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Calcium exchanges in two neotenic urodeles: Necturus maculosus and Ambystoma tigrinum. Role of the integument
Camp B,o
CALCIUM NECTURUS
031X%9629/81/090065-O4102.C4l/O Copyright 0 1981 Pergamon Press Ltd
EXCHANGES MACULOSUS ROLE
IN TWO NEOTENIC AND AMBYSTOMA
URODELES: TIGRINUM.
OF THE INTEGUMENT*
GERALDINEF. BALDWIN and P. J. BENTLEY~ Departments of Pharmacology and Ophthalmology, Mount Sinai School of Medicine of The City University of New York, NY 10029, U.S.A. (Receiaed 3 February 1981) Abstract-l. We have measured calcium exchanges across the integument of two neotenous urodele amphibians, mudpuppies, (Necturus maculosus) and tiger salamanders (Ambystoma tigrinum). We also studied adult tiger salamanders. 2. The integument was permeable to Ca, both an uptake and loss of the mineral may occur. In fasting mudpuppies and adult tiger salamanders integumental and urinary loss of Ca were similar to each other. The total Ca loss each day was equivalent to less than 0.5% of the total body calcium, though it represents 35 to 50% of the amount of Ca in the extracellular fluids. 3. Unidirectional fluxes of Ca (influx and efflux) across the skin of the mudpuppy were similar (in vitro) suggesting that an active transport of Ca is not occurring. 4. The permeability of the external gills of the neotenous animals appeared to be very low compared to that of the skin; there was no change in accumulation of 45Ca (in vim) when the gills were ligated.
INTRODUCTION
METHODS
Aquatic vertebrates may experience special problems with respect to their calcium metabolism which reflect the permeability of their integuments and the Ca content of the water in which they live. This situation has been studied in fish (see Dacke, 1979; Pang et al., 1980). These animals can exchange Ca across their gills so that in seawater they tend to become hypercalcemic, as a result of Ca uptake, while in freshwater they tend to lose the mineral and become hypocalcemic. Amphibians also possess a relatively permeable integument which, in this instance, is mainly skin, although larval and neotenic forms also possess gills. In 1937 Krogh showed that the skin of frogs (Rana esculenta) was permeable to Ca; an observation which has since been confirmed in other anurans (Watlington ef al., 1968; Zadunaisky & Lande, 1972; Baldwin & Bentley, 1981). The exchanges of calcium across the integument of urodeles has, however, not been previously studied. Some of these amphibians are of special interest as they are neotenous and possess prominent external gills so that Ca exchange may be occurring through both cutaneous and branchial pathways. In the present investigation we have measured the accumulation of 45Ca across the integument of two species of neotenous urodeles, the mudpuppy Necturus maculosus and the tiger salamander Ambystoma tigrinum. The permeability of their skins to Ca was similar to that described previously in Rana pipiens (Baldwin & Bentley, 1981) and in contrast to anuran tadpoles (Baldwin & Bentley, 1980) exchanges of Ca across their gills were not detectable.
Mudpuppies, Necturus maculosus, were obtained from a commercial supplier (Nasco, Fort Atkinson, Wisconsin) and kept, unfed, in a tapwater aquarium at room temperature (21-22°C). Tiger salamanders, Ambystoma rigrinum were obtained from Amphibians of Eastern North America, Nashville, Tennessee. A neotenic larval form (A. tigrinum) was kept in a tapwater aquarium at 16”C, and the adult on a bed of moistened foam chips in a terrarium at the same temperature. Artificial pondwater, with the following ionic composition (mM): I.0 NaCl; 0.1 KCI; 0.05 MgSO.,; 0.2 NaHCO, and 0.2 calcium gluconate was used as the experimental bathing medium. The animals were killed by pithing. In order to remove any Ca adhering to the outer surface of the body, the animals were first rinsed for 5 min, with three changes, in Ca-free pondwater. The calcium content of bathing medium, serum, urine, whole animals or tissue samples was determined using a Perkin-Elmer 290B atomic absorption spectrophotometer. Whole animals or tissue samples were dried, and ashed overnight at 500°C in a muffle furnace. The ash was dissolved in a small amount of concentrated HCl, and diluted with LaCI, solution to give a final concentration of 0.1 N HCI and 1% LaCI,. The accumulation of 45Ca in oiuo was determined by exposing the animals to 11 of pondwater containing the isotope (0.1 &i/ml) for 24 hr. In order to estimate what contribution the gills make to Ca accumulation, a group of animals were lightly anesthetized in icewater and ligatures were tied around the external gills, thereby cutting off their circulation. The gills were ligated on the day prior to the experimental measurement of Ca uptake. A control group of animals were also immersed in icewater and handled similarly to the experimental animals. At the end of the experimental period the animals were rinsed, anesthetized with 0.1% tricaine methane sulfonate (Sigma Chemical Co, St. Louis, Missouri) and divided into separate tissue components: skin, gill, gut, and the remaining carcass. For determination of %a the tissues were dried overnight at 115°C. and then ashed at 500°C. The ash was dissolved in a
* Supported by National Science Foundation Grant No. PCM78-21446. t To whom correspondence should be addressed.
G~KUI~INI- F. BAI DWIN and P. J. BFNTI I-Y
66
small amount of concentrated HCI. and aliquots of diluted solution were counted in a hquid scintillation counter (Beckman LSIOO). Although it has been shown that amphibians drink very little (Bentley & Yorio, 1979). some ingestion of the external medium may occur. We measured such drinking using [‘251]Na-iothalamate (GloFil. Abbott Laboratories, Chicago. Illinois) as a non-absorbable water maker. Animals were placed in pondwater containing 0.2 btCi;ml “‘1. and exposed to it for 8 to 16 hr. After brief rinsing, the animals were killed and the whole digestive tract was removed and counted in a Beckman autogramma counter (Baldwin & Bentley, in preparation). Calcium loss was estimated by exposing animals to a measured volume of pondwater with no added calcium (0.01 mM Ca). The mudpuppies or salamanders were first anesthetized with tricaine methane sulfonate. and a polyethylene cannula, emptying into a small rubber bag, was fitted into the cloaca and attached with a purse string suture. Calcium concentration of the urme and balhing medium was measured after 16 hr for mudpuppies, and 8 hr for salamanders. To assess the role of the skin in calcium exchange, bidirectional 45Ca transcutaneous fluxes were measured using paired pieces of ventral skin mounted in Ussing-type chambers (3 cm’ area) (Baldwin & Bentley, 1981). To minimize “edge damage” double parafilm “o-rings” and a layer of silicone grease were placed between the lucite chamber halves and the tissue. The preparations were continually short-circuited with an automatic voltage clamp. “5Ca (0.5 /&i;‘ml) was placed in one side of the chamber and samples were collected at intervals from the opposite or frtrns side. The specific radioactivity of the cis side was used to determine the flux across the tissue. The ““Ca used was obtained from New England Nuclear, Boston. Massachusetts.
Table
I. Calcium
content
Hk:SI
1.7s
The calcium concentrations puppies and tiger salamanders
in the
compared The total
the neotenous urodeles was less than that of the adult tiger salamander.
The exchanges of Ca between the urodeles and the external bathing media are shown in Table 2. In mudpuppies the measured uptake and losses of Ca are similar, indicating that the animals can maintain a C’a balance under these fasting conditions. The accumulation of ?a (in rioo) each day is equivalent to 0.04”,, of the total body Ca or about one-third of all that present in the extracellular fluids. Loss of Ca to a Ca-free solution was 0.12 mmol kg- ’ BW day 1 and it occurs in equal amounts in the urine and across the integument (i.e. both skin and gills). Neither the neotenic nor adult tiger salamanders appear to be able to maintain a Ca balance under these, fasting, conditions. In the neotenic animals daily accumulation was equivalent to 0.07”,, of total body Ca or about 50”,, of that in the extracellular fluids. The Ca loss was. however, about 4-times
of N. muculosus
and A. riyrimrm
Skin
N. mtrculo\~r.s (8)
324 _t 30
5.4 f 0.6
3.8 ) 0.4
2.5 + 0.3
I.6 _+ 0.1
A. tigrinum Neotenic (4) Adult (6)
151 f 5 250 + I5
3.9 + 0.2 3.6 f 0.4
6.6 + 1.8
9.2 f 3.1 7.9 f 0.8
1.05 ? 0.05 2.0 4 0.1
k SE. Number
Table 2. Calcium Calcium Urine (mmol kg-’ N. mclculosus
of animals
exchanges
Gills (mmol kg-
0.05 f 0.01 (7)
0.07 + 0.01 (7)
0.37 f 0.05 (9) 0.18 & 0.03 (12)
0.33 * 0.07(12)
Serum
in parentheses.
in fasting
loss Integument day-‘)
Gut
’ tissue)
urodele
(mmol kg-’
amphibians *‘Ca gain Integument day-‘) (nmolcm~L
hr-I)
0.1 I f 0.02 (9)
1.8 + 0.33 (9)
0.10 + 0.02(5) 0.05 + 0.02(5)
I.8 k 0.23 (5) 0.6 i 0.24(5)
A. figrinum
Neotenic Adult
of mud-
to 2.5”,, in frogs (Baldwin & Bentley. 1981). Ca content and serum Ca concentration of
Whole animal (mmol kg-’ BW)
Values are as mean
tissues
are given in Table I. In frogs and toads the skin contains relatively high concentrations of Ca but this was not seen in these urodeles. The Ca present in the skin of these amphibians was equivalent to 0.24 to 0.45”,, of the total body Ca,
*
* Not detectable. The gain of 45Ca is that accumulated in the carcass of the animals no! including the gut. skin 01 gills. Uptake experiments were performed in animals bathed with pondwater containing 0.2 mM Ca and loss was measured in a Ca-“free” media containing less than IO- ’ M Ca. Results are as means f SE. Number of experiments in parentheses.
Ca exchanges in urodeles greater than this uptake; loss across the integument was not detectable, urinary loss being predominant. In adult tiger salamanders the loss of Ca was about lo-times greater than uptake and the leakage from the skin exceeded the loss in the urine. The contribution of drinking to the uptake of Ca is negligible, under fasting conditions, in the larval animals and minor in the adult. The rate of drinking in neotenic tiger salamanders and mudpuppies was, respectively 0.014 and 0.016 ml 100 g-’ hr-’ (Baldwin & Bentley, in preparation) which, assuming complete absorption of the ingested Ca, would correspond to 0.6 and 0.7% of the total Ca accumulated. In the adult tiger salamanders drinking was 0.075 ml 100 g- 1hr - ’ which could provide a Ca uptake equivalent to as much as 7”~; of the total accumulation. A role for the gills in Ca exchange? The results of the experiments
on Ca accumulation
in oivo (Table 2) suggested that permeability of the adult A. tigrinum integument is less than that in the
neotenous animals. It is possible that this difference may reflect the presence of gills in the latter. However the uptake of 45Ca did not differ significantly between animals with their gills ligated and paired control animals; influx (mmol kg- ’ BW day- ‘) .was, respectively, 0.09 k 0.02 (9) vs 0.11 f 0.02 (9) in N. maculosus, and 0.08 _t 0.02 (5) vs 0.10 + 0.02 (5) in neotenic A. tigrinum. Transcutuneous Ca movement in vitro The movement of 45Ca was measured across electrically short-circuited pieces of skin (in oitro) of the mudpuppies. There was no significant difference between the influx (outside to inside) and efflux: influx was 1.8 2 0.21 (6) nmolcm-2hr-1 and efflux was 1.4 + 0.32 (6) nmolcm-2 hr-’ (1 mM Ca2+ both sides). Thus an active transport mechanism for transcutaneous Ca transport does not appear to be functioning under these conditions. The influx was reduced when the external Ca concentration was reduced from 1.0 to 0.2 mM, as it is in the pondwater. The results of Tables 2 and 3 can be used to make a comparison of the Ca influx observed in vitro and in ciuo. In mudpuppies, the permeability of the skin to Ca was not different in either situation. In neotenous
Table 3. Transcutaneous movement of ‘%a across urodele skin, in vitro. The outer surface was bathed by 0.2 mM Ca, which is the concentration used in uiuo 45Ca influx (nmol cm-‘hr-‘) N. macu/osus (March) (6)
1.0 f 0.13
61
tiger salamanders, however, the two sets of results were not consistent, Ca influx in vitro was only about half that observed in uiuo. On the other hand, in the adult tiger salamanders the Ca influx in uiuo was about double that seen in vitro. Clearly the experimental conditions vary considerably in uiuo and in vitro and presumably contribute to these inconsistencies. A comparison (in uitro) of Ca influx (Table 3) in the urodeles and anurans is also provided. The tadpole’s skin was less permeable to Ca than that of the urodeles. Frog skin showed seasonal variability but was similar in November to that of the urodeles in spring and summer. DISCUS!3ON
An exchange of calcium can occur across the integument of urodele amphibians. A permeable integument is considered to be a phyletic character of the Amphibia and is of considerable physiological significance with respect to respiration, and water and salt metabolism. The permeability of the skin to calcium is not great, uptake may, however, compensate for urinary losses. Leakage of Ca from the skin in mudpuppies and adult tiger salamanders was of a magnitude similar to that which is lost in the urine. In fish the gills are an important site for Ca exchanges (see Simmons, 1971; Dacke, 1979) and in teleosts this process may be regulated by the hormone calcitonin and extracts of the corpuscles of Stannius (Milhaud er al., 1977; Milet, Peignoux-Deville & Martelly, 1979; SO & Fenwick, 1979). Tadpole gills also appear to be permeable to Ca and uptake of this mineral is reduced by injections of calcitonin (Baldwin & Bentley, 1980). However, the external gills of the neotenic urodeles appeared to have a much lower permeability to Ca than the skin and was not measurable. This property of the gills may be fortunate, or an evolutionary adaptation, as neotenous urodeles normally live in solutions in which the Ca concentrations are much less than that in their body fluids. They thus would be prone to lose this mineral by diffusion, which, in the absence of any compensating “Ca pumps” in the integument, could accentuate the problem of their Ca homeostasis. The skin of anuran amphibians usually contains large amounts of Ca (see Baldwin & Bentley, 1981). The functions of this cutaneous Ca is unknown but it could have a structural role and even function, like bone, as a store for the mineral. The skins of the urodeles which we examined lacked such a prominent store of Ca. It is unknown whether this is a phyletic characteristic, though the levels are also low in an anuran, the toad Xenopus laeuis.
A. tigrinum
Neotenic form (July-August) (6) Adult (July-August) (6)
0.8 f. 0.17 1.5 + 0.1
Runu pipien.9
November (8) February (8)
0.9 & 0.17 0.2 + 0.07
Rum cutesbeiuna
Tadpoiest (12)
0.2 + 0.07
REFERENCES BALDWIN G.
F. & BENTLEYP. J. (1980) Calcium metabolism in bullfrog tadpoles (Ram catesbeianu) J. exp. Biol.
88, 357-365. G. F. & BENTLEY P. J. (1981) A role for skin in Ca metabolism of frogs? Comp. Biochem. Physiol. 68, 181-185. BENTLEY P. J. & YORIOT. (1979) Do frogs drink? J. exp. Biol. 79, 41-46.
BALDWIN
* From Baldwin & Bentley (1981). t From Baldwin & Bentley (1980). Results are as means f SE. Number of experiments in parentheses.
68
GERALDINE
F. BALDWIN and P. J. BENTLEY
DA~KE C. G. ( 1979) CtrlGrrm Reyulution in Sub-mammaliun Vc~rehrur~s, pp. 8488. Academic Press. New York. KROGH A. (1937) Osmotic regulation in the frog (R. escuIrntcr) by active absorption of chloride ions. Skund Arch. Physiol. 76, 6@74. MILET C.. PEIGNOUX-DEVILLF J. & MARTELLY E. (1979) Gill calcium fluxes in the eel Anynilltr trnguillu (L.) effects of Stannius corpuscles and ultimobranchial body. Comp. Bioc~ltrm. f%)~i~~l. 63A. 63 -70. M1LHAC.L) G.. RANKIN J. C.. B~LIS L. & BENSON A. A. (1977) Calcitonm: its hormonal action on the gill. Proc. nutn. A~rtl. .Qi. L’.S.A. 74, 469334696. PANG P. K. T., KENNY A. D. & OGURO C. (1980) Evolution of endocrine control of calcium regulation, In Er’oltrfion of Vutc+raft~ Endocrine Swetns (Edited by
PANG P. K. T. & EPPLE A.). pp. 3233356. Texas Tech. Press, Lubbock. SIMMONS D. J. (1971) Calcium and skeletal tissue phystology in teleost fishes. C/in. Orthopaedics 76, 244-280. So Y. P. & FENWICK J. C. (1979) In riw and in vitro effects of Stannius corpuscle extract on the branchial uptake of 4SCa in stanniectomized North American eels (Anyttilltr rostrutu) Gen. camp. Entlocr. 37, 143. 149. WATLINGTON C. 0.. BURKE P. K. & ESTEP H. L. (1968) Calcium flux in isolated frog skin: the effect of parathroid substances. Proc. Sot. cup. Biol. Med. 128, 853-856. ZADUNAISKY J. A. & LANDE M. (1972) Calcium content and exchange in amphibian skin and its isolated epithelium. Am. J. Ph!xiol. 222, 130991315.
CALCIUM NECTURUS
031X%9629/81/090065-O4102.C4l/O Copyright 0 1981 Pergamon Press Ltd
EXCHANGES MACULOSUS ROLE
IN TWO NEOTENIC AND AMBYSTOMA
URODELES: TIGRINUM.
OF THE INTEGUMENT*
GERALDINEF. BALDWIN and P. J. BENTLEY~ Departments of Pharmacology and Ophthalmology, Mount Sinai School of Medicine of The City University of New York, NY 10029, U.S.A. (Receiaed 3 February 1981) Abstract-l. We have measured calcium exchanges across the integument of two neotenous urodele amphibians, mudpuppies, (Necturus maculosus) and tiger salamanders (Ambystoma tigrinum). We also studied adult tiger salamanders. 2. The integument was permeable to Ca, both an uptake and loss of the mineral may occur. In fasting mudpuppies and adult tiger salamanders integumental and urinary loss of Ca were similar to each other. The total Ca loss each day was equivalent to less than 0.5% of the total body calcium, though it represents 35 to 50% of the amount of Ca in the extracellular fluids. 3. Unidirectional fluxes of Ca (influx and efflux) across the skin of the mudpuppy were similar (in vitro) suggesting that an active transport of Ca is not occurring. 4. The permeability of the external gills of the neotenous animals appeared to be very low compared to that of the skin; there was no change in accumulation of 45Ca (in vim) when the gills were ligated.
INTRODUCTION
METHODS
Aquatic vertebrates may experience special problems with respect to their calcium metabolism which reflect the permeability of their integuments and the Ca content of the water in which they live. This situation has been studied in fish (see Dacke, 1979; Pang et al., 1980). These animals can exchange Ca across their gills so that in seawater they tend to become hypercalcemic, as a result of Ca uptake, while in freshwater they tend to lose the mineral and become hypocalcemic. Amphibians also possess a relatively permeable integument which, in this instance, is mainly skin, although larval and neotenic forms also possess gills. In 1937 Krogh showed that the skin of frogs (Rana esculenta) was permeable to Ca; an observation which has since been confirmed in other anurans (Watlington ef al., 1968; Zadunaisky & Lande, 1972; Baldwin & Bentley, 1981). The exchanges of calcium across the integument of urodeles has, however, not been previously studied. Some of these amphibians are of special interest as they are neotenous and possess prominent external gills so that Ca exchange may be occurring through both cutaneous and branchial pathways. In the present investigation we have measured the accumulation of 45Ca across the integument of two species of neotenous urodeles, the mudpuppy Necturus maculosus and the tiger salamander Ambystoma tigrinum. The permeability of their skins to Ca was similar to that described previously in Rana pipiens (Baldwin & Bentley, 1981) and in contrast to anuran tadpoles (Baldwin & Bentley, 1980) exchanges of Ca across their gills were not detectable.
Mudpuppies, Necturus maculosus, were obtained from a commercial supplier (Nasco, Fort Atkinson, Wisconsin) and kept, unfed, in a tapwater aquarium at room temperature (21-22°C). Tiger salamanders, Ambystoma rigrinum were obtained from Amphibians of Eastern North America, Nashville, Tennessee. A neotenic larval form (A. tigrinum) was kept in a tapwater aquarium at 16”C, and the adult on a bed of moistened foam chips in a terrarium at the same temperature. Artificial pondwater, with the following ionic composition (mM): I.0 NaCl; 0.1 KCI; 0.05 MgSO.,; 0.2 NaHCO, and 0.2 calcium gluconate was used as the experimental bathing medium. The animals were killed by pithing. In order to remove any Ca adhering to the outer surface of the body, the animals were first rinsed for 5 min, with three changes, in Ca-free pondwater. The calcium content of bathing medium, serum, urine, whole animals or tissue samples was determined using a Perkin-Elmer 290B atomic absorption spectrophotometer. Whole animals or tissue samples were dried, and ashed overnight at 500°C in a muffle furnace. The ash was dissolved in a small amount of concentrated HCl, and diluted with LaCI, solution to give a final concentration of 0.1 N HCI and 1% LaCI,. The accumulation of 45Ca in oiuo was determined by exposing the animals to 11 of pondwater containing the isotope (0.1 &i/ml) for 24 hr. In order to estimate what contribution the gills make to Ca accumulation, a group of animals were lightly anesthetized in icewater and ligatures were tied around the external gills, thereby cutting off their circulation. The gills were ligated on the day prior to the experimental measurement of Ca uptake. A control group of animals were also immersed in icewater and handled similarly to the experimental animals. At the end of the experimental period the animals were rinsed, anesthetized with 0.1% tricaine methane sulfonate (Sigma Chemical Co, St. Louis, Missouri) and divided into separate tissue components: skin, gill, gut, and the remaining carcass. For determination of %a the tissues were dried overnight at 115°C. and then ashed at 500°C. The ash was dissolved in a
* Supported by National Science Foundation Grant No. PCM78-21446. t To whom correspondence should be addressed.
G~KUI~INI- F. BAI DWIN and P. J. BFNTI I-Y
66
small amount of concentrated HCI. and aliquots of diluted solution were counted in a hquid scintillation counter (Beckman LSIOO). Although it has been shown that amphibians drink very little (Bentley & Yorio, 1979). some ingestion of the external medium may occur. We measured such drinking using [‘251]Na-iothalamate (GloFil. Abbott Laboratories, Chicago. Illinois) as a non-absorbable water maker. Animals were placed in pondwater containing 0.2 btCi;ml “‘1. and exposed to it for 8 to 16 hr. After brief rinsing, the animals were killed and the whole digestive tract was removed and counted in a Beckman autogramma counter (Baldwin & Bentley, in preparation). Calcium loss was estimated by exposing animals to a measured volume of pondwater with no added calcium (0.01 mM Ca). The mudpuppies or salamanders were first anesthetized with tricaine methane sulfonate. and a polyethylene cannula, emptying into a small rubber bag, was fitted into the cloaca and attached with a purse string suture. Calcium concentration of the urme and balhing medium was measured after 16 hr for mudpuppies, and 8 hr for salamanders. To assess the role of the skin in calcium exchange, bidirectional 45Ca transcutaneous fluxes were measured using paired pieces of ventral skin mounted in Ussing-type chambers (3 cm’ area) (Baldwin & Bentley, 1981). To minimize “edge damage” double parafilm “o-rings” and a layer of silicone grease were placed between the lucite chamber halves and the tissue. The preparations were continually short-circuited with an automatic voltage clamp. “5Ca (0.5 /&i;‘ml) was placed in one side of the chamber and samples were collected at intervals from the opposite or frtrns side. The specific radioactivity of the cis side was used to determine the flux across the tissue. The ““Ca used was obtained from New England Nuclear, Boston. Massachusetts.
Table
I. Calcium
content
Hk:SI
1.7s
The calcium concentrations puppies and tiger salamanders
in the
compared The total
the neotenous urodeles was less than that of the adult tiger salamander.
The exchanges of Ca between the urodeles and the external bathing media are shown in Table 2. In mudpuppies the measured uptake and losses of Ca are similar, indicating that the animals can maintain a C’a balance under these fasting conditions. The accumulation of ?a (in rioo) each day is equivalent to 0.04”,, of the total body Ca or about one-third of all that present in the extracellular fluids. Loss of Ca to a Ca-free solution was 0.12 mmol kg- ’ BW day 1 and it occurs in equal amounts in the urine and across the integument (i.e. both skin and gills). Neither the neotenic nor adult tiger salamanders appear to be able to maintain a Ca balance under these, fasting, conditions. In the neotenic animals daily accumulation was equivalent to 0.07”,, of total body Ca or about 50”,, of that in the extracellular fluids. The Ca loss was. however, about 4-times
of N. muculosus
and A. riyrimrm
Skin
N. mtrculo\~r.s (8)
324 _t 30
5.4 f 0.6
3.8 ) 0.4
2.5 + 0.3
I.6 _+ 0.1
A. tigrinum Neotenic (4) Adult (6)
151 f 5 250 + I5
3.9 + 0.2 3.6 f 0.4
6.6 + 1.8
9.2 f 3.1 7.9 f 0.8
1.05 ? 0.05 2.0 4 0.1
k SE. Number
Table 2. Calcium Calcium Urine (mmol kg-’ N. mclculosus
of animals
exchanges
Gills (mmol kg-
0.05 f 0.01 (7)
0.07 + 0.01 (7)
0.37 f 0.05 (9) 0.18 & 0.03 (12)
0.33 * 0.07(12)
Serum
in parentheses.
in fasting
loss Integument day-‘)
Gut
’ tissue)
urodele
(mmol kg-’
amphibians *‘Ca gain Integument day-‘) (nmolcm~L
hr-I)
0.1 I f 0.02 (9)
1.8 + 0.33 (9)
0.10 + 0.02(5) 0.05 + 0.02(5)
I.8 k 0.23 (5) 0.6 i 0.24(5)
A. figrinum
Neotenic Adult
of mud-
to 2.5”,, in frogs (Baldwin & Bentley. 1981). Ca content and serum Ca concentration of
Whole animal (mmol kg-’ BW)
Values are as mean
tissues
are given in Table I. In frogs and toads the skin contains relatively high concentrations of Ca but this was not seen in these urodeles. The Ca present in the skin of these amphibians was equivalent to 0.24 to 0.45”,, of the total body Ca,
*
* Not detectable. The gain of 45Ca is that accumulated in the carcass of the animals no! including the gut. skin 01 gills. Uptake experiments were performed in animals bathed with pondwater containing 0.2 mM Ca and loss was measured in a Ca-“free” media containing less than IO- ’ M Ca. Results are as means f SE. Number of experiments in parentheses.
Ca exchanges in urodeles greater than this uptake; loss across the integument was not detectable, urinary loss being predominant. In adult tiger salamanders the loss of Ca was about lo-times greater than uptake and the leakage from the skin exceeded the loss in the urine. The contribution of drinking to the uptake of Ca is negligible, under fasting conditions, in the larval animals and minor in the adult. The rate of drinking in neotenic tiger salamanders and mudpuppies was, respectively 0.014 and 0.016 ml 100 g-’ hr-’ (Baldwin & Bentley, in preparation) which, assuming complete absorption of the ingested Ca, would correspond to 0.6 and 0.7% of the total Ca accumulated. In the adult tiger salamanders drinking was 0.075 ml 100 g- 1hr - ’ which could provide a Ca uptake equivalent to as much as 7”~; of the total accumulation. A role for the gills in Ca exchange? The results of the experiments
on Ca accumulation
in oivo (Table 2) suggested that permeability of the adult A. tigrinum integument is less than that in the
neotenous animals. It is possible that this difference may reflect the presence of gills in the latter. However the uptake of 45Ca did not differ significantly between animals with their gills ligated and paired control animals; influx (mmol kg- ’ BW day- ‘) .was, respectively, 0.09 k 0.02 (9) vs 0.11 f 0.02 (9) in N. maculosus, and 0.08 _t 0.02 (5) vs 0.10 + 0.02 (5) in neotenic A. tigrinum. Transcutuneous Ca movement in vitro The movement of 45Ca was measured across electrically short-circuited pieces of skin (in oitro) of the mudpuppies. There was no significant difference between the influx (outside to inside) and efflux: influx was 1.8 2 0.21 (6) nmolcm-2hr-1 and efflux was 1.4 + 0.32 (6) nmolcm-2 hr-’ (1 mM Ca2+ both sides). Thus an active transport mechanism for transcutaneous Ca transport does not appear to be functioning under these conditions. The influx was reduced when the external Ca concentration was reduced from 1.0 to 0.2 mM, as it is in the pondwater. The results of Tables 2 and 3 can be used to make a comparison of the Ca influx observed in vitro and in ciuo. In mudpuppies, the permeability of the skin to Ca was not different in either situation. In neotenous
Table 3. Transcutaneous movement of ‘%a across urodele skin, in vitro. The outer surface was bathed by 0.2 mM Ca, which is the concentration used in uiuo 45Ca influx (nmol cm-‘hr-‘) N. macu/osus (March) (6)
1.0 f 0.13
61
tiger salamanders, however, the two sets of results were not consistent, Ca influx in vitro was only about half that observed in uiuo. On the other hand, in the adult tiger salamanders the Ca influx in uiuo was about double that seen in vitro. Clearly the experimental conditions vary considerably in uiuo and in vitro and presumably contribute to these inconsistencies. A comparison (in uitro) of Ca influx (Table 3) in the urodeles and anurans is also provided. The tadpole’s skin was less permeable to Ca than that of the urodeles. Frog skin showed seasonal variability but was similar in November to that of the urodeles in spring and summer. DISCUS!3ON
An exchange of calcium can occur across the integument of urodele amphibians. A permeable integument is considered to be a phyletic character of the Amphibia and is of considerable physiological significance with respect to respiration, and water and salt metabolism. The permeability of the skin to calcium is not great, uptake may, however, compensate for urinary losses. Leakage of Ca from the skin in mudpuppies and adult tiger salamanders was of a magnitude similar to that which is lost in the urine. In fish the gills are an important site for Ca exchanges (see Simmons, 1971; Dacke, 1979) and in teleosts this process may be regulated by the hormone calcitonin and extracts of the corpuscles of Stannius (Milhaud er al., 1977; Milet, Peignoux-Deville & Martelly, 1979; SO & Fenwick, 1979). Tadpole gills also appear to be permeable to Ca and uptake of this mineral is reduced by injections of calcitonin (Baldwin & Bentley, 1980). However, the external gills of the neotenic urodeles appeared to have a much lower permeability to Ca than the skin and was not measurable. This property of the gills may be fortunate, or an evolutionary adaptation, as neotenous urodeles normally live in solutions in which the Ca concentrations are much less than that in their body fluids. They thus would be prone to lose this mineral by diffusion, which, in the absence of any compensating “Ca pumps” in the integument, could accentuate the problem of their Ca homeostasis. The skin of anuran amphibians usually contains large amounts of Ca (see Baldwin & Bentley, 1981). The functions of this cutaneous Ca is unknown but it could have a structural role and even function, like bone, as a store for the mineral. The skins of the urodeles which we examined lacked such a prominent store of Ca. It is unknown whether this is a phyletic characteristic, though the levels are also low in an anuran, the toad Xenopus laeuis.
A. tigrinum
Neotenic form (July-August) (6) Adult (July-August) (6)
0.8 f. 0.17 1.5 + 0.1
Runu pipien.9
November (8) February (8)
0.9 & 0.17 0.2 + 0.07
Rum cutesbeiuna
Tadpoiest (12)
0.2 + 0.07
REFERENCES BALDWIN G.
F. & BENTLEYP. J. (1980) Calcium metabolism in bullfrog tadpoles (Ram catesbeianu) J. exp. Biol.
88, 357-365. G. F. & BENTLEY P. J. (1981) A role for skin in Ca metabolism of frogs? Comp. Biochem. Physiol. 68, 181-185. BENTLEY P. J. & YORIOT. (1979) Do frogs drink? J. exp. Biol. 79, 41-46.
BALDWIN
* From Baldwin & Bentley (1981). t From Baldwin & Bentley (1980). Results are as means f SE. Number of experiments in parentheses.
68
GERALDINE
F. BALDWIN and P. J. BENTLEY
DA~KE C. G. ( 1979) CtrlGrrm Reyulution in Sub-mammaliun Vc~rehrur~s, pp. 8488. Academic Press. New York. KROGH A. (1937) Osmotic regulation in the frog (R. escuIrntcr) by active absorption of chloride ions. Skund Arch. Physiol. 76, 6@74. MILET C.. PEIGNOUX-DEVILLF J. & MARTELLY E. (1979) Gill calcium fluxes in the eel Anynilltr trnguillu (L.) effects of Stannius corpuscles and ultimobranchial body. Comp. Bioc~ltrm. f%)~i~~l. 63A. 63 -70. M1LHAC.L) G.. RANKIN J. C.. B~LIS L. & BENSON A. A. (1977) Calcitonm: its hormonal action on the gill. Proc. nutn. A~rtl. .Qi. L’.S.A. 74, 469334696. PANG P. K. T., KENNY A. D. & OGURO C. (1980) Evolution of endocrine control of calcium regulation, In Er’oltrfion of Vutc+raft~ Endocrine Swetns (Edited by
PANG P. K. T. & EPPLE A.). pp. 3233356. Texas Tech. Press, Lubbock. SIMMONS D. J. (1971) Calcium and skeletal tissue phystology in teleost fishes. C/in. Orthopaedics 76, 244-280. So Y. P. & FENWICK J. C. (1979) In riw and in vitro effects of Stannius corpuscle extract on the branchial uptake of 4SCa in stanniectomized North American eels (Anyttilltr rostrutu) Gen. camp. Entlocr. 37, 143. 149. WATLINGTON C. 0.. BURKE P. K. & ESTEP H. L. (1968) Calcium flux in isolated frog skin: the effect of parathroid substances. Proc. Sot. cup. Biol. Med. 128, 853-856. ZADUNAISKY J. A. & LANDE M. (1972) Calcium content and exchange in amphibian skin and its isolated epithelium. Am. J. Ph!xiol. 222, 130991315.