ABSTRACT
Calcium is present in amphibian blood at a concentration similar to that in other vertebrates, about 1–2mmol l−1. The fraction of free calcium in amphibians is lower than that in other tetrapod vertebrates because about 50% of the plasma Ca2+ is bound to plasma proteins and perhaps other molecules. Plasma [Ca2+] varies seasonally, increasing in spring and summer and decreasing in winter. Changes in plasma [Ca2+] also occur during larval development, as the concentration of this ion increases in larval forms as they approach metamorphosis. Calcium is exchanged at a variety of sites in animals. There is evidence for Ca2+ uptake across the skin and gills of larval anurans. It is also transported into the blood from the small intestine (especially the duodenum) and reabsorbed in renal tubules from the glomerular filtrate. The possibility of Ca2+ absorption from urine stored in the urinary bladder has not been confirmed, however. Calcium is stored in bone and in specialized endolymphatic sacs. This Ca2+ can be mobilized when the need arises. There are a number of endocrine and other humoral factors that appear to be involved in amphibian calcium metabolism. These include parathyroid hormone, calcitonin, vitamin D and prolactin.
INTRODUCTION
The study of amphibian calcium metabolism has lagged behind the study of other major ions such as Na+, K+ and Cl− for several reasons. The interest created in Na+, K+ and Cl− by the early work of Krogh (1937) and Ussing (1949) stimulated a large number of workers to study the transport of these ions in frog skin. Divalent ions are more difficult to work with than monovalent ions for a variety of reasons. The ease and inexpensiveness of flame photometry compared with atomic absorption spectroscopy probably caused a bias towards monovalent ions. The binding of divalent ions such as Ca2+ to plasma protein (Walser, 1973) complicates renal transport studies. Our knowledge of amphibian calcium transport is also poor compared with what is known about fish Ca2+ transport. This is probably because of the economic importance of fish and the generally larger pool of support for studies of fish biology. Nevertheless, this indifference to amphibian calcium metabolism is unwarranted. Amphibians, the first animals to make the water–air transition, occupy a pivotal place in evolution. The changes in Ca2+ metabolism that occurred when vertebrates began to abandon the aquatic habitat and a ready supply of dissolved ions at the interface between their epithelial exchange surfaces and the water in favour of a terrestrial existence may provide clues to a greater understanding of Ca2+ regulation in tetrapods in general and for human medicine in particular. Calcium ions, although at a low concentration in bodily fluid compared with Na+, Cl− or even K+, are critically important for a wide variety of physiological and biochemical functions, such as membrane stabilization, muscle contraction, nerve transmission, cell secretion and enzyme regulation.
The physical state of calcium in the blood
Total Ca2+ concentration in amphibian blood (Table 1) is very similar to that of other vertebrate groups. Total [Ca2+] does not begin to describe Ca2+ availability for biochemical function or even for transport of Ca2+ into cells where it can be used. Because of the physical nature of proteins and divalent ions, a significant portion of the calcium that exists in biological fluids is bound and therefore unavailable for ionic interactions. This binding of Ca2+ to plasma protein is greatest, among tetrapods, in the Amphibia (Table 2), where only half of the plasma calcium is ionized.
Seasonal and developmental changes in calcium metabolism
It would be a mistake to conclude that, once protein binding is accounted for, the description of amphibian blood calcium is complete. Plasma [Ca2+] fluctuates seasonally and during development. There are seasonal alterations in the structure and apparent function of the parathyroid glands (Dougherty, 1973). In the winter, the parathyroid cells shrink and the spaces between them enlarge. Histological evidence of secretory activity associated with, for example, Golgi complexes, is also reduced in the winter. The parathyroid glands of Notophthalmus viridenscens (Wittle and Dent, 1979) do not regress in the winter. Paralleling the histological changes in R. pipiens are annual cycles in plasma [Ca2+], with [Ca2+] falling in the winter and rising in the spring and summer in Rana pipiens (Fig. 1: Robertson, 1977).
During metamorphosis, changes in plasma [Ca2+] also occur. In Rana catesbeiana, plasma Ca2+ concentration increases steadily from less than 8mgdl−1 in tadpoles to near 12mgdl−1 in adults (Fig. 2: Oguro et al. 1975). Larval A. tigrinum have about 40% lower plasma [Ca 2+] than adults (Stiffler et al. 1987; Stiffler, 1991). Larval amphibians have cartilaginous skeletons that do not become ossified until metamorphosis. The increasing [Ca2+] may be supplying the osteoblasts with the minerals needed to ossify the skeleton. In anurans, a specialized collection of structures known as the paravertebral lime-sacs or endolymphatic sacs (see below) appear to supply Ca2+ for this purpose at metamorphosis (Pilkington and Simkiss, 1966). Larval urodeles lack parathyroid glands; these structures appear at metamorphosis (Clark, 1983).
Sites of calcium exchange
Amphibians possess a large number of exchange sites for calcium. These calcium exchanges occur between body fluid compartments and between the environment and body fluids. Uptake of calcium from the environment can occur across the skin (Watlington et al. 1968; Baldwin and Bentley, 1981a,b; Kingsbury and Fenwick, 1989), across gills, at least in anuran tadpoles (Baldwin and Bentley, 1980), and at the surface of the small intestine (primarily duodenum; Robertson, 1975, 1976).
Skin
In isolated, short-circuited Rana pipiens skin with Ringer’s solution bathing both sides, the influx of Ca2+ was consistently and significantly greater than the efflux, suggesting active transport (Watlington et al. 1968). These observations have not been replicated. A study by Baldwin and Bentley (1981a) found no significant difference between influx and efflux of Ca2+ in isolated R. pipiens skin. Seasonal differences were suggested as a possible explanation of this disparity because the latter study (Baldwin and Bentley, 1981 a) was performed in winter; however, season was not specified in the former study (Watlington et al. 1968). Investigation of unidirectional Ca2+ fluxes across isolated R. pipiens cutaneous epithelial sheets also failed to demonstrate significant net fluxes (Zadunaisky and Lande, 1972). Baldwin and Bentley (1981b) also studied cutaneous exchanges of Ca2+ in two urodeles, Ambystoma tigrinum and Necturus maculosus. As with their studies on frog skin, there were no significant differences between influx and efflux in isolated short-circuited salamander skin, suggesting that active transport was not involved. Since measurements were made in both winter and summer, season did not appear to play a role. However, Ca2+ influxes in whole animals (larval A. tigrinum) were much greater than those in isolated skin, suggesting that something supporting Ca2+ transport in the whole animal is missing from isolated skin. Calcium uptake in Ambystoma mexicanum was reported to be 0.079mmolkg−1 day−1 in July and 0.03 mmolkg−1 day−1 in August; however, effluxes were not given (Kingsbury and Fenwick, 1989). These conflicting results do not provide a clear indication of the possible role of amphibian skin in Ca2+ transport (Table 3). The studies of isolated skin were performed with Ringer’s solution bathing both sides of the skin. This non-physiological situation is known to obscure net Cl− uptake in frog skin (Kirschner, 1983). Obviously, further study of cutaneous Ca2+ exchange will be necessary before it is certain whether Ca2+ is, or is not, transported in net quantities across amphibian skin.
Gills
Rana catesbeiana tadpoles take up significant amounts of Ca2+ from the bathing medium (0.13mmol g−1 day−1) and about 75% of this can be shown to cross gill epithelia (Baldwin and Bentley, 1980). The gills of A. tigrinum and N. maculosus, however, appear not to support significant Ca2+ uptake (Baldwin and Bentley, 1981b).
Intestine
The final site of uptake for calcium in amphibians is the small intestine, which absorbs dietary Ca2+. In whole-gut preparations from Rana pipiens, the everted gut sac is able to concentrate Ca2+ 2.7-fold in the serosal medium (Robertson, 1975). When the duodenum is examined in isolation from the jejunum and ileum, the concentration ratio of that segment is approximately 3.0 while the jejunum–ileum combination produces a ratio of about 2.0 (Robertson, 1975). Duodenal Ca2+ transport is maximal at night (Robertson, 1976).
There are several sites where loss of calcium to the environment could occur. In addition to diffusion of calcium across the skin or the gills, urinary loss of calcium ions is potentially great.
Kidney
Renal tubular transport of calcium reduces Ca2+ loss in the urine of Rana pipiens (Cortelyou, 1967; Sasayama and Clark, 1984) and Notophthalamus viridenscens (Wittle and Dent, 1979). Urinary calcium concentration is approximately 4% of plasma calcium concentration in Rana pipiens (Cortelyou, 1967) and 3.9% in Notophthalamus viridenscens (Wittle and Dent, 1979). This suggests that large amounts of Ca2+ are reabsorbed from the filtered plasma and, after correction for bound (unfilterable) calcium in the plasma, it has been shown that 60% of filtered Ca2+ is reabsorbed by renal tubules in R. pipiens (Sasayama and Clark, 1984).
Urinary bladder
The amphibian urinary bladder is another possible site for calcium transport. This epithelium is capable of active sodium transport (Bentley, 1971) and the potential exists for active calcium transport from the urine into the blood. In a study of toad bladder (Bufo marinus) permeability to Ca2+, Walser (1970) found that resting calcium permeability is vanishingly low (4X10−9 cm s−1) but that a number of factors, including the presence of Cl− in the bathing medium, increased calcium permeability. It was suggested that calcium crosses the toad bladder as the ion CaCl+. A second study of Ca2+ flux ratios across toad bladder failed to show conclusive evidence of active Ca2+ transport; several individual bladders showed influxes to be significantly greater than effluxes, however (Walser, 1971).
In addition to exchanges of calcium between animals and their environments, there are exchanges of this ion between compartments within the body. Calcium exchanges between bone and extracellular fluid play an important role in amphibian calcium metabolism (Yoshida and Talmage, 1962). In addition to calcium storage depots in bone, amphibians (primarily anurans) have a novel Ca2+ storage site. This is the endolymphatic sac, which arises from the junction of the saculus and utriculus of the inner ear (Pilkington and Simkiss, 1966). This structure enlarges in anurans till it surrounds the brain in the cranial cavity and extends down the spinal canal, protruding as vertebral lime-sacs between the vertebrae. The name ‘lime-sacs’ derives from the fact that this structure is filled with calcium carbonate crystals that can be mobilized as Ca2+ as the need arises (Simkiss, 1968; Pilkington and Simkiss, 1966).
Hormonal control of calcium exchanges
Parathyroid hormone
Parathyroid glands are found in all anurans but are lacking in larval urodeles (Baldwin, 1918). In those amphibians that possess parathyroid glands, the parathyroid hormone (PTH) is usually hypercalcaemic. Parathyroidectomy (Fig. 3 and see Fig. 5) causes significant decreases in plasma [Ca2+] in R. pipiens (Cortelyou et al. 1960), larval R. catesbeiana (Sasayama and Oguro, 1975), Notophthalamus viridenscens (Wittle and Dent, 1979), Tylototriton andersoni (Oguro and Sasayama, 1978), Desmognathus monticola (Wittle, 1983) and Cynops pyrrhogaster (Oguro, 1969). Each of the calcium exchange sites discussed above is a potential target for PTH.
Calcium uptake by the skin of R. pipiens showed a transient increase shortly after treatment with parathyroid hormone (Watlington et al. 1968). This response was not seen in a later study of this species (Baldwin and Bentley, 1981a). Similarly, Ca2+ uptake by the gills of R. catesbeiana tadpoles was not stimulated by PTH (Baldwin and Bentley, 1980).
The renal responses to parathyroidectomy and administration of PTH are not clear. Injection of the hormone increases, rather than decreases, urinary [Ca2+] (Cortelyou, 1967). It was suggested that PTH mobilizes Ca2+ from bone and that the resulting hypercalcaemia increases the filtered Ca2+ load to an extent greater than can be accommodated by the tubular transport mechanism for the ion, even under the stimulus of PTH. Parathyroidectomy reduces urinary [Ca2+] (Wittle and Dent, 1979) and increases tubular Ca2+ transport in the short term (Fig. 4: Sasayama and Clark, 1984), which would imply an inhibition of tubular Ca2+ transport by PTH. In the latter study, there was no significant change in the calcium clearance ratio, indicating that fractional Ca2+ reabsorption did not change and that the increased rate of Ca2+ transport might be explained by an increased glomerular filtration rate, which did occur. The paradoxical dilution of urine calcium concentration following parathyroidectomy in N. viridenscens (Wittle and Dent, 1979) may be resolved by the possibility that parathyroidectomy leads to a transient hypercalcuria, which becomes reversed as other regulatory systems come on line to compensate for the loss of PTH. In support of this, there was an increase in urinary [Ca2+] following parathyroidectomy in R. pipiens that reversed after several days (Fig. 5: Cortelyou et al. 1960). The hypocalcuria in N. viridenscens was apparent 1 day after parathyroidectomy whereas the hypercalcuria in R. pipiens began on day two, suggesting that there may be species differences in the time courses of the possible compensatory responses. It is difficult to unravel these inconsistencies and paradoxes from whole-animal data; studies of responses of isolated tubules to PTH may be required to clarify the role of this hormone in renal tubular Ca2+ transport.
The role of parathyroid hormone in intestinal Ca2+ transport is similarly obscure. Five days after parathyroidectomy, there was no significant change in duodenal or jejunal–ileal Ca2+ transport in isolated preparations of R. pipiens intestine (Robertson, 1975).
The response of bone (and possibly other calcium deposits) to parathyroid hormone is much clearer (Yoshida and Talmadge, 1962). The approach of these workers was to perfuse (lavage) the body cavity with Ca2+-free Ringer’s solution to stimulate parathyroid hormone release and then to study the responses of bone to this treatment in intact and parathyroidectomized frogs ( Rana catesbeiana). Microscopic examination of bone cells showed a large increase in osteoclast frequency after 10h of Ca2+-free lavage that was abolished by parathyroidectomy (Fig. 6). Since osteoclasts are known to be the bone cell type that mobilizes Ca2+, these data suggest that parathyroid hormone stimulates the development of osteoclasts which, in turn, dissolve bone to liberate Ca2+. Ca2+-free lavage also increased the liberation of 45Ca2+ from calcareous deposits in this study and the response was abolished by parathyroidectomy (Fig. 7). Parathyroid extract causes the appearance of osteoclasts in bone of N. viridensens; these cells could not be located in control animals (Wittle and Dent, 1979).
Calcitonin
In amphibians, calcitonin is produced by the ultimobranchial glands, which are the evolutionary anlage of the C-cells of the mammalian thyroid. Calcitonin or ultimobranchial gland extracts cause decreases in plasma [Ca2+] in Rana pipiens (McWhinnie and Scopelliti, 1978), Cynops pyrrhogaster (Uchiyama, 1980), Bufo boreas and B. marinus (Boschwitz and Bern, 1971) and Rana catesbeiana tadpoles (Sasayama, 1978), but not Xenopus laevis (McWhinnie and Scopelliti, 1978) or Ambystoma mexicanum (Kingsbury and Fenwick, 1989). Ultimobranchialectomy (UBX; removal of the ultimobranchial glands) results in significant increases of the plasma [Ca2+] in R. pipiens (Robertson, 1975).
Furthermore, plasma calcitonin titres increase when R. pipiens are adapted to high-Ca2+ environments (Robertson, 1987). These results, taken together, suggest that calcitonin is a hypocalcaemic hormone, as it is in other vertebrate groups. Potential target organs for calcitonin are, of course, the same as those discussed above.
The skin, as a calcium uptake organ, is one potential site of calcitonin action, but experiments have given varying results. Calcium influx was unaffected by calcitonin in a study of R. pipiens skin (Baldwin and Bentley, 1981a). Experiments on intact A. mexicanum showed significant inhibition of Ca2+ influx (Kingsbury and Fenwick, 1989). Calcitonin was effective in decreasing branchial Ca2+ uptake by R. catesbeiana tadpoles (Baldwin and Bentley, 1980).
Urinary excretion of Ca2+ is also responsive to calcitonin. Ultimobranchialectomy increases renal calcium loss (Robertson, 1975), indicating that calcitonin stimulates renal tubular Ca2+ transport. This would be counter to all expectations, however, and it is possible that the UBX-induced hypercalcuria is an indirect effect of the increased plasma [Ca2+] under these conditions and of the consequent increase in the filtered load of Ca2+ in the nephrons.
Intestinal Ca2+ transport in R. pipiens is unaffected by UBX (Robertson, 1975) in vitamin-D-deficient preparations and in preparations containing vitamin D3. Intestinal segments from ultimobranchialectomized frogs did increase Ca2+ absorption when dihydrotachysterol, a vitamin D analogue, was present, however (Robertson, 1975).
Vitamin D
Vitamin D, or cholecalciferol, is known to be intimately involved in calcium metabolism in the vertebrates. Rana pipiens exhibits a small, but significant, increase in plasma [Ca2+] when treated with 2.5mg of vitamin D (Fig. 8: Robertson, 1975). Again several potential sites of Ca2+ exchange have been investigated as possible targets for vitamin D. The intestine of Rana pipiens clearly responds to vitamin D (Fig. 8: Robertson, 1975) as does the kidney (Fig. 9: Robertson, 1975). Baldwin and Bentley (1981a) could find no significant effect of vitamin D in Rana pipiens cutaneous Ca2+ exchange; however, they did find a stimulatory effect of this vitamin on Rana catesbeiana gill Ca2+ uptake (Baldwin and Bentley, 1980).
Prolactin
The anterior pituitary hormone prolactin has a hypercalcaemic effect in teleost fish (Pang et al. 1971). This peptide has been tested for calcium regulatory activity in a few species of amphibians. An early study of prolactin activity in Ca2+ regulation was unable to find a statistically significant response in Bufo boreas and B. marinus (Boschwitz and Bern, 1971). Later studies in Rana catesbeiana tadpoles (Sasayama and Oguro, 1982) and adults (Baksi et al. 1978) revealed a hypercalcaemic response to this peptide.
Hypophysectomy causes a hypocalcaemia in Necturus maculosus that is reversed by prolactin (Fig. 10: Pang, 1981). It may be that aquatic amphibians rely more on prolactin than on parathyroid hormone. Indeed, larval urodeles, which are aquatic, lack parathyroid glands (Clark, 1983).
Oestrogen
Oestrogen is known to affect calcium metabolism in mammals so it it interesting to know whether similar effects occur in amphibians. Baksi et al. (1978), working with Ranapipiens, demonstrated oestradiol-induced increases in plasma [Ca2+] and 1,25(OH)2 vitamin D3; oestradiol was given at 5mgday−1 for 6 days.
Interactions between calcium and acid–base balance
Amphibians, especially anurans, have significant stores of calcium phosphates and calcium carbonates in their bones and endolymphatic sacs. These salts, especially the carbonates, are potentially effective buffers during times of acidotic stress. Simkiss (1968) demonstrated that urinary Ca2+ excretion increases in Rana temporaria during respiratory acidosis, although there was no significant change in plasma [Ca2+]. Tufts and Toews (1985), working with Bufo marinus, reported increased plasma and urinary [Ca2+] during respiratory acidosis. Robertson (1972) showed that parathyroidectomized Rana pipiens have reduced acid–base compensatory capability during respiratory acidosis, suggesting that parathyroid hormone is involved. Parathyroid hormone stimulates H+ secretion in toad urinary bladder (Frazier, 1976). All of this has prompted Dacke (1979) to suggest that the primitive function of the parathyroid might be in acid–base balance.
Conclusions
After a period of considerable activity through the 1970s, study of amphibian calcium metabolism has diminished significantly. From the earlier studies, we have a reasonable idea about which organs and tissues are involved in calcium exchange. We know little, however, of the magnitudes of exchange. Many of the elementary forms of analysis that have been applied to the study of other ion-transporting systems, such as Michaelis–Menten kinetic analysis, have not been applied to calcium transport in any amphibian tissue. A similar situation exists with respect to our knowledge of the stoichiometry of calcium exchanges in amphibian tissues. Although a great deal of information relating to mechanisms of calcium exchange at the cell membrane level in fish tissues has accumulated (see Flick and Verbost, 1993), there has been no such progress in amphibian physiology. Finally, our knowledge of the role played by endocrine regulatory systems in Ca2+ metabolism is fragmentary at best. While considerable effort has been expended to understand the involvement of a number of humoral agents, the results have often been inconclusive and inconsistent.