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Calcium transport during skeletogenesis in hermatypic corals
CALCIUM
TRANSPORT DURING SKELETOGENESIS IN HERMATYPTC CORALS BRUCE E. CHALKEK
Roscnstiel
School of Marine 4600 Rickenbacker
and Atmospheric Science. University of Miami. Causeway. Miami. FL 33149, U.S.A.
(Rewired
26 AUJUS~
197.5)
I. Light-enhanced calcification in the hermatypic corals Acroportr crrricorni.s and A. /ortnos~~ results from the active transpo’rt of calcium ions. It shows saturation kinetics. 2. Dark calcificatmn. simulated by the addition of DCMU, results from enzyme mediated isotopic exchange. 3. Strontium is a competitive inhibitor of both light-enhanced and dark calcification. The K, is I.3 mM. 4. The data refutes the diffusional model for calcium movement in hermatypic corals. Abstract
IRTRODUCTION R&-building (hermatypic) corals are inevitably associated with the endosymbiotic dinoflagellate, G~jmrlodir7ium mic.roildricrricun7. Endosymbiont photosynthesis allows the corals to calcify may times faster in the light than in the dark. In contrast, ahermatypic corals and hermatypic corals which have lost their endosymbionts (aposymbiotic corals) calcify at rates which are slow and unaffected by illumination (Goreau, 1959. 1961a’; Goreau & Goreau, 1960~). If hermatypic corals are exposed to lo-’ M DCMU (3-(3,4-dichlorophenyl)-l.l-dimethyl urea), algal photosynthesis and light-enhanced calcification will be completely abolished (Vandermeulen, Davis, & Muscatine,
1972;
Chalker,
1975).
The biochemical mechanisms of calcium movement in corals are unknown. Goreau & Bowen (1955) demonstrated that the intracellular calcium concentration of the ahermatype Astrungiu dame is less than that of the surrounding sea water, implying the existence of active calcium transport. All present models of light-enhanced calcification ignore the energetic demands which would be placed upon the symbiotic association by high rates of calcium transport (for a review see Vandermeulen & Muscatine, 1974). Calcification processes are also important geochemically. The relative incorporation of calcium and strontium into biogenic carbonate has been extensively examined as a potential source of information about paleoenvironmental, paleoecological, and diagenie conditions (for reviews see Dodd, 1967; and Kinsman, 1969). The principal model for strontium incorporation postulates that strontium and calcium diffuse to the sites of skeletogenesis (Weber, 1973). ‘The accuracy of this model and thus its predictive value depends upon the validity of the diffusional assumption. I have examined and compared the kinetics of calcium transport in two species of hermatypic corals, 4croporu cwaicornis from the Atlantic and Acropora fimnosa from the Pacific. This comparison helps to identify the energy demands of light-enhanced calcifi-
cation. define the dark rate of calcification. the diffusional assumption.
and test
MATERIALS AND METHODS Acropmx crrricomis was collected by SCUBA diving at depths of 45 feet near Grand Bahama Island, Bahamas. Experiments were conducted aboard the University of Miami’s R!V CALANUS. ifuoporrr fhrrnmtr was collected at a depth of 6 feet in the “Quarry”. Eniwetak Island. Eniwetak Atoll. Marshall Islands. Experiments here conducted at the Mid-Pacific Marine Laboratory. Corals were maintained under illumination in aerated aquaria at 29 C, and used the day of collection. Terminal portions of branches were cut to 3 cm with surgical bone forceps, rinsed twice with calcium- and strontium-free Lyman & Fleming artificial sea water (Lyman & Fleming. 1940). and placed in I50 ml of modified Lyman & Fleming artificial sea hater in 350 ml polycarbonatc beakers. Calcium content of the medium was adjusted by adding I.0 M CaC12 before introducing the coral tips. In some experiments. algal photosynthesis and light-enhanced calcification were abolished by the addition of I.5 ml of IO-’ M DCMU in absolute ethanol to each beaker of A. wrriwruis and I.5 ml of 10~’ M DCMU to each beaker of A. fiwnwsu. Controls indicate that this concentration of ethanol affects neither the light nor the dark rate of calcification. Prior to the start of each experiment IO ml samples were withdrawn from each beaker to determine the calcium concentration by EDTA (ethylenediaminetetraacetic acid) titration (Pate & Robinson, 195X). Corals were preincubatcd for 12 hr prior to the addition of IO rtl of “CaCI, (30 uCi:ml). Incubations with the label lasted 2 hr at-29’C. Illumination at the surfilce of the medium was 10.500 lux. This was provided by an ovcrhead bank of cool white fluorescent bulbs. After 2 hr, the corals were removed, rinsed in calciumand strontium-free artificial seawater, and placed in 50”,, chlorox~50’~, tap water for I hr to remove tissue. The tips were then cut to 2 cm and stored at room temperature. Skeletons were dissolved in liquid scintillation vials with concentrated HCI plus 0.1 ml of I-butanol to retard foaming. The final volume was adjusted to 3 ml with distilled water, and 9 ml Aquasol (New England Nuclear) was added to produce a clear gel. Samples were counted in a Nuclear Chicago Mark II liquid scintillation counter.
456
BRUct:
1
0
E.
CHALKKK
t
I
20
IO MILLMOLAR
CALCIUM
30
I 40
CONCENTRATION
Fig. I. A plot of calcification rate vs mM calcium concentration for A. curvicorrlis incubated at an illumination intensity of 10.500 lux without (open circles) and with (closed circles) 1O--5 M DCMU. Mean + one SD for five individuals Calcification rates are expressed fixed per hour per l-cm tip.
as micromoles
of calcium
RESULTS
Calcification Calcification rates as a function of calcium concentration were measured at an illumination intensity of 10,500 lux in the absence and in the presence of DCMU for Acropora ceroicomis (Fig. 1) and A. ,firHIOS~(Fig. 2). The curves follow Michaelis-Menton erlzyme kinetics. These meas~trements were repeated for A. ,formosa incubated in the light with lo-’ M ethacrynic acid. The resultant curve (Fig. 3) is nearly identical to the corresponding DCMU curve. A double reciprocal replot was made of the data presented in Figs. l--3. All the lines were fit by computer using the method of least squares. The maximum 30
1
T
I Fig. 2. A plot of calcification rate vs mM calcium concentration for A. ,fix~osa incubated at an illurni~3~~tio~3intensity of 10,500 lux without (open circles) and with (closed circles) IO -’ M DCMU. Mean i one SD. for five
Fig. 3. A plot of c~lci~c~~tioll rate YS mM calcium concentration for A. ,fhrrnosc~ incubated at an illumination intensity of 10,500 lux with IO-’ M ethacrynic acid. Mean + one SD. for five individuals.
rates of ~Icification (V,,,) and the apparent K,‘s are presented in Table 1. Within experimental error, the K.,‘s are identical. For each species the maximal rate of calcification at 10,500 lux is dependent upon the nutritional status of the animals and the time of day during which the experiments are conducted. Regional variations are also possible (Glynn, 1973). As a control, a similar experiment was conducted with A. ccroicornis fixed for I8 hr in loo,{, formalin in sea water. The rate of calcification is nearly directly proportional to the calcium concentration (Fig. 4). These data are indicative of calcium diffusion from the medium to the skeleton and subsequent isotopic exchange. Similar results were obtained for A. jbriW)SLi.
Calcium
transport
during
skeletogenesis
in hermatypic
8
Table I. The maximal rates of calcification (V,,,) and apparent K,‘s for A. cervicornis and A. fornwsa. The rates are pmoles of calcium fixed/hr per 2-cm tip. The concentrations
and K,‘s
are expressed
(DCMI!)
457
corals
1
in mmoles/liter.
(Ethacrymc Xld)
v,., ,\
Apparent K.,.
Fig. 6. Competitive
inhibition of calcification in A. cerviReciprocal velocity vs reciprocal mM concentration. Mean for five individuals incubated at 10.500 lux with IO-’ M DCMU.
uw~~~s by stronttum.
calcium
Fig. 4. A plot of calcification rate vs mM calcium concentration for A. cercicornis fixed in 10% formalin in sea water for 18 hr. Mean i one S.D. for five individuals. Comprtiticeinhibition by
strontium
tration for A. certlicornis incubated at various strontium concentrations without and with lo-” M DCMU are shown in Fig. 5 and 6. Completely competitive inhibition is indicated in both cases by the constant y-intercept (l/V,,,) and the variable x-intercept. A replot of the slopes in Fig. 5 and 6 vs strontium concentration is shown in Fig. 7. The value of the inhibition constant (K,) is given by negative one times the value of the x-intercept. For incubations with and without DCMU, the K, is I.3 mM. DISCL’SSION
Inhibition of calcification by strontium was tested by the method of Plowman (1972). Plots of reciprocal calcification rate vs reciprocal mM calcium concen-
35-
Several observations are relevant to a discussion of skeletogenesis in scleractinians. First, coral tissues have a lower calcium concentration than that of seawater (Goreau & Bowen, 19.55). Second, lightenhanced calcification requires the energy derived from oxidative phosphorylation (Chalker & Taylor, 1975). Finally, light-enhanced calcification follows
m~lhmolor strontum concentratw Fig. 5. Competitive inhibition of calcification in A. cornis by strontium.
calcium
cerci-
Reciprocal velocity vs reciprocal mM concentration. Mean for five individuals incubated at 10.500 lux.
Fig. 7. Replot of the data in Figs. 5 and lines vs mM strontium concentration. The tion with (closed circles) and without (open is given by negative one times the value of
6. Slope of the K, for incubacircles) DCMU the x-intercept.
4%
BKUC~ E.
enzyme mediated kinetics. The light-enhanced incorporation of calcium into skeleton is thus an active, mediated process. The diffusional theory of calcium movement is refuted. Light-en~l~~llced c~llci~cation creates a significant energy demand which may possibly be satisfied by the oxidation of low molecular weight compounds translocated from the endosymbiotic algae to animal tissues. Previous studies. however. have shown that glucose, glycerol, and alanine which arc cvcreted hy the cndosymbiotic algae and readily assimilated and mct~~bolized by hermatypic corals (~usc~~titlc. 1973). do not increase the rates of caici~c~~ti~~i~in the dark. light. or light with DCMU (Vandermeulen &i Muscatine, 1974; Chalker. 1975). Other organic compounds should be examined. Calcification in the dark also appears to follow enzyme kinetics although most of the rate is not inhibited and uncouplers of oxidative ph~~sphory~~~tioll (Chalker & Taylor, 1975). It probably results from facilitated diffusion (Stein. 1964; Borle. 1973) and suhsequent isotopic exchange. Stein has given a general equation for facilitated diffusion: Net
Rate
=
v,,;,,
-_.“I_._- -3
s, i- K”,
Sz + Kwi
where S, and S2 are the concentrations of permeant on the outside and the inside of the system respectively. If the number of calcium-45 ions present in the skeleton is small in comparison to the total number of calcium ions on the surface of the skeleton, this equ~~tion simplifies to a form identical to MichaelisMenton kinetics. The relatively high rate of exchange contradicts the findings of Goreau & Goreau (196Oh: Goreau, 1961 h) and magnifies the significance of endosymbiotic algae in enhancing carbonate deposition on the reef. It also poses an interpretational problem for those investigators ~~lc~iiating light-dark ratios and comparing calcification rates as measured by calcium-45 and carbon- l4-bicarbonate. If this mediated exchange also occurs in the light, the customary calcification rates measured with calcium-45 would be too large. The portion of the dark calcification rate which is due to active transport and results in the accretion of calcium carbonate has not yet been accurately determined. Ethacrynic acid has been widely used as an inhibitor of cation transport and of N~‘.;KK ‘- and and Ca’ ‘,Mg’ ‘-ATPases (Askari & Rae, 1970; Duggan & Knoll. 1965: Schatzmann, 1970). Epstein (1972) emphasizes that ethacrynic acid inhibits ATP production by both oxidative phosphorylation and by gfycolysis. He states that ethacrynic acid affects the energy supply available for the active transport of any solute and cannot be used as a specific ATP-ase inhibitor. Whatever the mode of action, IO- ’ M cthacrynic acid completely inhibits light-enhanced calcification in A. ccwicnrr~i.s without affecting photosynthesis by the isolated endosymbiotic algae (Chalker. 1975). Figure 3 demonstrates that when A. fi~rttz~~suis inhibited with ethacrynic acid, the kinetics of calcification are nearly identical with those found during DCMU inhibition. This supports the contention that the dark rate of calcification results chiefly from facilitated diffusion
CtihLKEK
and subsequent isotopic exchange. The diffusional control (Fig. 4) demonstrates that similar kinetics do not result from simple diffusion and the resultant isotopic exchange. Strontium is a competitive i~~hibit~~rof both lightenhanced and dark calcification in A. ~~~~j~~~~?~~.s (Figs. 57). In both cases the Ki is 1.3 mM. When facilitated diffusion is inhibited by a substrate analogue, the net rate of flux is given by the equation: Net Rate =
_
‘5 -2
S2 + K, -t (R,:K,)K, where R, and R2 are the concentrations of the inhibitor on the outside and the inside of the system respectively and K, is the inhibition constant (Stein. 1964). Again if the number of calcium-45 ions present in the skeleton is small in comparison to the total number of calcium ions on the surface, the rquation simplifies to a form which is identical to that derived from Michaelis-Menton kinetics. These measurements are not sutticiently precise to reflect the small variations in the strontium/caicium ratio reported by geochemists but they demonsti-ante that any model that explains the strontium~c~~lcium ratios found in coral skeletons must consider the specificity of the transport sites. A~k,~o,r~/~,~~~e,~~~~i~rs---This work was supported by, NSF grants GA-43270 :tnd GA-33533 X. The work at Emwtak ~v:as conducted at the Mid-~~ci~c Marine Laborator? under the auspices of the University of Hawaii and the Atomic Energy Commission. The assistance of Dr. Phihp HelErich, Director. Mr. Phil Lamherson. Lab Mwxqer, nnd Mr. Robert Hall. Radiological Safety Oflicer. Universit) of Ha&i. is gratefully ncknowledged. The cthacrynic acid was supplied by Walter B. Gall. Mcrk. Sharp. and Dohme Research Laboratory. Rahwnq. New Jersoq. Mr. Akira J. ikehara helped to collect the .4. wrric~w~is. Dr. Dennis L. Taylor, University of Miomi, provided helpful commrtnt and criticism concerning the manuscript. REFERENC’ES
ASKAR~ A. & RAO S. N. (lY70) Drugs atrecting sodium transport in human erythrocyte ghosts. J, P~~[~rt~f~~.(‘\I’. Tker. 172, 21 l-x23. BOKLE A. B. (lY73) Calcium metabolism at the cellular level. Ferln. Proc. Fcd,~. ,401. S0c.s rup. Birjl. 32, 1944-1950. CHALKER B. E. (1975) Calcification. metabolism. and growth in the staghorn coral, .4croporrc crrricwwi,\. Ph.D.. Dissertation. University of Miami. CHALKCR B. E. & TAYLOR D. L. (1975) Light-elll~~nced calcification. and tbc role of oxidativc phosphor~l~~ti~~n in calcification of’ the coral Acrq.ww cr,rr~ic~orrzi.s.PWC. R. Sot. B. 190. 323.-331. Doou J. R. (lYh7) Magnesium and strontium 111c:dc;n-cons skeletons: A revicwy J. Pcrlwrrt. 41, 13I3- I 3%. DL’GGAN D. E. & NML R. M. (1965) The c%ct of ethncrynic acid, cardiac glycosidcs upon a membrane :rdenoBwsine tri~~~osp~~~lte of renal cortex. 4rcf1s. ~j~~~.~~~~/~l. f&lx 109. 3X8 396. EPSTI:IN R. W. (lY71) The eflects of’ cthacrynK xid on active transport of sugars and ions and on other metabolic processes in rabbit kidney cortex. Biochirn hioph,~,\. Acta 274, 12X 139.
Calcium
transport
during
skeletogenesis
GLYNN P. W. (1973) Aspects
of the ecology of coral reefs Atlantic region. In Biology and Geoloy,~ of Cowl Ree$~ (Edited by JONES 0. A. & ENDEAN R.). Kj/, II, Bioloyy I pp. 271 -324. Academic Press. New York. GOREAU T. F. (1959) The physiology of skeleton formation corals-- I. A method for measuring the rate of calcium deposition by corals under different conditions Biol Bull. mrrr’. hiol. Ltrh., Wads Hole. 116, 59-75. GOREA~ T. F. (1961~) On the relation of calcification to primary productivity in reef building organisms. In The Biology of Hydra and somr other Corlenterates (Edited by LENHOFFH. M. & LOOMIS W. F.), pp. 267-285. University of Miami Press. Coral Gables, Florida. GORIZAUT. F. (1961 h) Problems of growth and calcium deposition in reef corals. Endeucour 20, 32-39. GORFAU T. F. & BOWEN V. (1955) Calcium uptake by a coral. Scirwe, N. Y. 122, 1 188-l 189. GoI<~:~\~ T. F. & GOREAU N. 1. (1960~) The physiology of skeleton formation in corals~ll. Calcium deposition by hcrmatypic corals under various conditions in the reef. Biol. Bul/. mur. hiol. Lob.. Woods Hole. 117, 239-250. GOKIA,I T. F. & GoRt:AI, N. I. (1960b) The physiology of skclcton formation in corals IV. On isotopic equilibrium exchanges of calcium between corallum and environment in living and dead reef-building corals. Biol. in the Western
Bull. mar. bid. Luh.. Woods Hole. 119, 416-427. KINSMAN D. J. J. (1969) Interpretation of Sr’+ concen-
trations Pcvrol.
in carbonate 39.
minerals
and
rocks.
J.
srdim.
486-508.
LYMAN J. & FL~MINC; R. H. (1940) Composition water. J. Mar. Rcs. 3. l34- 146.
of sea
in hermatypic
corals
459
MUSCATINI: L. (1973) Nutrition in corals. In Bioloyy md Geology of’ Coral RwfS (Edited by JONES 0. A. & ENDEAN R.). C’ol. II. Biology I pp. 271-324. Aacademic Press. New York. PATE J. B. & ROBIMON R. J. (195X) The (ethylene dinitrilo) tetraacetate titration of calcium and magnesium in ocean waters I. Determination of calcium. J. )?1(1r. Rrs. 17. 390 402.
PLOWMAN K. M. (1972) E,r~~,nlr Kinc>rics p, 171. McGrawHill. New York. SCHATZMANN H. J. (1970) Transmembrane calcium movements. In Culciunt und Cellular Function (Edited by CU~HBERT A. W.). pp. X5-95. St. Martin’s Press, New York. SEIN W. D. (1964) Facilitated diffusion. In Recrnt Progress in Sw$ce Scirrm (Edited by DANIELLI J. R., PAUKHURST K. G. A. & RIDDIFORD A. C.), Vol. I. pp. 300 337. Academic Press, New York. VAN~ERMEULENJ. H., DAVIS N. D. & MUSCATINE L. (1972) The effect of inhibitors of photosynthesis on zooxanthellae in corals and other marine invertebrates. Mar. Biol. 16, IX5 191. VAUD~RMLI:LI‘N J. H. & MLIS(.ATIN~: L. (1974) Intluencc of symbiotic algae on calcification in reef corals: Critique and progress report. In S!wrbioais in t/w SW (Edited by V~RUIXRC~W. B.). pp. I 20. University of South Carolina Press. Columbus. South Carolina. WLB~R J. N. (1973) Incorporation of strontium into reef coral skeletal carbonate. Groclrin~. c~ostnocllinl. 4cto 37. 2 I 73-2 190.
TRANSPORT DURING SKELETOGENESIS IN HERMATYPTC CORALS BRUCE E. CHALKEK
Roscnstiel
School of Marine 4600 Rickenbacker
and Atmospheric Science. University of Miami. Causeway. Miami. FL 33149, U.S.A.
(Rewired
26 AUJUS~
197.5)
I. Light-enhanced calcification in the hermatypic corals Acroportr crrricorni.s and A. /ortnos~~ results from the active transpo’rt of calcium ions. It shows saturation kinetics. 2. Dark calcificatmn. simulated by the addition of DCMU, results from enzyme mediated isotopic exchange. 3. Strontium is a competitive inhibitor of both light-enhanced and dark calcification. The K, is I.3 mM. 4. The data refutes the diffusional model for calcium movement in hermatypic corals. Abstract
IRTRODUCTION R&-building (hermatypic) corals are inevitably associated with the endosymbiotic dinoflagellate, G~jmrlodir7ium mic.roildricrricun7. Endosymbiont photosynthesis allows the corals to calcify may times faster in the light than in the dark. In contrast, ahermatypic corals and hermatypic corals which have lost their endosymbionts (aposymbiotic corals) calcify at rates which are slow and unaffected by illumination (Goreau, 1959. 1961a’; Goreau & Goreau, 1960~). If hermatypic corals are exposed to lo-’ M DCMU (3-(3,4-dichlorophenyl)-l.l-dimethyl urea), algal photosynthesis and light-enhanced calcification will be completely abolished (Vandermeulen, Davis, & Muscatine,
1972;
Chalker,
1975).
The biochemical mechanisms of calcium movement in corals are unknown. Goreau & Bowen (1955) demonstrated that the intracellular calcium concentration of the ahermatype Astrungiu dame is less than that of the surrounding sea water, implying the existence of active calcium transport. All present models of light-enhanced calcification ignore the energetic demands which would be placed upon the symbiotic association by high rates of calcium transport (for a review see Vandermeulen & Muscatine, 1974). Calcification processes are also important geochemically. The relative incorporation of calcium and strontium into biogenic carbonate has been extensively examined as a potential source of information about paleoenvironmental, paleoecological, and diagenie conditions (for reviews see Dodd, 1967; and Kinsman, 1969). The principal model for strontium incorporation postulates that strontium and calcium diffuse to the sites of skeletogenesis (Weber, 1973). ‘The accuracy of this model and thus its predictive value depends upon the validity of the diffusional assumption. I have examined and compared the kinetics of calcium transport in two species of hermatypic corals, 4croporu cwaicornis from the Atlantic and Acropora fimnosa from the Pacific. This comparison helps to identify the energy demands of light-enhanced calcifi-
cation. define the dark rate of calcification. the diffusional assumption.
and test
MATERIALS AND METHODS Acropmx crrricomis was collected by SCUBA diving at depths of 45 feet near Grand Bahama Island, Bahamas. Experiments were conducted aboard the University of Miami’s R!V CALANUS. ifuoporrr fhrrnmtr was collected at a depth of 6 feet in the “Quarry”. Eniwetak Island. Eniwetak Atoll. Marshall Islands. Experiments here conducted at the Mid-Pacific Marine Laboratory. Corals were maintained under illumination in aerated aquaria at 29 C, and used the day of collection. Terminal portions of branches were cut to 3 cm with surgical bone forceps, rinsed twice with calcium- and strontium-free Lyman & Fleming artificial sea water (Lyman & Fleming. 1940). and placed in I50 ml of modified Lyman & Fleming artificial sea hater in 350 ml polycarbonatc beakers. Calcium content of the medium was adjusted by adding I.0 M CaC12 before introducing the coral tips. In some experiments. algal photosynthesis and light-enhanced calcification were abolished by the addition of I.5 ml of IO-’ M DCMU in absolute ethanol to each beaker of A. wrriwruis and I.5 ml of 10~’ M DCMU to each beaker of A. fiwnwsu. Controls indicate that this concentration of ethanol affects neither the light nor the dark rate of calcification. Prior to the start of each experiment IO ml samples were withdrawn from each beaker to determine the calcium concentration by EDTA (ethylenediaminetetraacetic acid) titration (Pate & Robinson, 195X). Corals were preincubatcd for 12 hr prior to the addition of IO rtl of “CaCI, (30 uCi:ml). Incubations with the label lasted 2 hr at-29’C. Illumination at the surfilce of the medium was 10.500 lux. This was provided by an ovcrhead bank of cool white fluorescent bulbs. After 2 hr, the corals were removed, rinsed in calciumand strontium-free artificial seawater, and placed in 50”,, chlorox~50’~, tap water for I hr to remove tissue. The tips were then cut to 2 cm and stored at room temperature. Skeletons were dissolved in liquid scintillation vials with concentrated HCI plus 0.1 ml of I-butanol to retard foaming. The final volume was adjusted to 3 ml with distilled water, and 9 ml Aquasol (New England Nuclear) was added to produce a clear gel. Samples were counted in a Nuclear Chicago Mark II liquid scintillation counter.
456
BRUct:
1
0
E.
CHALKKK
t
I
20
IO MILLMOLAR
CALCIUM
30
I 40
CONCENTRATION
Fig. I. A plot of calcification rate vs mM calcium concentration for A. curvicorrlis incubated at an illumination intensity of 10.500 lux without (open circles) and with (closed circles) 1O--5 M DCMU. Mean + one SD for five individuals Calcification rates are expressed fixed per hour per l-cm tip.
as micromoles
of calcium
RESULTS
Calcification Calcification rates as a function of calcium concentration were measured at an illumination intensity of 10,500 lux in the absence and in the presence of DCMU for Acropora ceroicomis (Fig. 1) and A. ,firHIOS~(Fig. 2). The curves follow Michaelis-Menton erlzyme kinetics. These meas~trements were repeated for A. ,formosa incubated in the light with lo-’ M ethacrynic acid. The resultant curve (Fig. 3) is nearly identical to the corresponding DCMU curve. A double reciprocal replot was made of the data presented in Figs. l--3. All the lines were fit by computer using the method of least squares. The maximum 30
1
T
I Fig. 2. A plot of calcification rate vs mM calcium concentration for A. ,fix~osa incubated at an illurni~3~~tio~3intensity of 10,500 lux without (open circles) and with (closed circles) IO -’ M DCMU. Mean i one SD. for five
Fig. 3. A plot of c~lci~c~~tioll rate YS mM calcium concentration for A. ,fhrrnosc~ incubated at an illumination intensity of 10,500 lux with IO-’ M ethacrynic acid. Mean + one SD. for five individuals.
rates of ~Icification (V,,,) and the apparent K,‘s are presented in Table 1. Within experimental error, the K.,‘s are identical. For each species the maximal rate of calcification at 10,500 lux is dependent upon the nutritional status of the animals and the time of day during which the experiments are conducted. Regional variations are also possible (Glynn, 1973). As a control, a similar experiment was conducted with A. ccroicornis fixed for I8 hr in loo,{, formalin in sea water. The rate of calcification is nearly directly proportional to the calcium concentration (Fig. 4). These data are indicative of calcium diffusion from the medium to the skeleton and subsequent isotopic exchange. Similar results were obtained for A. jbriW)SLi.
Calcium
transport
during
skeletogenesis
in hermatypic
8
Table I. The maximal rates of calcification (V,,,) and apparent K,‘s for A. cervicornis and A. fornwsa. The rates are pmoles of calcium fixed/hr per 2-cm tip. The concentrations
and K,‘s
are expressed
(DCMI!)
457
corals
1
in mmoles/liter.
(Ethacrymc Xld)
v,., ,\
Apparent K.,.
Fig. 6. Competitive
inhibition of calcification in A. cerviReciprocal velocity vs reciprocal mM concentration. Mean for five individuals incubated at 10.500 lux with IO-’ M DCMU.
uw~~~s by stronttum.
calcium
Fig. 4. A plot of calcification rate vs mM calcium concentration for A. cercicornis fixed in 10% formalin in sea water for 18 hr. Mean i one S.D. for five individuals. Comprtiticeinhibition by
strontium
tration for A. certlicornis incubated at various strontium concentrations without and with lo-” M DCMU are shown in Fig. 5 and 6. Completely competitive inhibition is indicated in both cases by the constant y-intercept (l/V,,,) and the variable x-intercept. A replot of the slopes in Fig. 5 and 6 vs strontium concentration is shown in Fig. 7. The value of the inhibition constant (K,) is given by negative one times the value of the x-intercept. For incubations with and without DCMU, the K, is I.3 mM. DISCL’SSION
Inhibition of calcification by strontium was tested by the method of Plowman (1972). Plots of reciprocal calcification rate vs reciprocal mM calcium concen-
35-
Several observations are relevant to a discussion of skeletogenesis in scleractinians. First, coral tissues have a lower calcium concentration than that of seawater (Goreau & Bowen, 19.55). Second, lightenhanced calcification requires the energy derived from oxidative phosphorylation (Chalker & Taylor, 1975). Finally, light-enhanced calcification follows
m~lhmolor strontum concentratw Fig. 5. Competitive inhibition of calcification in A. cornis by strontium.
calcium
cerci-
Reciprocal velocity vs reciprocal mM concentration. Mean for five individuals incubated at 10.500 lux.
Fig. 7. Replot of the data in Figs. 5 and lines vs mM strontium concentration. The tion with (closed circles) and without (open is given by negative one times the value of
6. Slope of the K, for incubacircles) DCMU the x-intercept.
4%
BKUC~ E.
enzyme mediated kinetics. The light-enhanced incorporation of calcium into skeleton is thus an active, mediated process. The diffusional theory of calcium movement is refuted. Light-en~l~~llced c~llci~cation creates a significant energy demand which may possibly be satisfied by the oxidation of low molecular weight compounds translocated from the endosymbiotic algae to animal tissues. Previous studies. however. have shown that glucose, glycerol, and alanine which arc cvcreted hy the cndosymbiotic algae and readily assimilated and mct~~bolized by hermatypic corals (~usc~~titlc. 1973). do not increase the rates of caici~c~~ti~~i~in the dark. light. or light with DCMU (Vandermeulen &i Muscatine, 1974; Chalker. 1975). Other organic compounds should be examined. Calcification in the dark also appears to follow enzyme kinetics although most of the rate is not inhibited and uncouplers of oxidative ph~~sphory~~~tioll (Chalker & Taylor, 1975). It probably results from facilitated diffusion (Stein. 1964; Borle. 1973) and suhsequent isotopic exchange. Stein has given a general equation for facilitated diffusion: Net
Rate
=
v,,;,,
-_.“I_._- -3
s, i- K”,
Sz + Kwi
where S, and S2 are the concentrations of permeant on the outside and the inside of the system respectively. If the number of calcium-45 ions present in the skeleton is small in comparison to the total number of calcium ions on the surface of the skeleton, this equ~~tion simplifies to a form identical to MichaelisMenton kinetics. The relatively high rate of exchange contradicts the findings of Goreau & Goreau (196Oh: Goreau, 1961 h) and magnifies the significance of endosymbiotic algae in enhancing carbonate deposition on the reef. It also poses an interpretational problem for those investigators ~~lc~iiating light-dark ratios and comparing calcification rates as measured by calcium-45 and carbon- l4-bicarbonate. If this mediated exchange also occurs in the light, the customary calcification rates measured with calcium-45 would be too large. The portion of the dark calcification rate which is due to active transport and results in the accretion of calcium carbonate has not yet been accurately determined. Ethacrynic acid has been widely used as an inhibitor of cation transport and of N~‘.;KK ‘- and and Ca’ ‘,Mg’ ‘-ATPases (Askari & Rae, 1970; Duggan & Knoll. 1965: Schatzmann, 1970). Epstein (1972) emphasizes that ethacrynic acid inhibits ATP production by both oxidative phosphorylation and by gfycolysis. He states that ethacrynic acid affects the energy supply available for the active transport of any solute and cannot be used as a specific ATP-ase inhibitor. Whatever the mode of action, IO- ’ M cthacrynic acid completely inhibits light-enhanced calcification in A. ccwicnrr~i.s without affecting photosynthesis by the isolated endosymbiotic algae (Chalker. 1975). Figure 3 demonstrates that when A. fi~rttz~~suis inhibited with ethacrynic acid, the kinetics of calcification are nearly identical with those found during DCMU inhibition. This supports the contention that the dark rate of calcification results chiefly from facilitated diffusion
CtihLKEK
and subsequent isotopic exchange. The diffusional control (Fig. 4) demonstrates that similar kinetics do not result from simple diffusion and the resultant isotopic exchange. Strontium is a competitive i~~hibit~~rof both lightenhanced and dark calcification in A. ~~~~j~~~~?~~.s (Figs. 57). In both cases the Ki is 1.3 mM. When facilitated diffusion is inhibited by a substrate analogue, the net rate of flux is given by the equation: Net Rate =
_
‘5 -2
S2 + K, -t (R,:K,)K, where R, and R2 are the concentrations of the inhibitor on the outside and the inside of the system respectively and K, is the inhibition constant (Stein. 1964). Again if the number of calcium-45 ions present in the skeleton is small in comparison to the total number of calcium ions on the surface, the rquation simplifies to a form which is identical to that derived from Michaelis-Menton kinetics. These measurements are not sutticiently precise to reflect the small variations in the strontium/caicium ratio reported by geochemists but they demonsti-ante that any model that explains the strontium~c~~lcium ratios found in coral skeletons must consider the specificity of the transport sites. A~k,~o,r~/~,~~~e,~~~~i~rs---This work was supported by, NSF grants GA-43270 :tnd GA-33533 X. The work at Emwtak ~v:as conducted at the Mid-~~ci~c Marine Laborator? under the auspices of the University of Hawaii and the Atomic Energy Commission. The assistance of Dr. Phihp HelErich, Director. Mr. Phil Lamherson. Lab Mwxqer, nnd Mr. Robert Hall. Radiological Safety Oflicer. Universit) of Ha&i. is gratefully ncknowledged. The cthacrynic acid was supplied by Walter B. Gall. Mcrk. Sharp. and Dohme Research Laboratory. Rahwnq. New Jersoq. Mr. Akira J. ikehara helped to collect the .4. wrric~w~is. Dr. Dennis L. Taylor, University of Miomi, provided helpful commrtnt and criticism concerning the manuscript. REFERENC’ES
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