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Hypocalcemic factor in the ultimobranchial gland of the frog, Rana rugosa
Cony. Biorhem. 0 F’ergamon
Phgsd
Vol.
68A,
pp
031%9629/81!0101-oO95802.C0/0
95 lo 97
Press Ltd1981. Printed !nGreatBritain
HYPOCALCEMIC GLAND
FACTOR IN THE ULTIMOBRANCHIAL OF THE FROG, RANA RUGOSA
CHITARU OGURO, KEN-ICHI NAGAI, HIDEO TARUI and YUICHI SASAYAMA Department
of Biology, Faculty of Science, Toyama Toyama 930, Japan (Rr~ric~d
University,
6 May 1980)
Abstract-l. The hypocalcemic activity of the ultimobranchial gland of the frog, Rana rugosa, was estimated using a rat bioassay method. 2. Extracts of the ultimobranchial gland showed a very high hypocalcemic activity. The value corresponded to 6,340 mU (MRC)/kg b.w. 3. Serum inorganic phosphorus values of rats received the extract decreased in proportion to the dose, although no changes were found in serum sodium concentration.
INTRODUCTION
ultimobranchial glands which are attached to the glottic constrictor muscles. They could be removed through cuts made on the buccal floor at both sides of the glottis. Before the removal of the ultimobranchial glands, the frogs were put in ice cubes for anesthesia and then pithed. The mouth was kept open and the ultimobranchial glands were removed through the mouth. The ultimobranchial glands removed were homogenized in a Potter homogenizer and then sonicated in 0.6% NaCl solution adjusted to pH 4.6 with HCl. The homogenate was centrifuged at 12,000 rpm for 10 min. The supernatant was used for the assay using male Wister rats aged 5 weeks which fasted overnight before the experiments. After being anesthetized with ether, they were fixed dorsally on a board and cannulated with polyethylene tubings in the iliac vein and the iliac artery. The former was used for the administration of the extract and the latter was the path for obtaining blood samples. Each assay rat received 0.4 ml of the extract, containing 0.5. 1, 2 or 4 ultimobranchial glands or the same amount of control saline. For comparison, 10 or 1OOmU (MRC) of synthetic salmon calcitonin dissolved in the same amount of saline was administered. Blood samples were obtained at intervals of 0.5, 1, 2 and 3 hr after the administration. Serum was separated in a microhematocrit centrifuge and calcium and sodium concentrations were determined by atomic absorption spectrophotometry. Inorganic phosphorus was measured according to the method of Fiske & Subbarow (1925).
Since Copp & Hirsch found a hypocalcemic factor in mammals and named it (thyro)calcitonin (Copp et al., 1962; Hirsch et al., 1963), numerous papers have been presented on calcitonin not only for its physiological effect on mammals but also for its biochemical nature. However, the physiological and biological significance of calcitonin is still not completely understood. Hirsch, who was one of the discoverers of this principle, presented an idea that the function of calcitonin does not directly involve bones or even calcium (Hirsch, 1971). In lower vertebrates, much needs to be clarified in the function of calcitonin, as pointed out recently (Oguro & Uchiyama, 1980). In non-mammalian vertebrates, the ultimobranchial glands are the site of calcitonin secretion. Histological and immunological similarities of cellular granules in some anuran ultimobranchial glands to the granules in mammalian C cells have been suggested (Coleman & Phillips, 1974; Coleman, 1975; van Noorden & Pearse, 1971). Robertson studied intensively on the function of the ultimobranchial gland of Rana p&ens and put forth the importance of the ultimobranchial gland in the regulation of calcium in this species (Robertson, 1969a,b, 1972). However, only one paper is available for showing the presence of calcitonin activity in the ultimobranchial gland of anuran amphibians (Copp & Parkes, 1968). They found very low hypocalcemic activity in the ultimobranchial gland of the bullfrog, Ram cates-
RESULTS
Ultimobranchial extracts of R. rugosa and salmon calcitonin caused a hypocalcemic response in the rats. Serum calcium concentrations in rats received the extract equivalent to four ultimobranchial glands were 2.5 mg/lOOml and 1.3 mg/lOOml lower than those in their respective controls at 30 min and 1 hr after the administration, respectively. The former corresponds to 22.1:/, decrease from the initial value. Serum calcium concentration in rats received one and two ultimobranchial gland extracts showed 16.8 and 20.2”/:, decreases from their respective initial value at 1 hr after the administration, respectively. Maximum
beiana.
In recent studies with the rat bioassay, a high hypocalcemic activity in terms of body weight was found in the ultimobranchial gland of the frog, Rana rugosa. The following is a brief description of the results. MATERIALS
AND
METHODS
Adults of the frog, Rana rugosa Schlegel, were collected in ditches in the suburbs of Toyama City during the summer and autumn. The present species bears a pair of the 95
CHITARUOGURO et al.
96
40
-40 t
t 1 0
I 0.5
I I
2
Hr after
injection
I 3
I 0
1 0.5
ti I
Hr after
2
1 3
injection
Fig. 1. Changes of serum calcium concentration in rats following administration of control saline (A), extracts of 1 (o), 2 (0) and 4 (0) ultimobranchial glands of R. rugosa, and 10 (0) and 100 (W) mU of salmon calcitonin. Values are shown by o/odifference from the respective initial value. Fig. 2. Changes of serum inorganic phosphorus concentration in rats following administration of control saline (A), extracts of 1 (o), 2 (8) and 4 (0) ultimobranchial glands of R. rugosa, and 10 (0) and 100 (B) mU of salmon calcitonin. Values are shown by ‘A difference from the respective initial value.
decreases caused by 10mU and 1OOmU of salmon calcitonin were 2.0 and 3.2 mg/lOO ml from their respective control values, respectively. The former corresponds to 18.9% decrease and the latter 30.4% decrease from their respective initial values. The maximum decreases caused by ultimobranchial extracts and salmon calcitonin were nearly proportional to the dose. The results are shown in Fig. 1. Comparison of the responses caused by ultimobranchial extracts to those caused by salmon calcitonin showed that the activity of one ultimobranchial gland of R. rugosa corresponds to 26 mU of salmon calcitonin. Since average body weight of the frogs used was 8.2 g, 52 mU per individual can be read 6,340 mu/kg b.w. Administration of ultimobranchial extracts and salmon calcitonin caused prominent decreases in serum phosphorus concentration. Serum phosphorus level in rats received four ultimobranchial gland extract was 2.7mg/lOOml lower than that of the control level. This was 21.2% decrease from the initial value. The magnitudes caused by ultimobranchial extracts and salmon calcitonin were nearly proportional to the dose. The results are shown in Fig. 2.
, 0
. 0.5
* I
I
2
3
Hr after injection Fig. 3. Changes of serum sodium concentrations in rats following administration of control saline (A), extracts of 1 (o), 2 (0) and 4 (0) ultimobranchial glands of R. rugosa, and 10 (0) and 100 m) mU of salmon calcitonin. Values are shown o/0difference from the respective initial value.
No changes were observed in serum sodium concentrations after the administration of ultimobranchial extracts, salmon calcitonin and control saline, although small fluctuations were found irrespective of the dose (Fig. 3). Hematocrit values decreased gradually in all rats used. This might be caused by repeated bleeding. DISCUSSION
It was reported in Ram pipiens that removal of the ultimobranchial glands resulted in increased osteolytic activity ‘and urinary calcium excretion and decreased calcium deposition in lymphatic sacs (Robertson, 1969a,b, 1972). These facts suggest that the ultimobranchial gland is directly involving in calcium regulation in anuran amphibians. Serum calcium concentration increased markedly when ultimobranchialectomized bullfrog tadpoles were kept in high calcium water. However, implantations of the ultimobranchial glands of the bullfrog tadpoles or of adult Ram nigromaculata into ultimobranchialectomized bullfrog tadpoles kept in high calcium water prevented the rise of serum calcium concentration (Sasayama & Oguro, 1976). Furthermore, injection of salmon calcitonin brought about similar results (Sasayama, 1978). These findings suggest the presence of a factor which may be a calcitonin and inhibits serum calcium increase in the ultimobranchial gland of anuran amphibians. Histological studies on frog ultimobranchial glands showed the presence of cellular granules which resemble those found in the C cell in mammals (Coleman & Phillips, 1974; Coleman, 1975). Furthermore, occurrence of a substance which crossreacts with antiportine calcitonin antibodies was reported in the ultimobranchial glands in two species of Raw (van Nooden & Pearse, 1971). In fact, Copp and Parkes reported calcitonin ac-
H~pocalcemic factor in frog UBG
97
tivity in ultimobranchial extracts of R. catesbeiana (Copp & Parkes, 1968). The activity reported was l-2mU/kg b.w. This value is extremely lpw in comparison with the activities found in mammals, birds, most reptiles and fishes (Clark, 1968; Copp ef ai., 1967; Copp & Parkes, 1968; Moseley ef al., 1968; Uchiyama et al., 1978). In a preliminary study with the frog, R. nigromaculata, the authors found that ultimobranchial extracts showed a hypocalcemic activity which corresponds to the activity elicited by several hundreds milliunits of salmon calcitonin (Oguro & Uchiyama, 1980). In the present study, a very high hypocalcemic activity (6,340mUjkg b.w.) was found in the ultimobranchiai gland of R. rugosa. This marks a prominent contrast to the very low hypocalcemic activity in the ultimobranchial gland of R. catesbeiana (Copp & Parkes, 1968). No animals have been known for such a high hypocalcemic activity in the ultimobranchial gland except the eel (Orimo et al., 1972). The activity found in the eel ultimobranchial gland was reported to be about, 30,000 mu/kg b.w. The function of the ultimobranchial gland of R. rugosa has not been studied. However, in related preliminary studies using R. nigromaculata, it was found that administration of ultimobranchial extracts caused a hypocalcemic response while serum sodium concentration was not affected (Oguro & Uchiyama, 1980). These findings may suggest that the hypocalcemic factor effective on rats is involving in serum calcium regulation in frogs. Very high hypocalcemic activity found in R. rugosa inspires further studies on the function of the ultimobranchial gland in the present species.
new hormone from the parathyroid that lowers blood calcium. Endocrinology 70, 638-649. COPP D. H., COCKCROFT D. W. & KUEH Y. (1967) Calcitonin from ultimobranchial glands of dogfish and chickens. Sciencr 158.924-925. COPP D. H. & PARKESC. 0. (1968) Extraction of calcitonin from ultimobranchial tissue. In Parar~~ra~d Hormone and Th~rorafcitonin (Calcironin) (Edited by TALMAGE,R. V. & B~LANGERL. F.), pp. 74-84. Excerpta Medica, Amsterdam. FISKEC. H. & SURRAROW Y. (1925) The calorimetric determination of phosphorus. J. Biol. Chem. 66, 375400. HIRSCHP. F. (1971) Thyrocalcitonin and its role in calcium regulation in mammals. f. PX~. Zoo!. 178, 139-149. HIRSCH P. F.. GAUTHIERG. F. & M~!NSONP. L. (1963) Thyroid hypocalcemic principle and recurrent laryngeal nerve injury as factors affecting the response to parathyroidectomy. Endocrinology 73, 244252. MOSELEYJ. M., MATHEWS,E. W., BREEDR. H., GALANTE L., TSE A. & MACINTYREI. (1968) The ultimobranchial origin of calcitonin. hrncct i, 108-l 10. MODEN S. VAN & PEARSEA. G. E. (1971) Immunofluorescent localization of calcitonin in the ultimobranchial gland of Rana temporaria and Rana pipirns. Histochemie
Ackno&dgements-The present study was in part sup ported by grants-in-aid from the Ministry of Education of Japan (Nos 274241, 334043). The authors are indebted to Drs Minoru Uchiyama and Masayoshi Yoshihara for their
ROBERTSON D. R. (1969b) The ultimobranchial body of Rana pipiens. IX. Eflects of extirpation and transplantation on urine calcium excretion. Endocrinology 48,
valuable
ROBERTSOND. R. (1972) Influence of the parathyroid glands and ultimobranchial glands in the frog (Rana pipiens) during respiratory acidosis. Gm. camp. Endocr.
26,95S97.
OWJROC. & UCHIYAMA M. (1980) Comparative
endocrinology of hypocalcemic regulation in lower vertebrates. In Hormones, A4apration and Erolurion (Edited by ISHI~S. et al.), pp. 113- 121. Japan Sci. Sot. Press. Tokyo/ Springer-Verlag, Berlin. ORIMOH., OHTA M., FUJITAT., YOSHIKAWA M., HIGASHI T., ABEJ., WATANABE S. & OTANI K. (1972) Ultimobranchial calcitonin of Anguilla japonica. In Endocrinology, 1971 (Edited by TAYLOR S.), pp. 48-54. Heineman, London. RORERTSON D. R, (1969a) The ultimobranchial body of Rana pjpi~~~is. VIII. Effects of extirpation upon calcium distribution and bone cell types. Gm. camp. Endocr. 12, 419-490.
advice.
1174-l
178.
suppl. 3, 421-429.
REFERENCES
CLARKN. B. (1968) Calcitonin studies in turtles. ~~docrinology 83, 1145-1148. COLEMANR. (1975) The development and fine structure of ultimobranchial glands in larval anurans. Cell 7%~. Res. 164, 215-232. COLEMAN R. & PHILLIPSA. D. (1974) The development and fine structure of the ultimobranchial glands in larval Rana temporaria L. Cell 7% Res. 148, 69-82. COPP D. C., CAMERON E. C., CHENEYB. A., DAVIDSONA. G. F. & HENZEK. G. (1962) Evidence for calcitonin-a
SASAYAMA Y. (1978) EfTects of implantation of the ultimobranchial glands and the administration of synthetic salmon calcitonin on serum Ca concentrations in ultimobranchialectomized bullfrog tadpoles. Cm. camp. Endocr. 34, 229-233.
SASAYAMA Y. & OGL!ROC. (1976) Etrects of ultimobranchialectomy on calcium and sodium concentrations of serum and coelomic fluid in bullfrog tadpoles under high calcium and high sodium environment. Camp. Biochem. Ph.wiol.
SSA, 35 -37.
UCH~YAMA M.. YOSHIHARA M., MURAKAMIT. & OG~IROC. (1978) Presence of a hypocalcemic factor in the ultimobranchial gland of the snake. GP~. con2~. Errtfocr. 36, 59 -62.
Phgsd
Vol.
68A,
pp
031%9629/81!0101-oO95802.C0/0
95 lo 97
Press Ltd1981. Printed !nGreatBritain
HYPOCALCEMIC GLAND
FACTOR IN THE ULTIMOBRANCHIAL OF THE FROG, RANA RUGOSA
CHITARU OGURO, KEN-ICHI NAGAI, HIDEO TARUI and YUICHI SASAYAMA Department
of Biology, Faculty of Science, Toyama Toyama 930, Japan (Rr~ric~d
University,
6 May 1980)
Abstract-l. The hypocalcemic activity of the ultimobranchial gland of the frog, Rana rugosa, was estimated using a rat bioassay method. 2. Extracts of the ultimobranchial gland showed a very high hypocalcemic activity. The value corresponded to 6,340 mU (MRC)/kg b.w. 3. Serum inorganic phosphorus values of rats received the extract decreased in proportion to the dose, although no changes were found in serum sodium concentration.
INTRODUCTION
ultimobranchial glands which are attached to the glottic constrictor muscles. They could be removed through cuts made on the buccal floor at both sides of the glottis. Before the removal of the ultimobranchial glands, the frogs were put in ice cubes for anesthesia and then pithed. The mouth was kept open and the ultimobranchial glands were removed through the mouth. The ultimobranchial glands removed were homogenized in a Potter homogenizer and then sonicated in 0.6% NaCl solution adjusted to pH 4.6 with HCl. The homogenate was centrifuged at 12,000 rpm for 10 min. The supernatant was used for the assay using male Wister rats aged 5 weeks which fasted overnight before the experiments. After being anesthetized with ether, they were fixed dorsally on a board and cannulated with polyethylene tubings in the iliac vein and the iliac artery. The former was used for the administration of the extract and the latter was the path for obtaining blood samples. Each assay rat received 0.4 ml of the extract, containing 0.5. 1, 2 or 4 ultimobranchial glands or the same amount of control saline. For comparison, 10 or 1OOmU (MRC) of synthetic salmon calcitonin dissolved in the same amount of saline was administered. Blood samples were obtained at intervals of 0.5, 1, 2 and 3 hr after the administration. Serum was separated in a microhematocrit centrifuge and calcium and sodium concentrations were determined by atomic absorption spectrophotometry. Inorganic phosphorus was measured according to the method of Fiske & Subbarow (1925).
Since Copp & Hirsch found a hypocalcemic factor in mammals and named it (thyro)calcitonin (Copp et al., 1962; Hirsch et al., 1963), numerous papers have been presented on calcitonin not only for its physiological effect on mammals but also for its biochemical nature. However, the physiological and biological significance of calcitonin is still not completely understood. Hirsch, who was one of the discoverers of this principle, presented an idea that the function of calcitonin does not directly involve bones or even calcium (Hirsch, 1971). In lower vertebrates, much needs to be clarified in the function of calcitonin, as pointed out recently (Oguro & Uchiyama, 1980). In non-mammalian vertebrates, the ultimobranchial glands are the site of calcitonin secretion. Histological and immunological similarities of cellular granules in some anuran ultimobranchial glands to the granules in mammalian C cells have been suggested (Coleman & Phillips, 1974; Coleman, 1975; van Noorden & Pearse, 1971). Robertson studied intensively on the function of the ultimobranchial gland of Rana p&ens and put forth the importance of the ultimobranchial gland in the regulation of calcium in this species (Robertson, 1969a,b, 1972). However, only one paper is available for showing the presence of calcitonin activity in the ultimobranchial gland of anuran amphibians (Copp & Parkes, 1968). They found very low hypocalcemic activity in the ultimobranchial gland of the bullfrog, Ram cates-
RESULTS
Ultimobranchial extracts of R. rugosa and salmon calcitonin caused a hypocalcemic response in the rats. Serum calcium concentrations in rats received the extract equivalent to four ultimobranchial glands were 2.5 mg/lOOml and 1.3 mg/lOOml lower than those in their respective controls at 30 min and 1 hr after the administration, respectively. The former corresponds to 22.1:/, decrease from the initial value. Serum calcium concentration in rats received one and two ultimobranchial gland extracts showed 16.8 and 20.2”/:, decreases from their respective initial value at 1 hr after the administration, respectively. Maximum
beiana.
In recent studies with the rat bioassay, a high hypocalcemic activity in terms of body weight was found in the ultimobranchial gland of the frog, Rana rugosa. The following is a brief description of the results. MATERIALS
AND
METHODS
Adults of the frog, Rana rugosa Schlegel, were collected in ditches in the suburbs of Toyama City during the summer and autumn. The present species bears a pair of the 95
CHITARUOGURO et al.
96
40
-40 t
t 1 0
I 0.5
I I
2
Hr after
injection
I 3
I 0
1 0.5
ti I
Hr after
2
1 3
injection
Fig. 1. Changes of serum calcium concentration in rats following administration of control saline (A), extracts of 1 (o), 2 (0) and 4 (0) ultimobranchial glands of R. rugosa, and 10 (0) and 100 (W) mU of salmon calcitonin. Values are shown by o/odifference from the respective initial value. Fig. 2. Changes of serum inorganic phosphorus concentration in rats following administration of control saline (A), extracts of 1 (o), 2 (8) and 4 (0) ultimobranchial glands of R. rugosa, and 10 (0) and 100 (B) mU of salmon calcitonin. Values are shown by ‘A difference from the respective initial value.
decreases caused by 10mU and 1OOmU of salmon calcitonin were 2.0 and 3.2 mg/lOO ml from their respective control values, respectively. The former corresponds to 18.9% decrease and the latter 30.4% decrease from their respective initial values. The maximum decreases caused by ultimobranchial extracts and salmon calcitonin were nearly proportional to the dose. The results are shown in Fig. 1. Comparison of the responses caused by ultimobranchial extracts to those caused by salmon calcitonin showed that the activity of one ultimobranchial gland of R. rugosa corresponds to 26 mU of salmon calcitonin. Since average body weight of the frogs used was 8.2 g, 52 mU per individual can be read 6,340 mu/kg b.w. Administration of ultimobranchial extracts and salmon calcitonin caused prominent decreases in serum phosphorus concentration. Serum phosphorus level in rats received four ultimobranchial gland extract was 2.7mg/lOOml lower than that of the control level. This was 21.2% decrease from the initial value. The magnitudes caused by ultimobranchial extracts and salmon calcitonin were nearly proportional to the dose. The results are shown in Fig. 2.
, 0
. 0.5
* I
I
2
3
Hr after injection Fig. 3. Changes of serum sodium concentrations in rats following administration of control saline (A), extracts of 1 (o), 2 (0) and 4 (0) ultimobranchial glands of R. rugosa, and 10 (0) and 100 m) mU of salmon calcitonin. Values are shown o/0difference from the respective initial value.
No changes were observed in serum sodium concentrations after the administration of ultimobranchial extracts, salmon calcitonin and control saline, although small fluctuations were found irrespective of the dose (Fig. 3). Hematocrit values decreased gradually in all rats used. This might be caused by repeated bleeding. DISCUSSION
It was reported in Ram pipiens that removal of the ultimobranchial glands resulted in increased osteolytic activity ‘and urinary calcium excretion and decreased calcium deposition in lymphatic sacs (Robertson, 1969a,b, 1972). These facts suggest that the ultimobranchial gland is directly involving in calcium regulation in anuran amphibians. Serum calcium concentration increased markedly when ultimobranchialectomized bullfrog tadpoles were kept in high calcium water. However, implantations of the ultimobranchial glands of the bullfrog tadpoles or of adult Ram nigromaculata into ultimobranchialectomized bullfrog tadpoles kept in high calcium water prevented the rise of serum calcium concentration (Sasayama & Oguro, 1976). Furthermore, injection of salmon calcitonin brought about similar results (Sasayama, 1978). These findings suggest the presence of a factor which may be a calcitonin and inhibits serum calcium increase in the ultimobranchial gland of anuran amphibians. Histological studies on frog ultimobranchial glands showed the presence of cellular granules which resemble those found in the C cell in mammals (Coleman & Phillips, 1974; Coleman, 1975). Furthermore, occurrence of a substance which crossreacts with antiportine calcitonin antibodies was reported in the ultimobranchial glands in two species of Raw (van Nooden & Pearse, 1971). In fact, Copp and Parkes reported calcitonin ac-
H~pocalcemic factor in frog UBG
97
tivity in ultimobranchial extracts of R. catesbeiana (Copp & Parkes, 1968). The activity reported was l-2mU/kg b.w. This value is extremely lpw in comparison with the activities found in mammals, birds, most reptiles and fishes (Clark, 1968; Copp ef ai., 1967; Copp & Parkes, 1968; Moseley ef al., 1968; Uchiyama et al., 1978). In a preliminary study with the frog, R. nigromaculata, the authors found that ultimobranchial extracts showed a hypocalcemic activity which corresponds to the activity elicited by several hundreds milliunits of salmon calcitonin (Oguro & Uchiyama, 1980). In the present study, a very high hypocalcemic activity (6,340mUjkg b.w.) was found in the ultimobranchiai gland of R. rugosa. This marks a prominent contrast to the very low hypocalcemic activity in the ultimobranchial gland of R. catesbeiana (Copp & Parkes, 1968). No animals have been known for such a high hypocalcemic activity in the ultimobranchial gland except the eel (Orimo et al., 1972). The activity found in the eel ultimobranchial gland was reported to be about, 30,000 mu/kg b.w. The function of the ultimobranchial gland of R. rugosa has not been studied. However, in related preliminary studies using R. nigromaculata, it was found that administration of ultimobranchial extracts caused a hypocalcemic response while serum sodium concentration was not affected (Oguro & Uchiyama, 1980). These findings may suggest that the hypocalcemic factor effective on rats is involving in serum calcium regulation in frogs. Very high hypocalcemic activity found in R. rugosa inspires further studies on the function of the ultimobranchial gland in the present species.
new hormone from the parathyroid that lowers blood calcium. Endocrinology 70, 638-649. COPP D. H., COCKCROFT D. W. & KUEH Y. (1967) Calcitonin from ultimobranchial glands of dogfish and chickens. Sciencr 158.924-925. COPP D. H. & PARKESC. 0. (1968) Extraction of calcitonin from ultimobranchial tissue. In Parar~~ra~d Hormone and Th~rorafcitonin (Calcironin) (Edited by TALMAGE,R. V. & B~LANGERL. F.), pp. 74-84. Excerpta Medica, Amsterdam. FISKEC. H. & SURRAROW Y. (1925) The calorimetric determination of phosphorus. J. Biol. Chem. 66, 375400. HIRSCHP. F. (1971) Thyrocalcitonin and its role in calcium regulation in mammals. f. PX~. Zoo!. 178, 139-149. HIRSCH P. F.. GAUTHIERG. F. & M~!NSONP. L. (1963) Thyroid hypocalcemic principle and recurrent laryngeal nerve injury as factors affecting the response to parathyroidectomy. Endocrinology 73, 244252. MOSELEYJ. M., MATHEWS,E. W., BREEDR. H., GALANTE L., TSE A. & MACINTYREI. (1968) The ultimobranchial origin of calcitonin. hrncct i, 108-l 10. MODEN S. VAN & PEARSEA. G. E. (1971) Immunofluorescent localization of calcitonin in the ultimobranchial gland of Rana temporaria and Rana pipirns. Histochemie
Ackno&dgements-The present study was in part sup ported by grants-in-aid from the Ministry of Education of Japan (Nos 274241, 334043). The authors are indebted to Drs Minoru Uchiyama and Masayoshi Yoshihara for their
ROBERTSON D. R. (1969b) The ultimobranchial body of Rana pipiens. IX. Eflects of extirpation and transplantation on urine calcium excretion. Endocrinology 48,
valuable
ROBERTSOND. R. (1972) Influence of the parathyroid glands and ultimobranchial glands in the frog (Rana pipiens) during respiratory acidosis. Gm. camp. Endocr.
26,95S97.
OWJROC. & UCHIYAMA M. (1980) Comparative
endocrinology of hypocalcemic regulation in lower vertebrates. In Hormones, A4apration and Erolurion (Edited by ISHI~S. et al.), pp. 113- 121. Japan Sci. Sot. Press. Tokyo/ Springer-Verlag, Berlin. ORIMOH., OHTA M., FUJITAT., YOSHIKAWA M., HIGASHI T., ABEJ., WATANABE S. & OTANI K. (1972) Ultimobranchial calcitonin of Anguilla japonica. In Endocrinology, 1971 (Edited by TAYLOR S.), pp. 48-54. Heineman, London. RORERTSON D. R, (1969a) The ultimobranchial body of Rana pjpi~~~is. VIII. Effects of extirpation upon calcium distribution and bone cell types. Gm. camp. Endocr. 12, 419-490.
advice.
1174-l
178.
suppl. 3, 421-429.
REFERENCES
CLARKN. B. (1968) Calcitonin studies in turtles. ~~docrinology 83, 1145-1148. COLEMANR. (1975) The development and fine structure of ultimobranchial glands in larval anurans. Cell 7%~. Res. 164, 215-232. COLEMAN R. & PHILLIPSA. D. (1974) The development and fine structure of the ultimobranchial glands in larval Rana temporaria L. Cell 7% Res. 148, 69-82. COPP D. C., CAMERON E. C., CHENEYB. A., DAVIDSONA. G. F. & HENZEK. G. (1962) Evidence for calcitonin-a
SASAYAMA Y. (1978) EfTects of implantation of the ultimobranchial glands and the administration of synthetic salmon calcitonin on serum Ca concentrations in ultimobranchialectomized bullfrog tadpoles. Cm. camp. Endocr. 34, 229-233.
SASAYAMA Y. & OGL!ROC. (1976) Etrects of ultimobranchialectomy on calcium and sodium concentrations of serum and coelomic fluid in bullfrog tadpoles under high calcium and high sodium environment. Camp. Biochem. Ph.wiol.
SSA, 35 -37.
UCH~YAMA M.. YOSHIHARA M., MURAKAMIT. & OG~IROC. (1978) Presence of a hypocalcemic factor in the ultimobranchial gland of the snake. GP~. con2~. Errtfocr. 36, 59 -62.