Translator Disclaimer
1 September 2002 Direct Raises in Blood Ca Levels by Infusing a High-Ca Solution into the Blood Stream Accelerate the Secretion of Calcitonin from the Ultimobranchial Gland in Eels
Author Affiliations +
Abstract

In eels, a CaCl2 solution was infused into the pneumatic duct vein. Plasma Ca levels were significantly increased during 3 hr and were followed by significant raises in plasma calcitonin levels. These results strongly suggest that, in eels, direct raises in blood Ca levels by infusion of a high-Ca solution via blood vessels can accelerate the secretion of calcitonin from the ultimobranchial gland.

INTRODUCTION

Calcitonin is a peptide hormone that is secreted from the C-cells of the thyroid gland in mammals or from the ultimobranchial gland in most vertebrates. In mammals, this hormone suppresses the activity of osteoclasts and decreases the mobilization of Ca from bones. As a result, blood Ca levels decline, and the hardness of bones is maintained (Azria, 1989).

On the other hand, except for anuran amphibians (Robertson 1971, 1988), the function of calcitonin is not clear in lower vertebrates (Chan et al., 1968; Hayslet et al., 1971; Sasayama, 1999). In the past, however, we suggested that, in chum salmon, calcitonin might work at early stages of body growth because the ultimobranchial gland in the fry before hatching was already immunostained with anti-calcitonin antiserum (Sasayama et al., 1989).

Recently, it was determined that plasma Ca levels and calcitonin levels in normally fed eels were significantly higher than those in starved eels (Sasayama et al., 1996). When a high-Ca solution or saline solution was infused into the stomach in goldfish, plasma Ca levels were significantly elevated only in the high-Ca group (Sasayama et al., 1996). In addition, the number of individuals with a high level of plasma calcitonin was significantly larger in the high-Ca group than in the saline group. Furthermore, we repeated a similar experiment in eels, in which plasma calcitonin levels in individuals administered a high-Ca solution into the stomach were significantly raised slightly after the elevation of plasma Ca levels (Suzuki et al., 1999). This result also demonstrates that, in teleosts, the ultimobranchial gland secretes calcitonin when blood Ca levels are raised. Taking these all results into consideration, it is strongly suggested that, in teleosts, calcitonin plays a physiological role on daily occurrences such as suppressing transitory hypercalcemia arising from feeding.

In teleosts, however, all evidence that we have submitted so far has been obtained by infusing Ca into the digestive tract. It is known that, in mammals, some gut hormones affect the secretion of calcitonin on the processes of digestion (Azria, 1989; Person et al., 1988; Sethi et al., 1983). For example, gastrin, CCK, glucagons, and secretin all accelerate the secretion of calcitonin. Therefore, when a high-Ca solution was infused into the stomach, it was not clear whether some gut hormones brought about the secretion of calcitonin from the ultimobranchial gland or the elevation of blood Ca levels did so directly.

The purpose of this study is to determine whether or not, in eels, raising blood Ca levels by infusing a high-Ca solution into the blood stream can accelerate the secretion of calcitonin from the ultimobranchial gland.

MATERIALS AND METHODS

Japanese eels (Anguilla japonica) were used. In eels, the ultimobranchial gland is located on the transverse septum that separates the heart from the visceral mass. Consequently, it is necessary to find a blood stream toward the transverse septum after bathing the ultimobranchial gland with a high-Ca solution. In this study, we selected the pneumatic duct vein, which immediately flows to the sinus venosus of the heart via the transverse septum from the swim bladder. A canula was set in the pneumatic duct vein to infuse the high Ca-solution. Another canula was put in the arterial bulb for collecting blood.

The eels were divided into two groups. In one group, 2 mM CaCl2 solution was infused at a rate of 0.3 ml/hr by an infusion pump. This concentration and rate were determined by considering the quantity of blood in eels and the results of the previous experiments (Sasayama et al., 1996; Suzuki et al., 1999). We intended to take blood successively at 0, 0.5, 1, and 3 hr after infusion of the high-Ca solution into the heparinized syringe. When the solution was actually infused to 6 individuals, however, most eels died as soon as infusion started. We could collect data from only 2 surviving individuals. One survived for 3 hr, while the other one died after 1 hr. Therefore, the CaCl2 concentration was decreased to 0.5 mM, and the infusion rate was changed to 0.4 ml/hr. Seven individuals were exposed to these conditions. Four of them survived for 3 hr, and 3 died after 1 hr. Consequently, the CaCl2 concentration was again decreased to 0.3 mM, and the infusion rate was readjusted to 0.3 ml/hr. Two individuals were exposed to these conditions, but both died after 1 hr. Therefore, we judged that direct infusion of CaCl2 solution to the sinus venosus affected the heart muscle toxically, although the solution passed through the transverse septum from the pneumatic duct vein. In this study, as a result, 3 kinds of CaCl2 concentrations were tried, and data of 11 individuals were obtained.

On the other hand, as a control group, we infused 1.0 mM or 0.6 mM NaCl solution in 4 individuals and 1 individual, respectively, when considering the chloride concentrations of CaCl2. Because no eels died in this experiment, we could obtain data from 5 individuals.

Plasma was preserved in a deep-freezer until examination for analyzing Ca and calcitonin levels. Total Ca levels in plasma were determined using a microplate reader (Sasayama et al., 1996) by a modified method of Gitelman (1967). Plasma calcitonin levels were measured by a sandwich method of ELISA (Sasayama et al., 1996).

For determining ionic Ca levels and Na levels in plasma, a large quantity of blood was taken at the final sampling at 3 hr. In samples taken at other times as well, plasma Na levels were determined when the blood volume was adequate to measure it. Furthermore, we checked the hematocrit value at every sampling time to determine whether the effects of blood samplings were different or not between the CaCl2 group and the NaCl group.

The significance of changes in data was statistically examined using the Friedman-test. In the CaCl2 group, we separately assayed the data from individuals from which blood samples could be taken until 3 hr and 1hr after infusion. The average body weights of the CaCl2 group and the NaCl group at the initial level were 189.2±2.02 g and 184.0±6.26 g, respectively. There was no significant difference between them.

RESULTS

Plasma Ca levels

All 3 kinds of CaCl2 solutions brought about raises in plasma Ca levels (Table 1). Although there were 6 times more differences in the Ca concentrations among those solutions infused, the rates of raises in plasma Ca levels did not correlate directly with the Ca concentrations of the solutions. Therefore, the average value in plasma Ca at each sampling time was calculated by putting the data together. The results indicated that the plasma Ca value was 2.9±0.23 mM just before the infusion of CaCl2 solutions, 5.6±0.51 mM at 0.5 hr after infusion, 7.1±0.65 mM at 1.0 hr after infusion, and 8.8±0.36 mM at 3.0 hr after infusion (Table 1) (Fig. 1). The final value was 3 times higher than the initial value. These changes in plasma Ca levels were statistically significant (p<0.01).

Table 1

Individual values in plasma Ca levels (μM) in eels infused with a high-Ca solution. Ca concentrations (μM) in the solutions infused and the infusion rates are also cited in this table.

i0289-0003-19-9-1039-t01.gif

Fig. 1

Changes in plasma Ca levels (mM) in eels infused with a high-Ca solution or NaCl solution. The number in parenthesis is the number of fish examined.

i0289-0003-19-9-1039-f01.tif

In the NaCl group, the initial value of plasma Ca was 2.5±0.19 mM, which was not significantly different from that in the CaCl2 group. After the infusion of the NaCl solution, plasma Ca levels were 2.6±0.12 mM at 0.5 hr, 2.3±0.15 mM at 1.0 hr, and 2.3±0.17 mM at 3 hr. These changes were not statistically significant (Fig. 1) (Table 2).

Table 2

Individual values in plasma Ca levels (μM) in eels infused with a NaCl solution.

i0289-0003-19-9-1039-t02.gif

Plasma calcitonin levels

Calcitonin values were rather variable in the initial levels from individual to individual in either group (Tables 3 and 4). Plasma calcitonin levels were not correlated with either plasma Ca levels or the CaCl2 concentrations infused as well. In Fig. 2, changes in plasma calcitonin levels during 3 hr in the high-Ca group are shown individually by converting each initial value to r1.0. Plasma calcitonin levels began to increase at 0.5 o 1 hr after infusion in most individuals and ascended to higher levels at 3 hr. These changes were statistically significant (p<0.01).

Table 3

Individual values in plasma calcitonin levels (pg/ml) in eels infused with a high-Ca solution.

i0289-0003-19-9-1039-t03.gif

Table 4

Individual values in plasma calcitonin levels (pg/ml) in eels infused with a NaCl solution.

i0289-0003-19-9-1039-t04.gif

Fig. 2

Individual changes in plasma calcitonin levels in eels infused with a high-Ca solution when converting each initial value 1.0.

i0289-0003-19-9-1039-f02.tif

On the other hand, changes in plasma calcitonin levels in the NaCl group are also shown in Fig. 3 as they are for the CaCl2 group. There was no significant change during 3 hr.

Fig. 3

Individual changes in plasma calcitonin levels in eels infused with a NaCl solution when converting each initial value 1.0.

i0289-0003-19-9-1039-f03.tif

Ionic Ca levels in plasma

Ionic Ca values in the CaCl2 group could be determined in 3 individuals that survived for 3 hr. The value was 6.3±0.38 mM, a level that corresponded to 69–76% of the total Ca levels. On the other hand, ionic Ca levels in the NaCl group were determined in 4 individuals. The value was 1.5±0.09 mM at 3 hr, a level that also corresponded to 61–73% of the total Ca levels. Notwithstanding the fact that the value in the NaCl group was conspicuously lower than that in the CaCl2 group, the rates of ionic Ca values to total Ca values were very similar in both groups.

Plasma Na levels

In the CaCl2 group, the initial level of plasma Na in 8 individuals was 150.3±2.15 mM. In the NaCl group, the initial level in 4 individuals was 152.8±1.89 mM. There was no significant difference between the 2 groups. At 3 hr after the infusion of either solution, however, plasma Na levels in 4 individuals in the CaCl2 group were slightly decreased to 144.8±2.50 mM; on the other hand, in 4 individuals in the NaCl group, it tended to increase to 160.8±2.63 mM. There was a significant difference between the 2 groups (p<0.05). In the NaCl group, therefore, NaCl infusion might affect plasma Na levels.

Hematocrit value

Although the initial value of the hematocrit in the CaCl2 group was 30.5±1.71, its level was acutely decreased to 17.0±2.28 at 3 hr after infusion. This decline was significant (p<0.05). In the NaCl group, the initial value was 28.8±2.89, a level which was not significantly different from that in the CaCl2 group. At 3 hr after infusion, the initial level in the NaCl group was also acutely decreased to 21.2±2.52 (p<0.01). There was no significant difference in the values at 3 hr between the 2 groups.

DISCUSSION

The present results clearly demonstrate that direct raises in blood Ca levels by infusing a high-Ca solution into the blood stream accelerate the secretion of calcitonin from the ultimobranchial gland.

In the previous experiment using eels that had been infused a CaCl2 solution into the stomach, plasma Ca concentrations were increased from 2.63 mM of the initial value to 6.20 mM at 0.5 hr, 7.45 mM at 1 hr, and 8.50 mM at 3 hr (Suzuki et al., 1999). These values were very similar to the results obtained in this study. On the other hand, in the previous experiment, plasma calcitonin levels were increased from 30 pg/ml of the initial value to 205.6 pg/ml at 0.5 hr, 697.5 pg/ml at 1 hr, and 1118.2 pg/ml at 3 hr. In contrast, the average values in this study were 1,907.8 pg/ml at 0 hr, 2,582.1 pg/ml at 0.5 hr, 4,320.7 pg/ml at 1 hr, and 14,005.6 pg/ml at 3 hr. The absolute values of plasma calcitonin were undoubtedly higher in the present experiment. The difference in the values must be due to the differences in the site from which blood samples were taken. In the previous experiment, blood was sampled at the caudal artery; on the other hand, in this study, blood just perfused from the ultimobranchial gland was taken at the arterial bulb.

In this study, as mentioned above, the rates of raises in plasma Ca levels were similar to those in the case of the infusion of a high-Ca solution into the stomach (Suzuki et al., 1999). In that experiment, no eels died. Therefore, the raise in plasma Ca levels itself does not seem to be the cause of death of fish. On the other hand, the rate of ionic Ca to total Ca was similar between the high-Ca group and the NaCl group. This fact indicates that ionic Ca infused into the blood stream was immediately combined with some proteins at the same rate as that in the NaCl group. It may be important for a living body to keep the rate of ionic Ca/total Ca constant. However, it is also a fact that the absolute quantity of ionic Ca was very high in the high-Ca group compared to that in the NaCl group. Therefore, an excessive number of Ca ions in the blood stream might result in toxicity. When plasma Ca levels are raised via the digestive tract, there may be unknown systems preventing the heart from stopping beating. In our previous experiment, therefore, we should determine the ionic Ca level in plasma. Recently, we knew that, in humans, hypercalcemia harms the function of the heart (Yang et al., 1997; Demers et al., 1998).

Acknowledgments

This work was supported in part by funds from the cooperative program (No. 20), in 2000 provided by the Ocean Research Institute, University of Tokyo.

REFERENCES

1.

M. Azria 1989. The calcitonins: physiology and pharmacology. Karger. Basel. pp. 21–65. Google Scholar

2.

D. K. O. Chan, I. Chester Jones, and R. N. Smith . 1968. The effect of mammalian calcitonin on the plasma levels of calcium and inorganic phosphate in the European eel (Anguilla anguilla L.). Gen Comp Endocrinol 11:243–354. Google Scholar

3.

C. Demers, J. L. Rouleau, T. K. Leung, and J. C. Tardif . 1998. Hypercalcemic cardiomyopathy associated with primary hyperparathyroidism mimicking primary obstructive hypertrophic cardiomyopathy. Can J Cardiol 14:1397–1400. Google Scholar

4.

H. J. Gitelman 1967. An improved automated procedure for the determination of calcium in biological specimens. Analytical Biochem 18:521–531. Google Scholar

5.

J. P. Hayslett, M. Epstein, D. Spector, J. D. Myers, H. V. Murdaugh, and F. H. Epstein . 1971. Effect of calcitonin on sodium metabolism in Squalus acanthias and Anguilla rostrata. Bull Mt Desert Is Biol Lab 11:33–35. Google Scholar

6.

P. Person, T. Grunditz, J. Axelson, F. Sundler, and R. Hakanson . 1988. Cholecystokinins but not gastrin-17 release calcitonin from thyroid C-cells in the rat. Regul Pept 21:45–56. Google Scholar

7.

D. R. Robertson 1971. Cytological and physiological activity of ultimobranchial gland in the premetamorphic anuran Rana catesbeiana. Gen Comp Endocrinol 16:329–341. Google Scholar

8.

D. R. Robertson 1987. Plasma immunoreacitve calcitonin in the frog (Rana pipiens). Comp Biochem Physiol A 88:701–705. Google Scholar

9.

Y. Sasayama 1999. Hormonal control of Ca homeostasis in lower vertebrates: considering the evolution. Zool Sci 16:857–869. Google Scholar

10.

Y. Sasayama, K. Matsuda, C. Oguro, and A. Kambegawa . 1989. Immunohistochemical study of the ultimobranchial gland in chum salmon fry. Zool Sci 6:607–610. Google Scholar

11.

Y. Sasayama, Y. Abe, N. Suzuki, and T. Hayakawa . 1996. Plasma calcium and calcitonin levels at food intake in eels and goldfish. Zool Sci 13:731–735. Google Scholar

12.

R. Sethi, S. C. Kukreja, E. N. Bowser, G. K. Hargis, W. J. Henderson, and G. A. Williams . 1983. Effect of meal on serum parathyroid hormone and calcitonin: possible role of secretin. J Clin Endocrinol Metab 56:549–552. Google Scholar

13.

N. Suzuki, D. Suzuki, Y. Sasayama, A. K. Srivastav, A. Kambegawa, and K. Asahina . 1999. Plasma calcium and calcitonin levels in eels fed a high calcium solution or transferred to seawater. Gen Comp Endocrinol 114:324–329. Google Scholar

14.

C. Y. Yang, C. C. Cheng, C. W. Chou, and H. M. Cheng . 1997. Primary hyper-parathyroidism with cardiac abnormalities: a case report. Chung Hua I Hsueh Tsa Chih (Taipei) 60:277–282. Google Scholar
Yuichi Sasayama, Yoshio Takei, Sanaé Hasegawa, and Daisuke Suzuki "Direct Raises in Blood Ca Levels by Infusing a High-Ca Solution into the Blood Stream Accelerate the Secretion of Calcitonin from the Ultimobranchial Gland in Eels," Zoological Science 19(9), 1039-1043, (1 September 2002). https://doi.org/10.2108/zsj.19.1039
Received: 15 February 2002; Accepted: 1 June 2002; Published: 1 September 2002
JOURNAL ARTICLE
5 PAGES


SHARE
ARTICLE IMPACT
Back to Top