maximum binding capacity; CT = calcitonin; cCT = chicken CT; ID50 = half-maximal inhibition dose; Kd = equilibrium dissociation constant;. K+1 = association ...
Presence of Calcitonin Receptors in Shell Gland of the Guineafowl and Changes in Binding Property During an Oviposition Cycle1 Hiroshi Ogawa,* Tetsuya Takahashi,† Takehito Kuwayama,* and Mitsuo Kawashima†,1,2 *Department of Animal Science, Tokyo University of Agriculture, Atsugi-shi, Kanagawa 243-0034, Japan; and †Department of Biological Diversity and Resources, Gifu University, Gifu 501-1193, Japan ABSTRACT Radioligand binding assays of membrane fraction of the shell gland endometrium of guineafowl revealed the presence of receptor for calcitonin. The equilibrium dissociation constant (Kd) obtained by Scatchard analyses was 0.50 to 1.25 nM in laying birds and 1.12 to 1.19 nM in nonlaying birds. The maximum binding
capacity (Bmax) per milligram of protein was 33.1 to 107.5 femtomoles in laying birds and 101.4 to 114.9 femtomoles in nonlaying birds. Both Kd and Bmax values changed during eggshell formation in laying birds. The results suggest that calcitonin receptor binding may be related to eggshell formation in guineafowl.
(Key words: guineafowl, calcitonin receptor, shell gland, oviposition cycle, eggshell formation) 2003 Poultry Science 82:1302–1306
INTRODUCTION Calcitonin (CT), the hormone secreted from the ultimobranchial gland, plays an important role in the maintenance of calcium homeostasis of the body (Taylor and Dacke, 1984). It acts on the bone to resorb calcium from the blood and on the kidney to modify mineral excretion (Taylor and Dacke, 1984). In the calvarial bone and the kidney, the presence of CT receptor has been reported in chickens (Yasuoka et al., 1998). The uterus is the portion of the avian oviduct where the eggshell is formed from calcium derived from blood (Nys et al., 1999). The present study was performed to verify the assumption that, in tissues where calcium transport is important, CT receptors must be present in relation to the physiological function of the tissue.
MATERIALS AND METHODS Birds and Tissues
on at 0400 h) with feed (17% CP; 2,850 kcal ME)4 and water available ad libitum were killed by decapitation at 1000 h to examine binding specificity, reversibility, affinity, and capacity of CT receptors (three birds in each sample). For laying hens, seven killing times were scheduled between 16 h before oviposition to 4 h after oviposition of the first egg in a laying sequence (five birds at each time) to elucidate the change in the binding affinity and capacity of CT receptor in relation to eggshell formation. For comparison, hens that had not laid an egg for more than 14 d prior to experiments were killed at five corresponding times (five birds at each time) of the day. The uterus was removed, and endometrium tissues were obtained. In the hens necropsied, the weight of the ovary and oviduct were 50.3 ± 1.7 g (x ± SEM, n = 35) and 59.8 ± 2.2 g (n = 35) for laying hens and 12.9 ± 2.0 g (n = 25) and 12.1 ± 2.1 g (n = 25) for nonlaying hens, respectively.
Preparation of Membrane Fractions
Egg-laying guineafowl hens (27 to 37 wk of age; 2.5 to 3.2 kg BW; Galor Strain)3 kept under 14L:10D (lights
2003 Poultry Science Association, Inc. Received for publication December 17, 2002. Accepted for publication March 24, 2003. 1 Financial support for this study was provided by a Grant-in-Aid for scientific research from the Ministry of Education, Science, Sports and Culture, Japan (No. 12660249). 2 To whom correspondence should be addressed: kawasima@cc. gifu-u.ac.jp. 3 Jafra Trading Co., Ltd., Dejima, Japan. 4 Heisei Feed Inc., Ibaraki, Japan. 5 Wako Pure Chemical Industries, Ltd., Osaka, Japan. 6 Ika Labortechnik, Janke & Kunkel GmbH & Co KG, Staufen, Germany.
Membrane fractions were prepared by using the method of Yasuoka et al. (1996) with slight modifications. All procedures were performed in a cold room at 4°C. The tissues were rinsed with ice-cold saline, blotted with a filter paper, weighed, and then minced. The tissues were homogenized in 3 vol/tissue weight in TE
Abbreviation Key: ACTH = adrenocorticotropic hormone; Bmax = maximum binding capacity; CT = calcitonin; cCT = chicken CT; ID50 = half-maximal inhibition dose; Kd = equilibrium dissociation constant; K+1 = association rate constant; K−1 = rate constant for dissociation; PTHrP = parathyroid hormone related peptide; CTGRP = calcitonin gene-related peptide; TE = Tris-EDTA.
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buffer (50 mM Tris5-HCl, 1 mM EDTA,6 pH 7.4) by using a Ultra-Turrax homogenizer.6 Strokes were repeated 10 times while cooling in an ice bath. Homogenates of each tissue were centrifuged at 700 × g for 10 min at 4°C, and the supernatant was obtained. The precipitate was resuspended in the buffer and centrifuged at 700 × g for 10 min at 4°C. The supernatants were combined and centrifuged at 30,000 × g for 30 min at 4°C. The precipitate was washed twice with TE buffer and then homogenized in the TE buffer (2 vol/tissue wt). The homogenate was divided into 1-mL aliquots, stored at −70°C, and used within 30 d as the membrane fraction. The protein concentration was measured by the method of Lowry et al. (1951) using BSA (Fraction V, pH5.6)7 as a standard.
Radioiodination of Chicken CT Chicken CT8 (cCT) was iodinated with Na[125I] using Iodogen9 (Yasuoka et al., 1998). The iodinated cCT was separated from free Na[125I] as described by Takahashi et al. (1992). The mixture was placed onto a 1 × 100cm Sephadex G-25 column10 pretreated with 1% BSA (Fraction V, pH 5.6) and eluted with 0.1 M acetic acid solution containing 0.1% BSA. The eluates were fractionated in each 1-mL volume, and the radioactivity was counted by a Packard Cobra gamma counter.11 A clearly separated peak of radioactivity prior to the peak of free Na[125I] was obtained. The peak bound well to the membrane fraction, and the percentage of nonspecific binding to total binding was less than 20%. The radioactive peak of the eluates was employed for binding assays. The specific activity of the radioligand was calculated (Ysuoka et al., 1996) to be 307 to 487 Ci/mmol. All experiments were performed within 40 d after radioiodination.
Binding Assay The membrane fractions (30 µg protein/tube) were incubated with various concentrations (0.07 to 3.5 nM) of [125I]cCT in the presence or absence of 1 µM of unlabeled cCT in a total volume of 300 µL. For experiments on competitive binding, 1 nM to 1,000 nM of unlabeled calcitonin gene-related peptide (CTGRP)9 and 10 nM to 1,000 nM of unlabeled parathyroid hormone related peptide (PTHrP),10 chicken angiotensin-II,12 chicken vasoactive intestinal peptide (VIP),9 human adrenocorticotropic hormone (ACTH),13 and human insulin (Ins)10 were used. Incubations were performed at 4°C for 2 h in duplicate. Polypropylene Eppendorf-type microfuge
7
Seikagaku Corp., Tokyo, Japan. Peninsula Laboratories, Inc., San Carlos, CA. 9 Sigma Chemical Co., St. Louis, MO. 10 Pharmacia LKB, Uppsala, Sweden. 11 Packard Instrument Co., Meriden, CT. 12 Bachem Inc., Torrence, CA. 13 Peptide Institute, Inc., Osaka, Japan. 14 Iwaki Glass Co., Ltd., Funahashi, Japan. 8
tubes (1.5 mL)14 pretreated with TE buffer supplemented with 2% BSA (fraction V, pH 7.0) for 3 d at 4°C were used for incubation. Immediately after incubation, the tubes were centrifuged at 10,000 × g for 10 min at 4°C. The precipitated pellet was rinsed with 0.5 mL of TE buffer, followed by centrifugation. Radioactivity was measured by using a Packard Cobra gamma counter; the counting efficiency for 125I was 70 to 87%. Specific binding was obtained by subtracting the nonspecific binding (in the presence of unlabeled hormone) from the total binding (in the absence of unlabeled hormone) and expressed as femtomoles per milligram of protein. The equilibrium dissociation constant (Kd) and the maximum binding capacity (Bmax) were determined by the method of Scatchard (1949). Means of duplicate determination were used as the points of the Scatchard analysis. Kinetic data were analyzed by the method of Bylund and Yamamura (1990). The pseudo-first-order condition was adopted to estimate the association rate constant (K+1), and the data obtained by the addition of a large excess of unlabeled ligand were used to estimate the rate constant for dissociation (K−1).
Statistical Analysis A half-maximal inhibition dose (ID50) of the [125I]cCT binding was estimated by the use of a log-logit linear regression (Finney, 1964). Statistical comparisons among groups during the oviposition cycle were performed by one-way ANOVA (Snedecor and Cochran, 1980a). When ANOVA was significant at the 5% level, Tukey’s multiple range test was applied for the posthoc comparisons (Snedecor and Cochran, 1980b).
RESULTS Relationship of Specific [125I]cCT Binding to Protein Concentration and Incubation Period The specific [125I]cCT binding increased linearly with the increase in the protein concentration from 20 to 80 µg per tube (Figure 1). The specific [125I]cCT binding increased within the first 1 h of incubation period at 4C and remained stable until 3 h (Figure 2).
Binding Specificity The binding to [125I]cCT was reduced by addition of 1-, 10-, 100-, and 1,000-fold molar excess of unlabeled cCT or CTGRP but not by the addition of insulin, Angiotensin-II, vasoactive intestinal peptide, or PTHrP. When a 1,000-fold molar excess of ACTH was added the binding was reduced to about 60% (Figure 3). The ID50 value calculated from the data of the dose-inhibition curve was 0.6 nM for cCT and 1,015 nM for ACTH.
Kinetic Analysis The specific [125I]cCT binding in the membrane fraction of shell gland of guineafowl hens reached a steady
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FIGURE 1. Relationship between protein concentration of the membrane fraction and specific [125I]chicken calcitonin ([125]IcCT) binding in the shell gland endometrium of guineafowl hens. Samples (20 to 80 µg protein per tube) were incubated at 4°C for 2 h with 1 nM [125I]cCT in the absence or presence of 1 µM unlabeled cCT. Each point represents the mean of three separate samples, and the vertical bars represent SEM. When not shown, SEM falls on the symbol.
state at 60 min and was stable for up to 180 min (Figure 4). Addition of a large excess of unlabeled cCT caused a decrease in specific [125I]cCT binding. The association rate constant (K+1) was 0.023 ± 0.001 nM/min (x ± SEM; n = 5). Specific [125I]cCT binding was reversed by the addition of unlabeled cCT [half time (t1/2) = 9.75 ± 0.43 min (n = 5)]. The K−1 determined from the pseudo-firstorder equation was 0.019 ± 0.001/min. The Kd for [125I]cCT binding, calculated from the ratio, K−1:K+1 was 0.81 ± 0.03 nM (n = 5).
FIGURE 3. Competition for [125I]chicken calcitonin ([125I]cCT) binding in the membrane fraction of the shell gland endometrium of guineafowl hens. Samples (30 µg protein per tube) were incubated at 4°C for 2 h with 1 nM [125I]cCT in the absence (control) or presence of various-fold molar excess of unlabeled competitors. The amount of [125I]cCT binding the in the control was 96.3 femtomol/mg of protein. Each point represents the mean of two separate samples. The competitors tested were cCT (䊉), parathyroid hormone related peptide (䊊), human adrenocorticotropic hormone (ACTH, 䊐), chicken angiotensinII (cAng-II, Υ), insulin (Ins: 䊏), chicken vasoactive intestinal peptide (VIP, ▲), and calcitonin gene-related peptide (CTGRP, ×).
ship between the amount of specific [125I]cCT binding and the ratio (B:F) of specific [125I]cCT binding to free [125I]cCT (Figure 5), indicating a single class of binding sites. The value of Kd and Bmax of the same five separate pools of samples as used for the measurement of kinetic analysis was 0.78 ± 0.04 nM (Kd, ± SEM; n = 5) and 82.5 ± 5.9 femtomoles/mg protein (Bmax, n = 5).
Binding Affinity and Capacity The specific [125I]cCT binding increased when increasing [125I]cCT were added, i.e., when the amount of free [125I]cCT was increased and was saturable at 1.6 nM (Figure 5). Scatchard analysis revealed a linear relation-
FIGURE 2. Time course of specific [125I]chicken calcitonin ([125I]cCT) binding in the shell gland endometrium of guineafowl hens. Samples (30 µg protein per tube) were incubated at 4°C for various times with 1 nM [125I]cCT in the absence or presence of 1 µM unlabeled cCT. Each point represents the mean of three separate samples, and the vertical bars represent SEM. When not shown, SEM falls on the symbol.
Changes in Binding Affinity and Capacity The Kd value in laying birds decreased at 12 h before oviposition and increased at 4 h after oviposition (P ≤
FIGURE 4. Time-course of the association (䊉) and dissociation (䊊) of [125I]chicken calcitonin ([125I]cCT) binding in the membrane fraction of the shell gland endometrium of guineafowl hens. Samples (30 µg protein per tube) were incubated at 4°C for various times with 1 nM [125I]cCT in absence or presence of 1 µM unlabeled cCT. The arrow indicates addition of 1 µM unlabeled cCT. Each point represents the mean of five separate samples and vertical bars represent SEM. When not shown, SEM falls on the symbol.
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DISCUSSION
FIGURE 5. Saturation curve and Scatchard plot of [125I]chicken calcitonin ([125I]cCT) binding in the membrane fraction of the shell gland endometrium of guineafowl hens. Samples (30 µg protein per tube) were incubated at 4°C for 2 h with various concentrations (0.07 to 3.5 nM) of [125I]cCT in the absence or presence of 1 µM unlabeled cCT. The value of equilibrium dissociation constant (Kd) and maximum binding capacity (Bmax) (calculated by the use of Scatchard analysis), and correlation coefficient (r) between B:F and specific binding was 0.85 nM (Kd), 89.0 femtomol per mg protein (Bmax) and −0.984 (r), respectively. Each point represents the mean of two experiments in duplicate determinations from one single sample. (䊉) Specific binding; (䊊) nonspecific binding; B = specific [125I]cCT bound; F = free [125I]cCT.
0.01). No appreciable change was observed in nonlaying birds (P > 0.05) (Figure 6). The Bmax value in laying birds decreased at 6 h before oviposition (P ≤ 0.01), decreased further at 1 h before and again at 1 h after oviposition, and then recovered at 4 h after oviposition. No change was observed in nonlaying birds (P > 0.05) (Figure 6).
The membrane fractions of the endometrium of the shellgland (uterus) of guineafowl were found to contain a binding component possessing specificity (Figure 3), saturation (Figure 5), and reversibility (Figure 4) of binding to [125I]cCT. These properties are requisites for being a receptor for cCT. The presence of CT receptor was already reported on the chicken shell gland (Ieda et al., 2001), calvaria, and kidney (Yasuoka et al., 1998, 2001). The Kd value obtained by Scatchard analysis was in close agreement with that obtained by the kinetic analysis and is very similar to that reported for chicken tissues. Both Kd and Bmax values changed during an oviposition cycle in laying birds but not in nonlaying birds during a 24-h d (Figure 6). A decrease in the Kd value was found 12 h before oviposition, and that in the Bmax value was at 6 h before oviposition. Both the Kd and Bmax values remained at reduced levels up to 1 h after oviposition. The decrease in the Kd value means an increase in the affinity of binding of hormone to its receptor and always follows a decrease in the Bmax value. During the period from the time at about 12 h before oviposition (about 6 h after entering the egg in the shell gland), a hard shell is formed by the deposition of calcium derived from the fluid from the shell gland on the egg surface (Nys et al., 1999). The change in the binding affinity and capacity of CT receptor may be related to the eggshell formation in the shell gland of the guineafowl.
REFERENCES
FIGURE 6. Equilibrium dissociation constant (Kd) and maximum binding capacity (Bmax) of the specific binding component of [125I]chicken calcitonin ([125I]cCT) binding in the membrane fraction of the shell gland endometrium of guineafowl hens at various times before and after oviposition (●) and of nonlaying guineafowl hens at corresponding times (䊊). Samples (30 µg per tube) were incubated at 4°C for 2 h with [125I]cCT (0.07 to 3.5 nM) in the absence or presence of 1 µM unlabeled cCT. The Kd and Bmax were calculated by the use of Scatchard analysis. The amount of protein in the membrane fraction was 11.31 ± 0.73 mg/ g of tissue (x ± SEM, n = 35) in laying hens and 10.32 ± 0.53 mg/g of tissue (n = 25) in nonlaying hens and was not significantly different among the hens at different times. Each point represents the mean of five separate samples of laying hens and of nonlaying hens. The vertical bars indicate SEM. When not shown, SEM falls on the symbol. *Significantly different (P ≤ 0.01) from the preceding value by Tukey’s test.
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