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Hypocalcemic potency of the ultimobranchial gland in some urodelan amphibians
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
Hypocalcemic
272-277 (1983)
Potency of the Ultimobranchial Urodelan Amphibians
CHITARU Department
51,
OGURO,
of Biology,
HIDEO
Faculty
TARUI,~
of Science,
AND YUICHI
Toyama
University,
Gland in Some SASAYAMA Toyama
930, Japan
Accepted September 24, 1982 The hypocalcemic
potencies of the ultimobranchial glands of two urodelans, Onychoand Hynobius nigrescens, were studied; according to the rat bioassay, their calcitonin values (MRC) were 30 and 18 mu/kg body wt, respectively. Various organs other than the ultimobranchial gland were also assayed in rats to see whether they had any hypocalcemic potency. Wowever, the ultimobranchial gland was the only organ examined with detectable hypocalcemic potencies in these urodelans. The hypocalcemic potencies of urodelan ultimobranchial glands are one order lower than those reported in the other vertebrate classes, contrasting with the potencies of anuran ultimobtanchial glands. The biological significance of this low potency is discussed. dactylus
japonicus
The occurrence of calcitonin, which is a hypocalcemic factor in mammals, has been reported in the ultimobranchial gland of some lower vertebrates (Copp and Parkes, 1968; Moseley et al., 1968; Clark, 1971; Oguro and Uchiyama, 1980). Piscine calcitonin showed a high hypocalcemic potency in rats compared with mammalian calcitonin (Copp et al., 1970; Orimo et al., 1972). The hypocalcemic potency of ultimobranchial extracts of reptiles and birds seems to be high in terms of body weight, as shown in the rat bioassay (Copp and Parkes, 1968; Uchiyama et al., 1978, 1981). Recently a high hypocalcemic potency was found in the ultimobranchial gland of some anuran amphibians (Oguro et al., 1981; Oguro and Sasayama, 1982). On the other hand, an extremely low hypocalcemic potency was reported in the ultimobranchial gland of the newt, Cynops pyrrhogaster, according to the rat bioassay (Uchiyama, 1980). The significance of the very low hypocalcemic potency reported in C. pyrrhogaster is not understood, since the physiI Present address: Department of Community Medicine, Toyama Medical and Pharmaceutical University, Toyama 930-01, Japan.
ological role of calcitonin in poikilothermic vertebrates is still a matter of discussion. Therefore, it is of interest whether the low hypocalcemic potency of the ultimobranchial gland is specific to C. pyrrhogaster or a common phenomenon among urodelan amphibians. In the present study, the hypocalcemic potency of the ultimobranchial gland extracts of two urodelan amphibians, Onychodactylus japonicus and Hynobius nigrescens, was examined using the rat bioassay. It is now accepted that C cells, which secrete calcitonin, originate in the neuroectodermal tissue at an early stage of development and later migrate to the organs which ultimately incorporate the C cells: Therefore, the possibility remains that the ultimobranchial gland is not the main C cellcontaining organ, but that other organs become the site of the C cells. In that case, the low hypocalcemic potency of the ultimobranchial gland of C. pyrrhogaster could be explained by a different distribution of C cells. Thus the hypocalcemic potencies of the brain, thyroid gland, thymus, stomach, small intestine, and kidney of the three species were also assayed.
272 0016-648OB3 $1.50 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
HYPOCALCEMIC
MATERIALS
POTENCY
OF URODELE
AND METHODS
In the present study, two urodelan species, 0. japonicus (Houttuyn) and H. nigrescens Stejneger, were used. C. pyrrhogaster (Boie) was also used for comparison. 0. japonicus and C. pyrrhogaster were obtained from commercial sources; specimens of H. nigrescens were collected in the mountainous area of Toyama Prefecture, Japan. In these three species, the ultimobranchial gland is very small in comparison with that of anurans and present only on the left side, located dorso-lateral to the pericardial cavity, as described previously (Sehe, 1960; Uchiyama, 1980). The right ultimobranchial gland is degenerated during development. The ultimobranchial glands of C. pyrrhogas&r were obtained throughout the year. No difference seemed to exist in the calcitonin activity of the ultimobranchial gland in the diierent season and sexual status. Those of H. nigrescens and 0. japonicus were removed during breeding and post-breeding season. The ultimobranchial glands removed were homogenized in 0.8% NaCl solution adjusted to pH 4.6 with HCl. The homogenate was then centrifuged at 11,000 rpm for 15 min. The supernatant was used as the ultimobranchial extract. Extracts of the brain, thyroid gland, thymus, stomach, small intestine, and kidney were also prepared as described above. All were assayed in rats for the presence of hypocalcemic potency. The calcitonin assay of the ultimobranchial extract was carried out as described previously (Oguro et a[., 1981). Each assay rat received 0.4 ml of the extract or vehicle. To obtain standard responses, 10 or 100 mU (MRC) of synthetic salmon calcitonin was used. Blood samples were taken from the assay rats through the cannula set in the sciatic artery before the extract or vehicle was administered and at intervals of 0.5, 1, 2, and 3 hr afterward. Serum Ca and Na concentrations were determined by atomic absorption spectrophotometry. The inorganic phosphorus (PI) concentration was measured according to a modified method of Fiske and Subbarow (1925). Determination of the activity was achieved by the method of Tauber (1967). Student’s t test was used for statistical analysis.
RESULTS Salmon calcitonin produced hypocalcemia in rats: 10 and 100 mU of salmon calcitonin caused 18 and 27% decrease in the serum Ca concentration, respectively, as shown in Fig. 1. The maximum decrease was found 30 min after administration with 10 mU and 1 hr with 100 mu. On the other hand, slight decreases (about 5%) were caused by the control saline. These results are essentially in accordance with those ob-
ULTIMOBRANCHIAL 8%
Ca
Pi
I i-L&--AI0
273
GLANDS
0.5
1
HOURS
2
0
0,5
1 HOURS
2
FIG. 1. Time courses of changes in serum Ca and Pi concentrations in rats after the administration of control saline (0) or 10 (0) or 100 mU (+) of salmon calcitonin. Mean r SE (5).
tained previously in this laboratory; thus these responses were adopted as the standard. Serum Pi concentrations were also decreased by salmon calcitonin. Salmon calcitonin, in amounts of 10 and ,100 mu, caused 19 and 32% decreases in the rat serum Pi concentration, respectively. The maximum decrease in serum Pi concentrations elicited by the control saline was about 8%.
Ten and twenty ultimobranchial glands brought about 4.5 and 9% decreases in serum Ca concentration 30 min after the administration, respectively. While the former value is not significantly different from the control value, higher doses elicited significant changes. Thirty and sixty n-rim& after the administration of an extract equivalent to 40 ultimobranchial glands, 15 and 12% decreases, respectively, were found, By comparing these results with the standard curves obtained with salmon calcitonin, the hypocalcemic potency of the ultimbbrancl&l gland of 0. japonkus was estimated to be about 0.19 mU/gland, or 30’ mU/kg bady wt. The serum Pi concentration was significantly decreased by the extract: 1 hr after t:he administration of 40 ultimobranchial glands, it dropped by 18%. Results are shown in Fig. 2. The serum Na concentration of the rats receiving the ,extract did not show significant differences from their ini-
274
OGURO,
‘- 0
0.5
1 HOURS
7.
’ ow
TARUI,
2 HOURS
AND
SASAYAMA
HOURS
FIG. 2. Time courses of changes in serum Ca and Pi concentrations in rats afer the administration of extracts corresponding to 10 (0), 20 (0), or 40 (+) ultimobranchial glands of Onychodactylus juponicus. Mean 2 SE (5).
FIG. 3. Time courses of changes in serum Ca and P, concentrations in rats after the administration of extracts corresponding to 10 (0) or 20 (0) ultimobranchial glands of Hynobius nigrescens. Mean + SE (5).
tial level nor from those of the rats receiving control saline.
decrease 30 min after the administration of the extract from 20 ultimobranchial glands, as shown in Fig. 4.
Ultirnobranchial
Gland of H. nigrescens
Because few specimens were available, only a minimum number of assays of the ultimobranchial gland of this species could be made. Administration of the ultimobranchial gland of H. nigrescens caused hypocalcemia in rats of a slightly greater magnitude than that caused by the same number of ultimobranchial gland of 0. japonicus. The potency of one ultimobranchial gland of this species corresponds to about 0.22 mU of salmon calcitonin, equivalent to 18 mu/kg body wt. Thirty minutes after the administration of 10 ultimobranchial glands, the serum Pi concentration decreased by 8%. Very little change was observed in the serum Na concentration after the administration of the extract (Fig. 3). Ultimobranchial
Gland of C. pyrrhogaster
Hypocalcemic Potency of Some Organs Other Than Ultimobranchial Glands
Extracts of the brain, thyroid gland, thymus, stomach, small intestine, and kidney from all three species were assayed for hypocalcemic potency and for their effects on the serum Pi concentrations. The results were rather diverse, but none of the extracts caused significant changes, as shown in Fig. 5. DISCUSSION In mammals, the thyroid gland is the main site of calcitonin secretion. The calcitonin activity of the thyroid gland has been reported to be 200 mu/kg body wt in man, 400 mu/kg body wt in the dog, and 500 mu/ kg body wt in the rat. In nonmammalian vertebrates, however, the ultimobranchial
Although the hypocalcemic activity of this A% Ca Pi species has been reported previously by Uchiyama (1980), it was assayed again in the present study for the purpose of comparison. Furthermore, the effect of the ultimobranchial gland extract on serum Pi concentration was determined. The hypo’ 005. 1 2 ’ 00.5 2 HOURS HOURS calcemic activity was 87 mu/kg body wt. FIG. 4. Time courses of changes in serum Ca and This is slightly higher than the value reP, concentrations in rats after the administration of ported by Uchiyama (1980), but not sub- extracts corresponding to 10 (0), 20 (O), or 40 (+) stantially different. ultimobranchial glands of Cyaops pyrrhogaster. Mean The serum Pi concentration showed a 13% IL SE (5).
HYPOCALCEMIC A%
ca
PI
Ca
POTENCY
Pi
Ca
OF URODELE
Pi
0
-10 1 +10
0.
-lo-
STOMACH(10)
S.INTESTINE(lO)
KIONEY(10)
5. Changes in serum Ca and Pi concentrations in rats 30 min after the administration of extracts of the brain, thyroid gland, tbymus, stomach, intestine, or kidney. Values represent the percentage of change relative to the initial value. Each column shows an Average of 3-5 doses. Vertical bars show standard error. The figure in parentheses show the numbers of organs in one dose. D, Cynops pyrrhogaster; FIG.
tylus japonicus;
0, Hynobius
nigrescens.
gland is the main site of calcitonin secretion. The ultimobranchial glands of the domestic fowl and the turkey have hypocalcemic potencies of 600 and 700 mu/kg body wt, respectively. Reptilian ultimobranchial glands contain hypocalcemic factors equivalent to 200-300 mU salmon calcitoninlkg body wt. Cod and salmon have been shown to have potencies of 300 and 500 mu/kg body wt, respectively. All this information suggests that the thyroid glands or the ultimobranchial glands of mammals, birds, reptiles, and fish contain hypocalcemic factors equivalent to several hundred mu/kg body wt of calcitonin. Although a function for the ultimobranchial gland of amphibians has been suggested (Robertson, 1969a, b; Sasayama, 1978; Qguro and Uchiyama, 1980), little has been known about its hypocalcemic potencies in amphibians until recently. In Rana nigromaculata, the hypocalcemic potency of the ultimobranchial gland has been reported to be 780 mu/kg body wt (Oguro and Sasayama, 1982). In preliminary studies in this laboratory using the rat bioassay, the
ULTIMOBRANCHIAL
GLANDS
275
ultimobranchial glands of Rana catesbeiana and Bufo bufo japoniclrs have been shown to have a hypocalcemic potency equivalent to about 560 and 300 mu/kg body wt, respectively. It was recently found that the hypocalcemic potency of the ultimobranchial gland of Raw rugosa is very high, about 7600 mu/kg body wt (Oguro et al., 1981). In general, from these results, the hypocalcemic potency of the anuran ultimobranchial gland seems to be in the same range of several hundred mu/kg body wt, as reported in the other vertebrate groups. Only one report concerns the occurrence of a hypocalcemic factor, probably a calcitonin, in urodelan amphibians (Uchiy&ma, 1980). According to Uchiyama, the hypocalcemic potency of the ultim~bra~~hial gland of the newt, C. pyrrhogn,ster, corresponds to 20-30 mu/kg body wt, an extremely low value in comparison with those observed in anuran amphibiaas and other vertebrate classes. The question then arises whether this very low hypocalcemic potency of ,C. pyrrhogaster is specific to this species or whe it is common to urodelans. The present study indicates the later: the Value for the ultimobranchial glands of 0. japonicgs and H. nigrescens were 30 and 18 mu/kg body wt, respectively, one order b,elow those found in anuran amphibians. Thus the present results support the observation of Uchiyama (1980). It is concluded in the preserJrt study that the ultimobranchial glands of uroddaE amphibians contain a hypocalGemic factor, probably a calcitonin, but its potency as revealed by rat bioassay is very low. However, it is not certain whether this is due to the low content of calcitonin in the ultimobranchial gland or to the loti activity of urodelan calcitonin in rats. It is ,,possibl& that the factor may lose its hypo@alcemic activity very rapidly. Whatever the cause, anuran and urodelan amphibians can be dis-
276
OGURO,
TARUI,
tinguished on the basis of the hypocalcemic potencies of their ultimobranchial glands in the rat bioassay. In anuran amphibians, the well-developed endolymphatic sacs serve as reservoirs of Ca, in which a large amount of Ca salts is deposited. Calcitonin promotes the deposition of Ca into the endolymphatic sacs, as suggested previously (Robertson, 1972; Sasayama and Oguro, 1982). However, endolymphatic sacs are very poorly developed in urodelan species. This fact implies that Ca homeostasis may be different in these two groups. Therefore, the very low hypocalcemic potency of the ultimobranchial gland in urodelan species found in the present study may reflect a difference in Ca homeostasis in these two amphibian groups. In mammals, the thyroid gland is the site of calcitonin secretion. However, it has been reported that in some species, the presence of calcitonin is not restricted to the thyroid gland (Copp and Parkes, 1968; Galante et al., 1968). Calcitonin has been found in the ultimobranchial gland of a variety of nonmammalian species. However, the extra-ultimobranchial origin of calcitonin has been established in some species: in chickens and in skinks the thyroid gland is one source of calcitonin in addition to the ultimobranchial gland. Furthermore, the thyroid gland and the ultimobranchial gland show nearly the same hypocalcemic potency in pigeons (Moseley et al., 1968). It is known that C cells, which are calcitonin secretory cells, originate in the neuroectodermal tissue at an early stage of development and later migrate to the organs which are their final site. Therefore, it is quite possible that C cells are distributed into more than one organ. Thus the extremely low hypocalcemic potency in the ultimobranchial gland of urodelan species suggested a search for organs which might show hypocalcemic potency. In fact, the observation that some cells in the brain of
AND
SASAYAMA
the bullfrog contain granules which are immunoreactive to anti-calcitonin antibody (Yui et al., 1981) raises the possibility that at least in some urodelan species the ultimobranchial gland is not the major site for C cells. Neither the brain, thyroid gland, thymus, stomach, small intestine, nor kidney showed a significant hypocalcemic potency, as revealed by the rat bioassay. The present results tend to exclude the possibility that calcitonin-secreting C cells have settled, not in the ultimobranchial gland, but in other organs. ACKNOWLEDGMENTS The writers would like to thank Mr. Ken-ichi Nagai, Department of Biochemistry, Teikyo University School of Medicine, for his willing cooperation. They are also indebted to Dr. Minoru Uchiyama and Dr. Masayoshi Yoshihara, Department of Oral Physiology, Niigata Faculty, Nippon Dental University, for their valuable suggestion. Salmon calcitonin used for the standard was donated by Dr. Peter K. T. Pang, Department of Pharmacology and Therapeutics, Texas Tech University, to whom the writers are much obliged. The present study was supported in part by grants-in-aid from the Ministry of Education of Japan (Nos. 334043, 56340038 to C.O.).
REFERENCES Clark, N. B. (1971). The ultimobranchial body of reptiles. J. Exp. Zooi. 178, 115-121. Copp, D. H., Brooks, C. E., Low, B. S., Newsome, E, O’Dor, R. K., Parkes, C. O., Walker, V., and Watts, E. G. (1970). Calcitonin and ultimobranchial function in lower vertebrates. ln “Calcitonin. Proceedings, II International Symposium, 1969” (S. Taylor, ed.), pp. 281-294. Heinemann Medical Books, London. Copp, D. H., and Pakes, C. 0. (1968). Extraction of calcitonin from ultimobranchial tissue. In “Parathyroid Hormone and Thyrocalcitonin (Calcitonin)” (R. V. Talmage and L. E Belanger, eds.), pp. 74-84. Excerpta Medical Foundation, Amsterdam. Fiske, C. H., and Subbarow, Y. (1925). The colorimetric determination of phosphorus. J. Biol. Chem. 66, 375-400. Galante, L., Matthews, E. W., Tse, A., Williams, E. D., Woodhouse, N. J. Y., and MacIntyre, I. (1968). Thymic and parathyroid origin of calcitonin in man. Lancer ii, 537-539.
HYPOCALCEMIC
POTENCY
OF URODELE
Moseley, J. M., Matthews, E. W., Breed, R. H., Galante, L., Tse, A., and MacIntyre, I. (1968). The ultimobranchial origin of calcitonin. Lancer i, 108-110. Oguro, C., Nagai, K.-I., Tarui, H., and Sasayama, Y. (1981). Hypocalcemic factor in the ultimobranchial gland of the frog, Rana rugosa. Comp. Biochem.
Physiol.
A 68, 95-97.
ULTIMOBRANCHIAL
GLANDS
277
Sasayama, Y. (1978). Effects of implantation of the ultimobranchial glands and the administration of synthetic salmon calcitonin of serum Ca concentrations in ultimobranchialectomized bullfrog tadpoles. Gen. Comp. Endocrinol. 34, 229-233. Sasayama, Y., and Oguro, C. (1982). The role of ultimobranchial glands on Ca balance in bullfrog tadpoles. In “Proceedings 1X.I.S.C.E. (B. Loft, ed.), Hong Kong Univ. Press, Hong Kong, in press. Sehe, C. T. (1960). Radioautographic studies on the ultimobranchial body and thyroid gland in vertebrates, fishes and amphibians. Endocrinology 67,
Oguro, C., and Sasayama, Y. (1982). Endocrinology of bypocalcemic regulation in anuran amphibians. In “Proceedings, IX I.S.C.E.” (B. Loft, ed.), Hong Kong Univ. Press, Hong Kong, In press. 674-684. Oguro, C., and Uchiyama, M. (1980). Comparative endocrinology of hypocalcemic regulation in lower Tauber, S. D. (1967). The ultimobranchial origin of thyrocalcitonin. Proc. Nat. Acad. Sci. USA 58, vertebrates. In “Hormones, Adaptation and Evolution” (S. Ishii, T. Hirano, and M. Wada, eds.), 1684-1687. pp. 113-121. Japan Sci. Sot. Press, Tokyo/Springer Uchiyama, M. (1980). Hypocalcemic factor in the ulVerlag, Berlin. timobranchial glands of the newt, Cynops pyrrhoOrimo, H., Fujita, T., Yoshikawa, M., Watanabe, S., gaster. Comp. Biochem. Physiol. A 66, 330-334. Otani, M., and Abe, J. (1972). Ultimobranchial Uchiyama, M., Yoshihara, M., Murakami, T., and calcitonin of the eel. Endocrinol. Japon. 19, Ognro, C. (1978). Presence of a hypocalcemic 299-302. factor in the ultimobranchial gland of the snake. Robertson, D. R. (1969a). The ultimobranchial body Gen. Camp. Endocrinol. 36, 59-62. of Rana pipiens. VIII. Effecs of extirpation upon Uchiyama, M., Yoshihara, M., Murakami, T., and calcium distribution and bone cell types. Gen. Oguro, C. (1981). Calcitonin content in the ultiComp. Endocrinol. 12, 479-490. mobranchial gland of the snake, Elaphe climacoRobertson, D. R. (1969b). The nltimobranchial body phora: Comparison of adults, young, and hatchof Rana pipiens. X. Effect of glandular extirpation lings. Gen. Comp. Endocrinol. 43, 259-261. on fracture healing. J. Exp. Zool. 172,425-442. Yui, R., Yamada, Y., Kayamori, R., and Fujita, T. Robertson, D. R. (1972). Calcitonin in amphibians and (1981). Calcitonin-immunoreactive neurons in the the relationship of the paravertebral lime sacs with brain of the bullfrog, Rana catesbeiana with specarbonic anhydrase. In “Calcium, Parathyroid cial reference to their liquor-contacting and neuHormone and the Calcitonins” (R. V. Talmage and rosecretory nature. An immunochemical and P. L. Munson, eds.), pp. 21-28. Excerpta Medica, immunohistochemical study. Biome”. Res. 2, Amsterdam. 208-216.
AND
COMPARATIVE
ENDOCRINOLOGY
Hypocalcemic
272-277 (1983)
Potency of the Ultimobranchial Urodelan Amphibians
CHITARU Department
51,
OGURO,
of Biology,
HIDEO
Faculty
TARUI,~
of Science,
AND YUICHI
Toyama
University,
Gland in Some SASAYAMA Toyama
930, Japan
Accepted September 24, 1982 The hypocalcemic
potencies of the ultimobranchial glands of two urodelans, Onychoand Hynobius nigrescens, were studied; according to the rat bioassay, their calcitonin values (MRC) were 30 and 18 mu/kg body wt, respectively. Various organs other than the ultimobranchial gland were also assayed in rats to see whether they had any hypocalcemic potency. Wowever, the ultimobranchial gland was the only organ examined with detectable hypocalcemic potencies in these urodelans. The hypocalcemic potencies of urodelan ultimobranchial glands are one order lower than those reported in the other vertebrate classes, contrasting with the potencies of anuran ultimobtanchial glands. The biological significance of this low potency is discussed. dactylus
japonicus
The occurrence of calcitonin, which is a hypocalcemic factor in mammals, has been reported in the ultimobranchial gland of some lower vertebrates (Copp and Parkes, 1968; Moseley et al., 1968; Clark, 1971; Oguro and Uchiyama, 1980). Piscine calcitonin showed a high hypocalcemic potency in rats compared with mammalian calcitonin (Copp et al., 1970; Orimo et al., 1972). The hypocalcemic potency of ultimobranchial extracts of reptiles and birds seems to be high in terms of body weight, as shown in the rat bioassay (Copp and Parkes, 1968; Uchiyama et al., 1978, 1981). Recently a high hypocalcemic potency was found in the ultimobranchial gland of some anuran amphibians (Oguro et al., 1981; Oguro and Sasayama, 1982). On the other hand, an extremely low hypocalcemic potency was reported in the ultimobranchial gland of the newt, Cynops pyrrhogaster, according to the rat bioassay (Uchiyama, 1980). The significance of the very low hypocalcemic potency reported in C. pyrrhogaster is not understood, since the physiI Present address: Department of Community Medicine, Toyama Medical and Pharmaceutical University, Toyama 930-01, Japan.
ological role of calcitonin in poikilothermic vertebrates is still a matter of discussion. Therefore, it is of interest whether the low hypocalcemic potency of the ultimobranchial gland is specific to C. pyrrhogaster or a common phenomenon among urodelan amphibians. In the present study, the hypocalcemic potency of the ultimobranchial gland extracts of two urodelan amphibians, Onychodactylus japonicus and Hynobius nigrescens, was examined using the rat bioassay. It is now accepted that C cells, which secrete calcitonin, originate in the neuroectodermal tissue at an early stage of development and later migrate to the organs which ultimately incorporate the C cells: Therefore, the possibility remains that the ultimobranchial gland is not the main C cellcontaining organ, but that other organs become the site of the C cells. In that case, the low hypocalcemic potency of the ultimobranchial gland of C. pyrrhogaster could be explained by a different distribution of C cells. Thus the hypocalcemic potencies of the brain, thyroid gland, thymus, stomach, small intestine, and kidney of the three species were also assayed.
272 0016-648OB3 $1.50 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
HYPOCALCEMIC
MATERIALS
POTENCY
OF URODELE
AND METHODS
In the present study, two urodelan species, 0. japonicus (Houttuyn) and H. nigrescens Stejneger, were used. C. pyrrhogaster (Boie) was also used for comparison. 0. japonicus and C. pyrrhogaster were obtained from commercial sources; specimens of H. nigrescens were collected in the mountainous area of Toyama Prefecture, Japan. In these three species, the ultimobranchial gland is very small in comparison with that of anurans and present only on the left side, located dorso-lateral to the pericardial cavity, as described previously (Sehe, 1960; Uchiyama, 1980). The right ultimobranchial gland is degenerated during development. The ultimobranchial glands of C. pyrrhogas&r were obtained throughout the year. No difference seemed to exist in the calcitonin activity of the ultimobranchial gland in the diierent season and sexual status. Those of H. nigrescens and 0. japonicus were removed during breeding and post-breeding season. The ultimobranchial glands removed were homogenized in 0.8% NaCl solution adjusted to pH 4.6 with HCl. The homogenate was then centrifuged at 11,000 rpm for 15 min. The supernatant was used as the ultimobranchial extract. Extracts of the brain, thyroid gland, thymus, stomach, small intestine, and kidney were also prepared as described above. All were assayed in rats for the presence of hypocalcemic potency. The calcitonin assay of the ultimobranchial extract was carried out as described previously (Oguro et a[., 1981). Each assay rat received 0.4 ml of the extract or vehicle. To obtain standard responses, 10 or 100 mU (MRC) of synthetic salmon calcitonin was used. Blood samples were taken from the assay rats through the cannula set in the sciatic artery before the extract or vehicle was administered and at intervals of 0.5, 1, 2, and 3 hr afterward. Serum Ca and Na concentrations were determined by atomic absorption spectrophotometry. The inorganic phosphorus (PI) concentration was measured according to a modified method of Fiske and Subbarow (1925). Determination of the activity was achieved by the method of Tauber (1967). Student’s t test was used for statistical analysis.
RESULTS Salmon calcitonin produced hypocalcemia in rats: 10 and 100 mU of salmon calcitonin caused 18 and 27% decrease in the serum Ca concentration, respectively, as shown in Fig. 1. The maximum decrease was found 30 min after administration with 10 mU and 1 hr with 100 mu. On the other hand, slight decreases (about 5%) were caused by the control saline. These results are essentially in accordance with those ob-
ULTIMOBRANCHIAL 8%
Ca
Pi
I i-L&--AI0
273
GLANDS
0.5
1
HOURS
2
0
0,5
1 HOURS
2
FIG. 1. Time courses of changes in serum Ca and Pi concentrations in rats after the administration of control saline (0) or 10 (0) or 100 mU (+) of salmon calcitonin. Mean r SE (5).
tained previously in this laboratory; thus these responses were adopted as the standard. Serum Pi concentrations were also decreased by salmon calcitonin. Salmon calcitonin, in amounts of 10 and ,100 mu, caused 19 and 32% decreases in the rat serum Pi concentration, respectively. The maximum decrease in serum Pi concentrations elicited by the control saline was about 8%.
Ten and twenty ultimobranchial glands brought about 4.5 and 9% decreases in serum Ca concentration 30 min after the administration, respectively. While the former value is not significantly different from the control value, higher doses elicited significant changes. Thirty and sixty n-rim& after the administration of an extract equivalent to 40 ultimobranchial glands, 15 and 12% decreases, respectively, were found, By comparing these results with the standard curves obtained with salmon calcitonin, the hypocalcemic potency of the ultimbbrancl&l gland of 0. japonkus was estimated to be about 0.19 mU/gland, or 30’ mU/kg bady wt. The serum Pi concentration was significantly decreased by the extract: 1 hr after t:he administration of 40 ultimobranchial glands, it dropped by 18%. Results are shown in Fig. 2. The serum Na concentration of the rats receiving the ,extract did not show significant differences from their ini-
274
OGURO,
‘- 0
0.5
1 HOURS
7.
’ ow
TARUI,
2 HOURS
AND
SASAYAMA
HOURS
FIG. 2. Time courses of changes in serum Ca and Pi concentrations in rats afer the administration of extracts corresponding to 10 (0), 20 (0), or 40 (+) ultimobranchial glands of Onychodactylus juponicus. Mean 2 SE (5).
FIG. 3. Time courses of changes in serum Ca and P, concentrations in rats after the administration of extracts corresponding to 10 (0) or 20 (0) ultimobranchial glands of Hynobius nigrescens. Mean + SE (5).
tial level nor from those of the rats receiving control saline.
decrease 30 min after the administration of the extract from 20 ultimobranchial glands, as shown in Fig. 4.
Ultirnobranchial
Gland of H. nigrescens
Because few specimens were available, only a minimum number of assays of the ultimobranchial gland of this species could be made. Administration of the ultimobranchial gland of H. nigrescens caused hypocalcemia in rats of a slightly greater magnitude than that caused by the same number of ultimobranchial gland of 0. japonicus. The potency of one ultimobranchial gland of this species corresponds to about 0.22 mU of salmon calcitonin, equivalent to 18 mu/kg body wt. Thirty minutes after the administration of 10 ultimobranchial glands, the serum Pi concentration decreased by 8%. Very little change was observed in the serum Na concentration after the administration of the extract (Fig. 3). Ultimobranchial
Gland of C. pyrrhogaster
Hypocalcemic Potency of Some Organs Other Than Ultimobranchial Glands
Extracts of the brain, thyroid gland, thymus, stomach, small intestine, and kidney from all three species were assayed for hypocalcemic potency and for their effects on the serum Pi concentrations. The results were rather diverse, but none of the extracts caused significant changes, as shown in Fig. 5. DISCUSSION In mammals, the thyroid gland is the main site of calcitonin secretion. The calcitonin activity of the thyroid gland has been reported to be 200 mu/kg body wt in man, 400 mu/kg body wt in the dog, and 500 mu/ kg body wt in the rat. In nonmammalian vertebrates, however, the ultimobranchial
Although the hypocalcemic activity of this A% Ca Pi species has been reported previously by Uchiyama (1980), it was assayed again in the present study for the purpose of comparison. Furthermore, the effect of the ultimobranchial gland extract on serum Pi concentration was determined. The hypo’ 005. 1 2 ’ 00.5 2 HOURS HOURS calcemic activity was 87 mu/kg body wt. FIG. 4. Time courses of changes in serum Ca and This is slightly higher than the value reP, concentrations in rats after the administration of ported by Uchiyama (1980), but not sub- extracts corresponding to 10 (0), 20 (O), or 40 (+) stantially different. ultimobranchial glands of Cyaops pyrrhogaster. Mean The serum Pi concentration showed a 13% IL SE (5).
HYPOCALCEMIC A%
ca
PI
Ca
POTENCY
Pi
Ca
OF URODELE
Pi
0
-10 1 +10
0.
-lo-
STOMACH(10)
S.INTESTINE(lO)
KIONEY(10)
5. Changes in serum Ca and Pi concentrations in rats 30 min after the administration of extracts of the brain, thyroid gland, tbymus, stomach, intestine, or kidney. Values represent the percentage of change relative to the initial value. Each column shows an Average of 3-5 doses. Vertical bars show standard error. The figure in parentheses show the numbers of organs in one dose. D, Cynops pyrrhogaster; FIG.
tylus japonicus;
0, Hynobius
nigrescens.
gland is the main site of calcitonin secretion. The ultimobranchial glands of the domestic fowl and the turkey have hypocalcemic potencies of 600 and 700 mu/kg body wt, respectively. Reptilian ultimobranchial glands contain hypocalcemic factors equivalent to 200-300 mU salmon calcitoninlkg body wt. Cod and salmon have been shown to have potencies of 300 and 500 mu/kg body wt, respectively. All this information suggests that the thyroid glands or the ultimobranchial glands of mammals, birds, reptiles, and fish contain hypocalcemic factors equivalent to several hundred mu/kg body wt of calcitonin. Although a function for the ultimobranchial gland of amphibians has been suggested (Robertson, 1969a, b; Sasayama, 1978; Qguro and Uchiyama, 1980), little has been known about its hypocalcemic potencies in amphibians until recently. In Rana nigromaculata, the hypocalcemic potency of the ultimobranchial gland has been reported to be 780 mu/kg body wt (Oguro and Sasayama, 1982). In preliminary studies in this laboratory using the rat bioassay, the
ULTIMOBRANCHIAL
GLANDS
275
ultimobranchial glands of Rana catesbeiana and Bufo bufo japoniclrs have been shown to have a hypocalcemic potency equivalent to about 560 and 300 mu/kg body wt, respectively. It was recently found that the hypocalcemic potency of the ultimobranchial gland of Raw rugosa is very high, about 7600 mu/kg body wt (Oguro et al., 1981). In general, from these results, the hypocalcemic potency of the anuran ultimobranchial gland seems to be in the same range of several hundred mu/kg body wt, as reported in the other vertebrate groups. Only one report concerns the occurrence of a hypocalcemic factor, probably a calcitonin, in urodelan amphibians (Uchiy&ma, 1980). According to Uchiyama, the hypocalcemic potency of the ultim~bra~~hial gland of the newt, C. pyrrhogn,ster, corresponds to 20-30 mu/kg body wt, an extremely low value in comparison with those observed in anuran amphibiaas and other vertebrate classes. The question then arises whether this very low hypocalcemic potency of ,C. pyrrhogaster is specific to this species or whe it is common to urodelans. The present study indicates the later: the Value for the ultimobranchial glands of 0. japonicgs and H. nigrescens were 30 and 18 mu/kg body wt, respectively, one order b,elow those found in anuran amphibians. Thus the present results support the observation of Uchiyama (1980). It is concluded in the preserJrt study that the ultimobranchial glands of uroddaE amphibians contain a hypocalGemic factor, probably a calcitonin, but its potency as revealed by rat bioassay is very low. However, it is not certain whether this is due to the low content of calcitonin in the ultimobranchial gland or to the loti activity of urodelan calcitonin in rats. It is ,,possibl& that the factor may lose its hypo@alcemic activity very rapidly. Whatever the cause, anuran and urodelan amphibians can be dis-
276
OGURO,
TARUI,
tinguished on the basis of the hypocalcemic potencies of their ultimobranchial glands in the rat bioassay. In anuran amphibians, the well-developed endolymphatic sacs serve as reservoirs of Ca, in which a large amount of Ca salts is deposited. Calcitonin promotes the deposition of Ca into the endolymphatic sacs, as suggested previously (Robertson, 1972; Sasayama and Oguro, 1982). However, endolymphatic sacs are very poorly developed in urodelan species. This fact implies that Ca homeostasis may be different in these two groups. Therefore, the very low hypocalcemic potency of the ultimobranchial gland in urodelan species found in the present study may reflect a difference in Ca homeostasis in these two amphibian groups. In mammals, the thyroid gland is the site of calcitonin secretion. However, it has been reported that in some species, the presence of calcitonin is not restricted to the thyroid gland (Copp and Parkes, 1968; Galante et al., 1968). Calcitonin has been found in the ultimobranchial gland of a variety of nonmammalian species. However, the extra-ultimobranchial origin of calcitonin has been established in some species: in chickens and in skinks the thyroid gland is one source of calcitonin in addition to the ultimobranchial gland. Furthermore, the thyroid gland and the ultimobranchial gland show nearly the same hypocalcemic potency in pigeons (Moseley et al., 1968). It is known that C cells, which are calcitonin secretory cells, originate in the neuroectodermal tissue at an early stage of development and later migrate to the organs which are their final site. Therefore, it is quite possible that C cells are distributed into more than one organ. Thus the extremely low hypocalcemic potency in the ultimobranchial gland of urodelan species suggested a search for organs which might show hypocalcemic potency. In fact, the observation that some cells in the brain of
AND
SASAYAMA
the bullfrog contain granules which are immunoreactive to anti-calcitonin antibody (Yui et al., 1981) raises the possibility that at least in some urodelan species the ultimobranchial gland is not the major site for C cells. Neither the brain, thyroid gland, thymus, stomach, small intestine, nor kidney showed a significant hypocalcemic potency, as revealed by the rat bioassay. The present results tend to exclude the possibility that calcitonin-secreting C cells have settled, not in the ultimobranchial gland, but in other organs. ACKNOWLEDGMENTS The writers would like to thank Mr. Ken-ichi Nagai, Department of Biochemistry, Teikyo University School of Medicine, for his willing cooperation. They are also indebted to Dr. Minoru Uchiyama and Dr. Masayoshi Yoshihara, Department of Oral Physiology, Niigata Faculty, Nippon Dental University, for their valuable suggestion. Salmon calcitonin used for the standard was donated by Dr. Peter K. T. Pang, Department of Pharmacology and Therapeutics, Texas Tech University, to whom the writers are much obliged. The present study was supported in part by grants-in-aid from the Ministry of Education of Japan (Nos. 334043, 56340038 to C.O.).
REFERENCES Clark, N. B. (1971). The ultimobranchial body of reptiles. J. Exp. Zooi. 178, 115-121. Copp, D. H., Brooks, C. E., Low, B. S., Newsome, E, O’Dor, R. K., Parkes, C. O., Walker, V., and Watts, E. G. (1970). Calcitonin and ultimobranchial function in lower vertebrates. ln “Calcitonin. Proceedings, II International Symposium, 1969” (S. Taylor, ed.), pp. 281-294. Heinemann Medical Books, London. Copp, D. H., and Pakes, C. 0. (1968). Extraction of calcitonin from ultimobranchial tissue. In “Parathyroid Hormone and Thyrocalcitonin (Calcitonin)” (R. V. Talmage and L. E Belanger, eds.), pp. 74-84. Excerpta Medical Foundation, Amsterdam. Fiske, C. H., and Subbarow, Y. (1925). The colorimetric determination of phosphorus. J. Biol. Chem. 66, 375-400. Galante, L., Matthews, E. W., Tse, A., Williams, E. D., Woodhouse, N. J. Y., and MacIntyre, I. (1968). Thymic and parathyroid origin of calcitonin in man. Lancer ii, 537-539.
HYPOCALCEMIC
POTENCY
OF URODELE
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Sasayama, Y. (1978). Effects of implantation of the ultimobranchial glands and the administration of synthetic salmon calcitonin of serum Ca concentrations in ultimobranchialectomized bullfrog tadpoles. Gen. Comp. Endocrinol. 34, 229-233. Sasayama, Y., and Oguro, C. (1982). The role of ultimobranchial glands on Ca balance in bullfrog tadpoles. In “Proceedings 1X.I.S.C.E. (B. Loft, ed.), Hong Kong Univ. Press, Hong Kong, in press. Sehe, C. T. (1960). Radioautographic studies on the ultimobranchial body and thyroid gland in vertebrates, fishes and amphibians. Endocrinology 67,
Oguro, C., and Sasayama, Y. (1982). Endocrinology of bypocalcemic regulation in anuran amphibians. In “Proceedings, IX I.S.C.E.” (B. Loft, ed.), Hong Kong Univ. Press, Hong Kong, In press. 674-684. Oguro, C., and Uchiyama, M. (1980). Comparative endocrinology of hypocalcemic regulation in lower Tauber, S. D. (1967). The ultimobranchial origin of thyrocalcitonin. Proc. Nat. Acad. Sci. USA 58, vertebrates. In “Hormones, Adaptation and Evolution” (S. Ishii, T. Hirano, and M. Wada, eds.), 1684-1687. pp. 113-121. Japan Sci. Sot. Press, Tokyo/Springer Uchiyama, M. (1980). Hypocalcemic factor in the ulVerlag, Berlin. timobranchial glands of the newt, Cynops pyrrhoOrimo, H., Fujita, T., Yoshikawa, M., Watanabe, S., gaster. Comp. Biochem. Physiol. A 66, 330-334. Otani, M., and Abe, J. (1972). Ultimobranchial Uchiyama, M., Yoshihara, M., Murakami, T., and calcitonin of the eel. Endocrinol. Japon. 19, Ognro, C. (1978). Presence of a hypocalcemic 299-302. factor in the ultimobranchial gland of the snake. Robertson, D. R. (1969a). The ultimobranchial body Gen. Camp. Endocrinol. 36, 59-62. of Rana pipiens. VIII. Effecs of extirpation upon Uchiyama, M., Yoshihara, M., Murakami, T., and calcium distribution and bone cell types. Gen. Oguro, C. (1981). Calcitonin content in the ultiComp. Endocrinol. 12, 479-490. mobranchial gland of the snake, Elaphe climacoRobertson, D. R. (1969b). The nltimobranchial body phora: Comparison of adults, young, and hatchof Rana pipiens. X. Effect of glandular extirpation lings. Gen. Comp. Endocrinol. 43, 259-261. on fracture healing. J. Exp. Zool. 172,425-442. Yui, R., Yamada, Y., Kayamori, R., and Fujita, T. Robertson, D. R. (1972). Calcitonin in amphibians and (1981). Calcitonin-immunoreactive neurons in the the relationship of the paravertebral lime sacs with brain of the bullfrog, Rana catesbeiana with specarbonic anhydrase. In “Calcium, Parathyroid cial reference to their liquor-contacting and neuHormone and the Calcitonins” (R. V. Talmage and rosecretory nature. An immunochemical and P. L. Munson, eds.), pp. 21-28. Excerpta Medica, immunohistochemical study. Biome”. Res. 2, Amsterdam. 208-216.