INTRODUCTION

Calcitonin (CT), a 32-amino acid peptide hormone, is secreted from C-cells of the thyroid glands in mammals and from parenchymal cells of the ultimobranchial glands in non-mammals [4]. It is well known that in mammals, this hormone functions to suppress the activity of osteoclasts, resulting in hypocalcemia and the mineralization of bones [14].

It has been reported that cartilaginous fish, as well as other vertebrates, possess ultimobranchial glands (UBG), and that administration of shark ultimobranchial extract (UBE) to rats induces hypocalcemia, as does homologous CT [4].

Recently, we reported that the stingray Dasyatis akajei possesses a pair of large UBG which contains abundant CT [21]. Furthermore, administration of synthetic stingray CT induces hypocalcemia in rats, similar to the effect of UBE from shark [19]. Thus, it is clear that cartilaginous fish has the ability to produce CT which can affect mineral balances in mammals, although the stingray itself does not possess bones. On the other hand, in mammals, bile has been shown to contain higher levels of Ca than serum [25]. Therefore, the gall-bladder is regarded as an important Ca excretory organ. In addition, it has been reported that in thyroparathyroidectomized rats, CT remarkably accelerates Ca excretion via bile [24]. In cartilaginous fish, the gall-bladder also contains bile in which the Ca level is 3–4 times higher than in serum [1, 2]. In the present study, to elucidate the physiological role of CT in cartilaginous fish, the effects of ultimobranchialectomy (UBX) on plasma and bile mineral balances in the stingray were examined.

MATERIALS AND METHODS

Seventeen stingray Dasyatis akajei, ranging 240–2,800 g in body weight and caught in Toyama Bay during the period from May to July 1993 were used. Prior to experiments, fish were acclimatized in an aquarium (120 cm × 60 cm × 45 cm) for 1 week after collection. UBX was then performed according to the following procedures. Fish were anesthetized with tricaine methanesulfonate in chilled seawater (4°C) at a dilution of 1/10,000. As the stingray possesses one pair of UBG just dorsal to the heart, an incision was made in the portion ventral to the heart. The heart was then pushed to the left side with a cotton, and the UBG of the right side was removed. The heart was next pushed to the right side, and the UBG of the left side was also removed. Thereafter, the incision was sutured with surgical thread. The same procedure was performed in the shamoperated (SHAM), but the UBGs were not removed. To allow recovery from anesthesia, fish were kept in physiological saline for elasmobranchus (380.0 mM NaCl, 6.9 mM KCl, 3.0 mM CaCl₂, 2.3 mM MgCl₂, 2.4 mM NaHCO₃ and 483.0 mM NH₂CONH₂) for 20 min, and then returned to normal seawater. During the experimental period of one week, seawater was exchanged at least once a day to prevent accumulation of NH₄⁺. Nine individuals (4 males and 5 females) were used for the UBX group and 8 individuals (5 males and 3 females) for the SHAM group. One week after the operation, both groups were anesthetized again, and blood was collected from the heart with heparinized syringes. Blood was centrifuged at 25,000×g for 10 min at 4°C, and hematocrit values were measured. The plasma samples were immediately frozen and kept at −50°C until analysis. In parallel with this procedure, bile was also taken from the gall-bladder with syringes, and the volume was examined.

A competitive inhibition enzyme-linked immunosorbent assay (ELISA) was used for measurement of plasma CT level in the stingray according to the method of Robertson [15, 16].

Ca, Mg, K and Na levels were determined by atomic absorption spectrophotometry (180–70 type; Hitachi-Zeeman; Hitachi Co. Ltd., Tokyo). Inorganic phosphate level was measured by the method of Fiske and Subbarow [7]. Urease-indophenol [20] and murotaseglucoseoxidase methods [13] were used for measurement of urea and glucose, respectively. Cl level was measured with a chloridemeter (C-50 type, Jookoo Sangyo Co., Ltd., Tokyo). Data were analyzed by Student's t-test.

RESULTS

Plasma CT level in the UBX group (457.6 ± 77.8 ng/ml) was significantly lower than that in the SHAM group (1321.2 ± 319.0 ng/ml) 1 week after the operation (P < 0.05) (Fig. 1).

Fig. 1

Plasma calcitonin levels in the ultimobranchialectomized group (UBX) and the sham-operated group (SHAM) in the stingray. The numbers of stingrays used were 9 in UBX and 8 in SHAM. Asterisk (*) indicates significant difference from the value for SHAM. P < 0.05.

Plasma mineral, urea and glucose concentrations and hematocrit values in both groups 1 week after the operation are shown in Table 1. There were no significant differences in values of any of these parameters between the UBX and SHAM groups.

Table 1

Plasma mineral, urea and glucose concentrations (mM) and hematocrit values (%) in the ultimobranchialectomized (UBX) and the sham-operated (SHAM) groups in the stingray

The results regarding bile minerals and bile volume are shown in Table 2. Bile Ca concentration in the UBX group was significantly lower than that in the SHAM group (P < 0.05). In contrast, bile K and Cl concentrations in the UBX group were significantly higher than those in the SHAM group (P < 0.05). There was no significant differences in bile levels of other minerals between the two groups. Bile volume in the UBX group was significantly (P < 0.05) greater than that in the SHAM group.

Table 2

Bile mineral concentrations (mM) and bile volume (ml/Kg BW) in the ultimobranchialectomized (UBX) and sham-operated (SHAM) groups in the stingray

DISCUSSION

At 1 week after the operation, plasma CT level in the UBX group was significantly lower than that in the SHAM group. However, the CT value in the UBX group was not decreased to near zero, but to about one-third of the level in the SHAM group. This leads us to consider that certain extra-ultimobranchial source(s) of CT may be present in the stingray. In the lizards [8] and salamander [12], such extraultimobranchial sources were demonstrated. On the other hand, Idler et al. [10] reported that in the skate Raja erinacea, hypophysectomy or interrenalectomy does not bring about significant changes in the glycogen levels in the liver when compared to sham-operated controls. It is possible that in cartilaginous fish, turnover rate of the hormone itself may be slower than in other vertebrates. On the other hand, it has been known that in lower vertebrates such as some teleosts and amphibians, plasma CT levels are so high (ng/ml order), when compared to those in mammals (pg/ml order) [15, 26]. In addition, also in shark, Triakis semifasciata, plasma CT level is ranged in ng/ml order [9]. Therefore, it may be possible that in these animals, all CT molecules present in blood do not function as a hormone. To clarify this problem, it may be helpful to determine the level of CT receptor in lower vertebrates, or to examine the effect of removal of all CT molecules by adding antibody against CT into blood.

It has been reported that in bullfrog tadpoles, serum Ca levels are increased significantly at 1 week after UBX when kept in Ca-rich water [18]. It was predicted that similar change may occur also in the stingray. However, there was no significant difference in the plasma Ca levels between UBX and SHAM groups at 1 week after the operation. CT is known to affect the ionic type of Ca, but not the proteinbound type of Ca [6]. It is known that in cartilaginous fish, the percentage of plasma ionic Ca is so high (about 80%) when compared to the value (50%) of most other vertebrates [5]. In the stingray, plasma Ca level could not be increased after UBX, because plasma was already saturated with ionic Ca. On the other hand, Chan and Wong [3] reported that in the lip-shark Hemiscyllium plagiosum, plasma Ca levels were more precisely regulated than Na, Cl and Mg levels when transferred to 70% or 35% seawater. It is possible that in the stingray, other mechanisms for regulation of plasma Ca level began to work immediately after UBX.

On the other hand, cartilarginous fish does not have “chondroclasts” which correspond to osteoclasts as a target for CT in bony vertebrates. Therefore, it is possible that in cartilaginous fish, CT plays some roles in Ca homeostasis by mechanism different from that on bones.

In higher vertebrates, the gall-bladder is an important Ca excretory organ [1, 2, 24, 25]. In hagfish, it is reported that Ca contained in the bile is 12 times higher than the level in plasma [17]. Also in amphibians, Ca contents in the gallbladder are higher than those in other soft tissues [22]. In the present study, the bile Ca concentration in the UBX group was significantly lower than that in the SHAM group. In the UBX group, Ca excretion via bile might be inhibited due to the lack of UBG. On the other hand, bile volume was greater in the UBX group than in the SHAM group. Therefore, in the UBX group, the fish might compensate for Ca excretion by increasing the bile volume. Consequently, bile Cl⁻ and K⁺ which are usually reabsorbed with water in gall-bladder, might be increased [11, 23].

The present results suggest that in cartilaginous fish, the gall-bladder may be one of the important targets for CT. This function of CT has been recognized so far only in mammals. Therefore, this action of CT could be ascended to at least this animal group. In addition, it seems of interest to consider from the physiological point of view that the CT action is not related to the presence of bones or cartilages.