GENERAL
AND
COMPARATIVE
involvement
KOUHEI
ENDOCRINOLOGY
81,442446
(1!?91)
of Prolactin in the Regulation of Plasma Callcium Levels in the Newt, Cynops pyrrhogaster
MATSUDA,
CHITARU
OGURO,* YUICHI
SASAYAMA,*
AND SAKAE KIKUYAMA’
Department of Biology, School of Education, Waseda University, Shinjuku-ku, Tokyo 169, Japan; and *Department of Biology, Faculty of Science, Toyama University, Toyama 930, Japan Accepted May 2, 1990 In the newt, Cynops pyrrhogaster, parathyroidectomy (PTX) brought about a marked decrease in the concentration of plasma calcium. The animals recovered from the hypocalcemia by 15 days after the operation if the pituitary gland was left intact. After PTX, no significant changes in the plasma sodium concentration were observed. Experiments were then conducted to obtain direct evidence that endogenous prolactin (PRL) is involved in this recovery process. Recovery of the calcium level after PTX was blocked by administration of an antiserum raised against newt PRL. In newts deprived of both the pituitary and parathyroid glands, no recovery from hypocalcemia was observed. Administration of newt or ovine PRL to parathyroidectomized-hypophysectomized newts significantly elevated the blood calcium level. After PTX, the concentration of immunoassayable PRL in the blood rose to 10 times the value in sham-operated animals. These results indicate the involvement of PRL in calcium homeostasis in newts with a shortage of parathormone. 0 1991 Academic press, Inc.
It has been generally accepted that the parathyroid gland is one of the principal organs responsible for regulation of blood calcium levels in tetrapod animals (Hurwitz, 1989). Amphibians were probably the first animals to evolve parathyroid glands (Pang et al., 1980). In urodeles (Oguro and Uchiyama, 1975) as well as anurans (Oguro et al., 1975), parathyroidectomy (PTX) produces a marked decline of blood calcium levels. However, there are some exceptions. In Megalobatrachus davidianus and Hynobius nigrescens, PTX has no effect. In contrast, hypophysectomy (HX) in the latter species and in several other urodeles (Cryptobranchus alleganiensis and Necturus maculosus), which lack parathyroid glands, is known to elicit hypocalcemia (Oguro and Uchiyama, 1975; Oguro et al., 1978). In the newts Cynops pyrrhogaster and Tylototriton andersoni, a marked recovery from hypocalcemia after PTX is ob’ To whom all correspondence should be addressed.
served (Oguro and Uchiyama, 1975; Oguro and Sasayama, 1978). This recovery, however, is blocked by HX (Oguro et al., 1978). Thus, the participation of pituitary hormone(s) in the homeostatic control of plasma calcium concentration in amphibians has been postulated (see Wendelaar Bonga and Pang, 1989). At present, very little information is available on the control of calcium levels by pituitary hormones in amphibians. Only one previous study found that injections of mammalian prolactin (PRL) corrected hypocalcemia resulting from the hypophysectomy in the urodele, C. alleganiensis (Oguro et al., 1978). Recently, we have obtained a purified newt PRL and an antiserum against it (Matsuda et al., 199Oa, b). Using the homologous hormone and its antiserum, experiments were conducted to obtain definite evidence that endogenous PRL is involved in calcium homeostasis in the newt. Part of this work has been reported in abstract form (Matsuda et al., 1989). 442
0016~6480/91 $1.50 Copyright All rights
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
PRL
MATERIALS
AND
CALCIUM
HOMEOSTASIS
3.21
AND METHODS
Animals. Adult males of the newt, C. pyrrhogaster (5-7 g body wt), were captured in July in the suburbs of Murakami city, Niigata prefecture. After 1-2 weeks of adaptation to laboratory conditions, they were used for the experiments. No food was offered to the animals during the experiments. PTX, HX, and sham operations were performed under anesthesia with MS222. Treatment
Newts deprived of the hypophysis and parathyroid glands received injections of 2 lxg of newt PRL (Matsuda et al., l!@Oa), ovine PRL (NIH, NIADDK-oPRL-18) dissolved in 50 l.~l of saline containing 0.1% BSA, or vehicle only every other day for 15 days (eight injections), commencing on the day after the operation. Parathyroidectomized newts also received injections of 50 J of rabbit antiserum against newt PRL or normal rabbit serum (NRS) every other day for 15 days. Two hours after the last injection, blood was collected from the hearts of individual animals into heparinized capillaries under anesthesia with MS-222. Plasma samples were prepared by centrifugation at 3,000 rpm for 10 min. Measurement concentrations.
with PRL
and its antiserum.
of plasma
calcium,
sodium,
IN
and PRL
Calcium and sodium concentrations in the plasma samples obtained from every experimental animal were measured by an atomic absorption photometer. Plasma samples from parathyroidectomized newts and newts given a sham operation were prepared 0, 5, 10, and 15 days after the operation. They were subjected to homologous radioimmunoassay according to the procedure described elsewhere (Matsuda et al., 19!4Ob).
NEWT
443
A
2.8
% -
2.6
S24
. 2.2
,100, E \, 80, 4 60, g 40. 01
RESULTS
FIG. 1. Effects of PTX on plasma calcium (A), sodium (B) and PRL (C) concentrations in the newt. Each point and vertical bar represents the mean of 10 determinations and SEM, respectively. Clear circles, sham operation; solid circles, PTX. *Significantly different from the value in the sham-operated group at the 5% level by Student’s I test.
PTX in the newt caused a significant decrease in plasma calcium concentration 5 days after the operation. A gradual recovery from the hypocalcemia was observed thereafter. Fifteen days after surgery, the levels were close to those in the shamoperated animals (Fig. 1A). On the other hand, plasma sodium levels were not affected by PTX (Fig. 1B). Plasma PRL levels increased gradually during the first 10 days and rose sharply thereafter (Fig. 1C). In the parathyroidectomized newts treated with antiserum against newt PRL, no recovery from hypocalcemia was observed, and the average calcium level remained considerably low when measured 15 days
after the operation. In the parathyroidectomized newts which had received injections of NRS, calcium levels were significantly higher than in the antiserum-treated animals (Fig. 2). There was no difference in plasma sodium concentrations between the NRS-injected and the antiserum-treated animals. Newts that had been deprived of both the pituitary and parathyroid glands received injections of newt PRL, ovine PRL, or saline. In the animals treated with newt PRL or ovine PRL, calcium levels were significantly higher than in the salineinjected animals (Fig. 3). The average plasma sodium concentration was higher in
444
MATSUDA
2.61
ET AL.
pocalcemia following PTX was reported by earlier workers (Oguro and Uchiyama, 2.4, 1975; Oguro et al., 1978). Administration of 2.2. -120 antiserum against PRL blocked the restoration of calcium levels. HX in addition to 32.0, .llO PTX in the newt induces severe hypocalced mia (Oguro et al., 1978). In this case, no 3.6, .lOOH recovery from the hypocalcemic state oc9 1.6 ,90 5 curs. In the present experiment, we demonstrated that administration of newt or 1.4 .60 ovine PRL to hypophysectomized-para1.2-70 thyroidectomized newts induced an elevaNRS AntinPRL tion of calcium levels. Moreover, our radioimmunoassay results indicated that PRL FIG. 2. Plasma calcium (0) and sodium (U) concentrations of parathyroidectomized newts treated with levels were markedly elevated by 15 days NRS or antiserum against newt (n) PRL. Each column after PTX. Accordingly, we conclude that and vertical bar represents the mean of nine determithe secreted PRL contributes to the recovnations and SEM, respectively. a vs b significantly ery of calcium levels. However, the mechdifferent at the 5% level by Student’s t test. anism by which PRL levels are elevated following PTX remains to be clarified. the prolactin-treated group than in the conIn the present experiment, PRL administrol group, but the difference was not sigtered to the hypophysectomized-parathyn&ant. roidectomized newts restored the calcium levels considerably but not completely. The DISCUSSION dose of newt PRL was comparable to that of the same PRL preparation which was efThe present study is the first to demontransepistrate that endogenous PRL is involved in fective in reducing integumental thelial potential in the eft, Notophthaimus the regulation of plasma calcium levels in amphibians. In the newt, recovery from hy- viridescens. (Brown et al., 1991). It is probable that some pituitary factor(s) other than PRL is also required for complete recov120 2.2 ery. In this respect, it is of interest to note that in the rat, growth hormone stimulates 2.a 110 intestinal calcium uptake as PRL does (Yeh and Aloia, 1984) and that in the fish, PRL, s 1.8 100 f ACTH, and a putative hormone produced .E 2 by the PAS-positive cells of the pars inter; 1.6 a0 d media (Olivereau et al., 1986) have been considered as candidates for the hypercal1.4 BO cemic action of the pituitary gland (see Wendelaar Bonga and Pang, 1989). 1.2 70 Saline The present experiment was carried out FIG. 3. Plasma calcium (0) and sodium (D) concenin July and August, when the basal PRL trations of parathyroidectomized-hypophysectomized levels are considerably lower than those of newts injected with saline, 2 pg newt (n) PRL, or 2 pg the same animals in March, as used in the ovine (0) PRL. Each column and vertical bar repreexperiment (Matsuda et al., sents the mean of 6-10 determinations and SEM, re- previous spectively. a vs b and a vs c significantly different at 199Ob). This seems to be due to seasonal fluctuation. We have noticed that PRL levthe 5% level by Duncan’s multiple range test. I La
d
PRL AND CALCIUM
HOMEOSTASIS
els in the male newt are relatively high in early spring, whereas the levels in summer are low and rather constant (Matsuda et al., unpublished data). In urodeles such as N. maculosus and C. alleganiensis, which lack parathyroid glands (Oguro et al., 1978), and H. nigrescens, in which the parathyroid glands are not important for the regulation of calcium concentration (Oguro and Uchiyama, 1973, HX causes a decline of plasma calcium levels. Administration of mammalian PRL to hypophysectomized C. alleganiensis restores the calcium level to normal (Oguro et al., 1978). Injection of mammalian PRL also corrects the decreased calcium concentration in the plasma of hypophysectomized N. maculosus (Pang, unpublished data). In these species, PRL secreted from the pituitary gland might be the principal factor for the maintenance of plasma calcium levels. In teleosts, which have no parathyroid glands, plasma calcium levels may also be controlled by PRL (see Wendelaar Bonga and Pang, 1989). In the present experiment as well as a previous one (Oguro and Uchiyama, 1973, PTX in the newt (C. pyrrhogaster) caused transient hypocalcemia but did not affect the plasma sodium concentration. On the other hand, the decrease of plasma calcium due to HX was less pronounced than the decrease of sodium (Oguro and Uchiyama, 1975). In the newt, therefore, parathormone seems to play a major role in calcium homeostasis. However, once these animals are confronted with a shortage of parathormone, hypersecretion of PRL occurs, and this acts to restore the reduced calcium levels. In teleosts, PRL is known to act on the gill to stimulate high-affinity Ca2+-ATPase (Flik et al., 1986). This enzyme activity is regarded as the driving force for transmembrane calcium transport (Flik et al., 1985a, b). In mammals, 1,25-dihydroxyvitamin D,, an active form of vitamin D, enhances intestinal and renal absorption of calcium
IN NEWT
445
ions (see Laverty and Clark, 1989). The stimulatory action of PRL on calcium absorption has been reported to be mediated by vitamin D (Robinson et al., 1982), although a vitamin D-independent mechanism of action also seems to exist (Pahuja and DeLuca, 1981). The mechanism of the calcium-homeostatic action of PRL in amphibians remains to be clarified. ACKNOWLEDGMENTS Ovine PRL (NIADDK-oPRL-18) was kindly supplied by the National Institute of Health. The authors appreciate the advice and interest of Dr. K. Yamamoto of Waseda University and Dr. S. Tanaka of Gunma University. Part of this study was done at the Ocean Research Institute, University of Tokyo. This work was supported by a Grant-in-Aid from the Ministry of Education, and a grant from Waseda University to SK.
REFERENCES Brown, S. C., Brown, P. S., Yamamoto, K., Matsuda, K., and Kikuyama, S. (1991). Amphibian prolactin: Activity in the eft skin TEP bioassay. Gen. Comp. Endocrinol., in press. Flik, G., Fenwick, J. C., Kolar, Z., Mayer-Gostan, N., and Wendelaar Bonga, S. E. (1986). Effects of ovine prolactin on calcium uptake and distribution in the freshwater cichlid teleost fish, Oreochromis mossambicus. Amer. .Z. Physiol. 250, Rl61-R166. Flik, G., van Rijs, J. H., and Wendelaar Bonga, S. E. (1985a). Evidence for high-affinity Ca*+-ATPase activity and ATP-driven Ca*+ transport in membrane preparations of the gill epithelium of the cichlid fish, Oreochromis mossambicus. J. Exp. Biol. 119, 335-347. Flik, G., Wendelaar Bonga, S. E., and Fenwick, J. C. (1985b). Active Ca*+ transport in plasma membranes of branchial epithelium of the North American eel, Anguilla rostrata LeSueur. Biol. Cell 55, 265-272.
Hurwitz, S. (1989). Parathyroid hormone. In “Vertebrate Endocrinology: Fundamentals and Biomedical Implications” (P. K. T. Pang and hi. P. Schreibman, Eds.), pp. 45-77. Academic Press, San Diego. Laverty, G., and Clark, N. B. (1989). The kidney. In “Vertebrate Endocrinology: Fundamentals and Biomedical Implications” (P. K. T. Pang and M. P. Schreibman, Eds.), pp. 298-300. Academic Press, San Diego. Matsuda, K., Kikuyama, S., Sasayama, Y., and
446
MATSUDA
Oguro, C. (1989). Involvement of prolactin in the regulation of blood calcium levels in the parathyroidectomized newt. Zoof. Sci. 6, 1189. [Abstract] Matsuda, K., Yamamoto, K., and Kikuyama, S. (1990a). Purification and properties of newt prolactin. Gen. Comp. Endocrinol. II, 63-69. Matsuda, K., Yamamoto, K., and Kikuyama, S. (1990b). Development and application of homologous radioimmunoassay for newt prolactin. Gen. Comp. Endocrinol. 19, 83-88. Oguro, C., and Sasayama, Y. (1978). Function of the parathyroid gland in serum calcium regulation in the newt, Tylototriton andersoni Boulenger. Gen. Comp. Endocrinol. 35, 10-15. Oguro. C., Sasayama, Y., and Katoh, M. (1975). Occurrence of tetanic convulsions in totally parathyroidectomized frogs, Rana nigromaculata. Zoo/. Mag. 84, 262-265. Oguro, C., and Uchiyama, M. (1975). Control of serum calcium concentration by parathyroid gland in two species of urodele amphibians. Gen. Comp. Endocrinol. 21, 531-537. Oguro, C., Uchiyama, M. Pang, P. K. T., and Sasayama, Y. (1978). Serum calcium homeostasis in urodele amphibians. In “Comparative Endocrinology” (P. J. Gaillard and H. H. Boar, Eds.), pp. 269-272. Elsevier/North-Holland, Amsterdam. Olivereau, M., Cbambolle, P., Dubourg, P., and Oliv-
ET AL.
ereau, J. (1986). Cytological and ultrastructural changes in the pituitary pars distalis of the goldfish kept in calcium-free environments. Biol. Cell 57, 77-88. Pahuja, D. N., and DeLuca, H. F. (1981). Stimulation of intestinal calcium transport and bone calcium mobilization by prolactin in vitamin D-deficient rats. Science 214, 1038-1039. Pang, P. K. T., Kenny, A. D., and Oguro, C. (1980). Evolution of endocrine control of calcium regulation. In “Evolution of Vertebrate Endocrine Systems” (P. K. T. Pang and A. Epple, Eds.), pp. 327-331. Texas Tech. Press, Lubbock. Robinson, C. J., Spanos, E., James, M. F., Pike, J. W., Haussler, M. R., Makeen, A. M., Hillyard, C. J., and MacIntyre, I. (1982). Role of prolactin in vitamin D metabolism and calcium absorption during lactation in the rat. J. Endocrinok 94, 443453. Wendelaar Bonga, S. E., and Pang, P. K. T. (1989). Pituitary Hormones. In “Vertebrate Endocrinology: Fundamentals and Biomedical Implications” (P. K. T. Pang and M. P. Schreibman, Eds.), pp. 105-137. Academic Press, San Diego. Yeh, J. K., and Aloia, J. F. (1984). Effect of hypophysectomy and 1,25-dihydroxyvitamin D on duodenal calcium absorption. Endocrinology 114, 171 l1717.
AND
COMPARATIVE
involvement
KOUHEI
ENDOCRINOLOGY
81,442446
(1!?91)
of Prolactin in the Regulation of Plasma Callcium Levels in the Newt, Cynops pyrrhogaster
MATSUDA,
CHITARU
OGURO,* YUICHI
SASAYAMA,*
AND SAKAE KIKUYAMA’
Department of Biology, School of Education, Waseda University, Shinjuku-ku, Tokyo 169, Japan; and *Department of Biology, Faculty of Science, Toyama University, Toyama 930, Japan Accepted May 2, 1990 In the newt, Cynops pyrrhogaster, parathyroidectomy (PTX) brought about a marked decrease in the concentration of plasma calcium. The animals recovered from the hypocalcemia by 15 days after the operation if the pituitary gland was left intact. After PTX, no significant changes in the plasma sodium concentration were observed. Experiments were then conducted to obtain direct evidence that endogenous prolactin (PRL) is involved in this recovery process. Recovery of the calcium level after PTX was blocked by administration of an antiserum raised against newt PRL. In newts deprived of both the pituitary and parathyroid glands, no recovery from hypocalcemia was observed. Administration of newt or ovine PRL to parathyroidectomized-hypophysectomized newts significantly elevated the blood calcium level. After PTX, the concentration of immunoassayable PRL in the blood rose to 10 times the value in sham-operated animals. These results indicate the involvement of PRL in calcium homeostasis in newts with a shortage of parathormone. 0 1991 Academic press, Inc.
It has been generally accepted that the parathyroid gland is one of the principal organs responsible for regulation of blood calcium levels in tetrapod animals (Hurwitz, 1989). Amphibians were probably the first animals to evolve parathyroid glands (Pang et al., 1980). In urodeles (Oguro and Uchiyama, 1975) as well as anurans (Oguro et al., 1975), parathyroidectomy (PTX) produces a marked decline of blood calcium levels. However, there are some exceptions. In Megalobatrachus davidianus and Hynobius nigrescens, PTX has no effect. In contrast, hypophysectomy (HX) in the latter species and in several other urodeles (Cryptobranchus alleganiensis and Necturus maculosus), which lack parathyroid glands, is known to elicit hypocalcemia (Oguro and Uchiyama, 1975; Oguro et al., 1978). In the newts Cynops pyrrhogaster and Tylototriton andersoni, a marked recovery from hypocalcemia after PTX is ob’ To whom all correspondence should be addressed.
served (Oguro and Uchiyama, 1975; Oguro and Sasayama, 1978). This recovery, however, is blocked by HX (Oguro et al., 1978). Thus, the participation of pituitary hormone(s) in the homeostatic control of plasma calcium concentration in amphibians has been postulated (see Wendelaar Bonga and Pang, 1989). At present, very little information is available on the control of calcium levels by pituitary hormones in amphibians. Only one previous study found that injections of mammalian prolactin (PRL) corrected hypocalcemia resulting from the hypophysectomy in the urodele, C. alleganiensis (Oguro et al., 1978). Recently, we have obtained a purified newt PRL and an antiserum against it (Matsuda et al., 199Oa, b). Using the homologous hormone and its antiserum, experiments were conducted to obtain definite evidence that endogenous PRL is involved in calcium homeostasis in the newt. Part of this work has been reported in abstract form (Matsuda et al., 1989). 442
0016~6480/91 $1.50 Copyright All rights
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
PRL
MATERIALS
AND
CALCIUM
HOMEOSTASIS
3.21
AND METHODS
Animals. Adult males of the newt, C. pyrrhogaster (5-7 g body wt), were captured in July in the suburbs of Murakami city, Niigata prefecture. After 1-2 weeks of adaptation to laboratory conditions, they were used for the experiments. No food was offered to the animals during the experiments. PTX, HX, and sham operations were performed under anesthesia with MS222. Treatment
Newts deprived of the hypophysis and parathyroid glands received injections of 2 lxg of newt PRL (Matsuda et al., l!@Oa), ovine PRL (NIH, NIADDK-oPRL-18) dissolved in 50 l.~l of saline containing 0.1% BSA, or vehicle only every other day for 15 days (eight injections), commencing on the day after the operation. Parathyroidectomized newts also received injections of 50 J of rabbit antiserum against newt PRL or normal rabbit serum (NRS) every other day for 15 days. Two hours after the last injection, blood was collected from the hearts of individual animals into heparinized capillaries under anesthesia with MS-222. Plasma samples were prepared by centrifugation at 3,000 rpm for 10 min. Measurement concentrations.
with PRL
and its antiserum.
of plasma
calcium,
sodium,
IN
and PRL
Calcium and sodium concentrations in the plasma samples obtained from every experimental animal were measured by an atomic absorption photometer. Plasma samples from parathyroidectomized newts and newts given a sham operation were prepared 0, 5, 10, and 15 days after the operation. They were subjected to homologous radioimmunoassay according to the procedure described elsewhere (Matsuda et al., 19!4Ob).
NEWT
443
A
2.8
% -
2.6
S24
. 2.2
,100, E \, 80, 4 60, g 40. 01
RESULTS
FIG. 1. Effects of PTX on plasma calcium (A), sodium (B) and PRL (C) concentrations in the newt. Each point and vertical bar represents the mean of 10 determinations and SEM, respectively. Clear circles, sham operation; solid circles, PTX. *Significantly different from the value in the sham-operated group at the 5% level by Student’s I test.
PTX in the newt caused a significant decrease in plasma calcium concentration 5 days after the operation. A gradual recovery from the hypocalcemia was observed thereafter. Fifteen days after surgery, the levels were close to those in the shamoperated animals (Fig. 1A). On the other hand, plasma sodium levels were not affected by PTX (Fig. 1B). Plasma PRL levels increased gradually during the first 10 days and rose sharply thereafter (Fig. 1C). In the parathyroidectomized newts treated with antiserum against newt PRL, no recovery from hypocalcemia was observed, and the average calcium level remained considerably low when measured 15 days
after the operation. In the parathyroidectomized newts which had received injections of NRS, calcium levels were significantly higher than in the antiserum-treated animals (Fig. 2). There was no difference in plasma sodium concentrations between the NRS-injected and the antiserum-treated animals. Newts that had been deprived of both the pituitary and parathyroid glands received injections of newt PRL, ovine PRL, or saline. In the animals treated with newt PRL or ovine PRL, calcium levels were significantly higher than in the salineinjected animals (Fig. 3). The average plasma sodium concentration was higher in
444
MATSUDA
2.61
ET AL.
pocalcemia following PTX was reported by earlier workers (Oguro and Uchiyama, 2.4, 1975; Oguro et al., 1978). Administration of 2.2. -120 antiserum against PRL blocked the restoration of calcium levels. HX in addition to 32.0, .llO PTX in the newt induces severe hypocalced mia (Oguro et al., 1978). In this case, no 3.6, .lOOH recovery from the hypocalcemic state oc9 1.6 ,90 5 curs. In the present experiment, we demonstrated that administration of newt or 1.4 .60 ovine PRL to hypophysectomized-para1.2-70 thyroidectomized newts induced an elevaNRS AntinPRL tion of calcium levels. Moreover, our radioimmunoassay results indicated that PRL FIG. 2. Plasma calcium (0) and sodium (U) concentrations of parathyroidectomized newts treated with levels were markedly elevated by 15 days NRS or antiserum against newt (n) PRL. Each column after PTX. Accordingly, we conclude that and vertical bar represents the mean of nine determithe secreted PRL contributes to the recovnations and SEM, respectively. a vs b significantly ery of calcium levels. However, the mechdifferent at the 5% level by Student’s t test. anism by which PRL levels are elevated following PTX remains to be clarified. the prolactin-treated group than in the conIn the present experiment, PRL administrol group, but the difference was not sigtered to the hypophysectomized-parathyn&ant. roidectomized newts restored the calcium levels considerably but not completely. The DISCUSSION dose of newt PRL was comparable to that of the same PRL preparation which was efThe present study is the first to demontransepistrate that endogenous PRL is involved in fective in reducing integumental thelial potential in the eft, Notophthaimus the regulation of plasma calcium levels in amphibians. In the newt, recovery from hy- viridescens. (Brown et al., 1991). It is probable that some pituitary factor(s) other than PRL is also required for complete recov120 2.2 ery. In this respect, it is of interest to note that in the rat, growth hormone stimulates 2.a 110 intestinal calcium uptake as PRL does (Yeh and Aloia, 1984) and that in the fish, PRL, s 1.8 100 f ACTH, and a putative hormone produced .E 2 by the PAS-positive cells of the pars inter; 1.6 a0 d media (Olivereau et al., 1986) have been considered as candidates for the hypercal1.4 BO cemic action of the pituitary gland (see Wendelaar Bonga and Pang, 1989). 1.2 70 Saline The present experiment was carried out FIG. 3. Plasma calcium (0) and sodium (D) concenin July and August, when the basal PRL trations of parathyroidectomized-hypophysectomized levels are considerably lower than those of newts injected with saline, 2 pg newt (n) PRL, or 2 pg the same animals in March, as used in the ovine (0) PRL. Each column and vertical bar repreexperiment (Matsuda et al., sents the mean of 6-10 determinations and SEM, re- previous spectively. a vs b and a vs c significantly different at 199Ob). This seems to be due to seasonal fluctuation. We have noticed that PRL levthe 5% level by Duncan’s multiple range test. I La
d
PRL AND CALCIUM
HOMEOSTASIS
els in the male newt are relatively high in early spring, whereas the levels in summer are low and rather constant (Matsuda et al., unpublished data). In urodeles such as N. maculosus and C. alleganiensis, which lack parathyroid glands (Oguro et al., 1978), and H. nigrescens, in which the parathyroid glands are not important for the regulation of calcium concentration (Oguro and Uchiyama, 1973, HX causes a decline of plasma calcium levels. Administration of mammalian PRL to hypophysectomized C. alleganiensis restores the calcium level to normal (Oguro et al., 1978). Injection of mammalian PRL also corrects the decreased calcium concentration in the plasma of hypophysectomized N. maculosus (Pang, unpublished data). In these species, PRL secreted from the pituitary gland might be the principal factor for the maintenance of plasma calcium levels. In teleosts, which have no parathyroid glands, plasma calcium levels may also be controlled by PRL (see Wendelaar Bonga and Pang, 1989). In the present experiment as well as a previous one (Oguro and Uchiyama, 1973, PTX in the newt (C. pyrrhogaster) caused transient hypocalcemia but did not affect the plasma sodium concentration. On the other hand, the decrease of plasma calcium due to HX was less pronounced than the decrease of sodium (Oguro and Uchiyama, 1975). In the newt, therefore, parathormone seems to play a major role in calcium homeostasis. However, once these animals are confronted with a shortage of parathormone, hypersecretion of PRL occurs, and this acts to restore the reduced calcium levels. In teleosts, PRL is known to act on the gill to stimulate high-affinity Ca2+-ATPase (Flik et al., 1986). This enzyme activity is regarded as the driving force for transmembrane calcium transport (Flik et al., 1985a, b). In mammals, 1,25-dihydroxyvitamin D,, an active form of vitamin D, enhances intestinal and renal absorption of calcium
IN NEWT
445
ions (see Laverty and Clark, 1989). The stimulatory action of PRL on calcium absorption has been reported to be mediated by vitamin D (Robinson et al., 1982), although a vitamin D-independent mechanism of action also seems to exist (Pahuja and DeLuca, 1981). The mechanism of the calcium-homeostatic action of PRL in amphibians remains to be clarified. ACKNOWLEDGMENTS Ovine PRL (NIADDK-oPRL-18) was kindly supplied by the National Institute of Health. The authors appreciate the advice and interest of Dr. K. Yamamoto of Waseda University and Dr. S. Tanaka of Gunma University. Part of this study was done at the Ocean Research Institute, University of Tokyo. This work was supported by a Grant-in-Aid from the Ministry of Education, and a grant from Waseda University to SK.
REFERENCES Brown, S. C., Brown, P. S., Yamamoto, K., Matsuda, K., and Kikuyama, S. (1991). Amphibian prolactin: Activity in the eft skin TEP bioassay. Gen. Comp. Endocrinol., in press. Flik, G., Fenwick, J. C., Kolar, Z., Mayer-Gostan, N., and Wendelaar Bonga, S. E. (1986). Effects of ovine prolactin on calcium uptake and distribution in the freshwater cichlid teleost fish, Oreochromis mossambicus. Amer. .Z. Physiol. 250, Rl61-R166. Flik, G., van Rijs, J. H., and Wendelaar Bonga, S. E. (1985a). Evidence for high-affinity Ca*+-ATPase activity and ATP-driven Ca*+ transport in membrane preparations of the gill epithelium of the cichlid fish, Oreochromis mossambicus. J. Exp. Biol. 119, 335-347. Flik, G., Wendelaar Bonga, S. E., and Fenwick, J. C. (1985b). Active Ca*+ transport in plasma membranes of branchial epithelium of the North American eel, Anguilla rostrata LeSueur. Biol. Cell 55, 265-272.
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