the calcareous part increased in proportion to the concentration of external bicarbonate, both in the groups treated and untreated with acetazolamide. The acetazolamidetreated group had a lower rate of incorporation (average 40%) than the untreated group (Fig. 4). The 14C-activity found in the calcareous part of the tooth is plotted against that in the soft tissue in Fig. 5. At the same level of 14C-activity in the soft tissue, the incorporation of 14Cinto the calcareous part was higher in the untreated group than the CA-inhibited group, indicating that CA promotes the transport of DIC between the soft tissue and the calcareous part (Fig. 5). When CA was not inhibited, the incorporation of 14C into the calcareous part increased proportionaIly to the increase of the 14C-activity of the soft tissue, indicating that the incorporation of 14Coccurs at an unsaturated CA-mediated transport. When CA was inhibited, the incorporation of 14Cinto the calcareous part became hyperbolic to the increase of the 14C-activity of the soft tissue, revealing that the incorporation of 14Cdoes not occur only by a physical process such as diffusion.
the soft tissue In sea-urchin teeth, CO, crosses the external plumula membrane, entering into the soft tissue from the external medium. Within the plumula CO, reacts with
H,O to form H&O,, or reacts with OH- to form HCO,. The former reaction is important at pH < 8, and the latter dominates at pH > 10. In the absence of CA and at pH < 8, the diffusion of CO2 and the uncatalyzed hydration~ehydration are rate-limiting in the biological transport process. In the presence of CA the hydration-dehydration of CO, is not ratelimiting (Gutknecht et al., 1977). Some aquatic plants can take up exogeous HCO; for photosynthetic
= 3 cl,
i
5
.
.control
0acetarolamide
0'
7
__’
l
,’
<,’
l
,
40
80
120
lb0 200
240
(cpmlpg-protein/h) 14c -activity of soft tissue
Fig. 5. The relationship between the “C activity of the soft tissue and that of the cakareous part of the tooth of sea urchin Lytechinus variegates. ‘The straight line (Y = -0.30 + 0.2070 X) is fitted by least squares method (r = 0.8382). The curve (Y = -3.91 + 0.2340 X - 0.0004 X2) is fitted significantly (r = 0.9639).
330
CHANG-POCHEN and JOHN M. LAWRENCE
assimilation (Raven, 1970; Lycas, 1983). In coccolithophorids, the carbon source for coccolith formation is HCO; in the medium rather than CO, or CO:-, produced internally or provided externally. In this case, a CA has only a minor role in the supply
of DIC (Sikes and Wheeler, 1983). In the present study, CA facilitates the entry of CO* in extremely low concentrations. The concentrations of DIC used in the experimental medium are lower than that of the perivisceral fluid, 2.2 mM in most of echinoids (Shick, 1983). This seems to suggest that CA is not too important in the transport of CO, between the coelomic fluid and the teeth. However, the teeth are not surrounded by perivisceral fluid (Hyman, 1955). Each tooth is enclosed by a little dental sac, which is connected to the peripharyngeal cavity. The peripharyngeal cavity is a minor coelomic compartment completely or partly closed off from the perivisceral coelomic cavity. In addition, there are gills at the edge of the peristome. The gill lumen opens into the peripharyngeal coelomic cavity. Thus, the concentration of CO2 in the dental sacs may be different from the perivisceral coelomic fluid. Thus, the importance of CA in the transport of DIC from the coelomic fluid to the tooth is still not clear. 3. The transport of CO? from calcareous part of the tooth
the soft tissue to the
In the plumula of the sea urchin Lytechinus uarieCA is localized adjacent to or in the vacuolar membrane of the calcareous part (Chen and Lawrence, 1986b). Between the soft tissue and the calcareous part, CA is unsaturated, suggesting a very low concentration of COz. This low level of CO, may be due to an alkaline microenvironment. The biological meaning of this is that precipitation of CaCO, occurs in an alkaline environment. For example, in the developing teeth of young mice, the cellular layers have pH values of 7.1 and the pH values in enamel. predentine and dentine range from 7.3 to 8.5 (Lyman and Waddell, 1977). In addition, the pH of the shell fluid compartment is maintained above pH 8 and at 0.330.5 pH units above the blood during mineralization of the blue crab following molting (Cameron and Wood, 1985). The results of the present study suggests that an external alkaline environment favors the incorporation of 14C into the calcareous part of the seaurchin tooth. At the same pH, the incorporation of 14Cinto the calcareous part increased proportionally with activity of 14Cin the soft tissue. Thus the ratio between the 14C-activity of the calcareous part to that of the soft tissue is constant. However, the ratios obtained from the teeth treated with different pH’s increase from 0.15 at pH 7.0 to 0.25 at pH 8.4, indicating that relatively more i4C is being incorporated into the calcareous part in an alkaline environment. The pH adjacent to the vacuolar membrane of the sea-urchin tooth is not known. However, the pH at the choroid plexus, which secretes the cerebrospinal fluid (consisting of HCO,) is thought to be about 9 (Maren and Vogh, 1980; Vogh et al., 1985). In this cerebrospinal fluid system, inhibition of CA reduces secretion by 50%. These authors, based on experimental data and theoretical calculation, concluded gatus,
that if the pH were above 10, the uncatalyzed reaction of OH- and COZ would be so rapid that catalysis would be unnecessary for generation of HCO; and inhibitors of CA would have no effect on secretion. If the pH were 7-8, the uncatalyzed rate would be nearly zero, and there would be no residual rate after inhibition of CA. There are some similarities between the cerebrospinal fluid secretion and the sea-urchin calcification: CA is required and the inhibition of CA reduces calcification by 4&60% (Heatfield, 1970; the present study), suggesting that the pH adjacent to the mineralizing may be 8-9. One possible explanation for the maintenance of an alkaline environment adjacent to the vacuolar membrane involves the characteristics of the membrane-bound CA. Wistrand (1984) suggested that the membrane-bound CA forms an ion-channel, which has negative charges on one end to separate the reactants Hi and HCO; to opposite directions. The accumulation of HCO; inside the vacuolar membrane makes the microenvironment alkaline (Atkinson and Camien, 1982). The reaction is HCO, + H,O-+H$O, + OH-. The relatively alkaline pH of sea-water is a consequence of this type of reaction (Skirrow, 1965). Acknowledgements-We thank Dr Clinton J. Dawes for the “‘C-NaHCO,, and Mr Thomas G. Ferguson for the use of the liquid scintillation counter. Chen thanks both the Academia Sinica and the National Science Council of the Republic of China for financial support.
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