Properties of Vasoactive Intestinal Polypeptide Receptors in the Anterior Pituitary of Female Chickens

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1 Journal of Reproduction and Development, Vol. 40, No. 3, 1994 Properties of Vasoactive Intestinal Polypeptide Receptors in the Anterior Pituitary of Female Chickens Sylvia Mae T. GONZALES, Mitsuo KAWASHIMA1), Michiharu KAMIYOSHI1), Katsuhide TANAKA and Kenji ICHINOE Department of Zootechnical Science, Tokyo University of Agriculture, Sakuragaoka, Setagaya, Tokyo 156, and 1 ')Department of Animal Science and Technology, Gifu University, 1-1 Yanagido, Gifu , Japan Abstract. The membrane fraction of anterior pituitary in laying hens was found to possess a vasoactive intestinal polypeptide (VIP)-binding component having the properties of a receptor. Scatchard analysis of saturation studies revealed that the binding component had a single class of binding site with an equilibrium dissociation constant (Kd) of } (mean } SEM; n=5) nm and the maximum binding capacity (Bmax) of 631 } 41 fmol/mg protein. The Kd value determined from kinetic analysis (0.091 } nm; n=3) was very similar to that obtained from Scatchard analysis. The Kd value obtained from Scatchard analysis was not significantly different between the laying and nonlaying hens. The Bmax in laying hens was greater than that in nonlaying hens. The administration of chicken VIP to nonlaying hens caused a marked decrease in the VIP receptor binding in the anterior pituitary with a concomitant increase in the blood serum concentrations of prolactin. The results may provide an evidence for a direct action of VIP on the anterior pituitary of female chickens. Key words: Vasoactive intestinal polypeptide receptor, Female chicken, Pituitary, Prolactin. (J. Reprod. Dev. 40: , 1994) asoactive intestinal polypeptide (VIP) has been U found in the hypothalamus of mammals [1] and birds [2-4], and in the mammalian anterior pituitary [5]. The chemical structure of this peptide in chickens [6] is different in its amino acid constituent from that of porcine or human VIP [7] at the position of 11, 13, 26 and 28. In mammals [8, 9] and birds [10-15], an administration of VIP in vivo or an incubation of isolated pituitary cells with VIP in vitro causes an increase of prolactin (PRL) release. If VIP exerts a direct action on the avian anterior pituitary, receptors for VIP must be Accepted for Publication: May 2, 1994 Correspondence: K. Tanaka present in this tissue as has been found in the mammal [16] and turkey [17]. The present study was performed to demonstrate the presence of VIP receptor in the anterior pituitary of the female chicken. Hormones Materials and Methods and chemicals Chicken vasoactive intestinal polypeptide (cvip) was purchased from Applied Biosystem Co. (San Francisco, CA, USA). Mammalian VIP (mvip), chicken secretin (Secretin), rat peptide histidine iso-

2 214 GONZALES et al. leucine (PHI), human glucagon (Glucagon) and rat growth hormone releasing factor (GRF) were obtained from Peninsula Lab. Inc. (Belmont, CA, USA). They were dissolved in 0.01 M acetic acid at a concentration of 300 AM. For dilution to a desired concentration, a binding assay's buffer was used. Bacitracin, bovine serum albumin (BSA, fraction V) and phenylmethylsulfonyl fluoride (PMSF), iodogen (1, 3, 4, 6-tetrachloro-3ƒ, 6ƒ -diphenylglycouril) were obtained from Sigma Chemical Co. (St Louis MO, USA). Polyethylenimine was obtained from Nacalai Tesque, Inc. (Kyoto). Na-125I (100 mci/ ml) was obtained from Amersham International Plc (Buckinghamshire, UK). Sephadex G-25 (super fine type) was obtained from Pharmacia LKB (Uppsala, Sweden). All other reagents were of analytical grade obtained from commercial source. Animals and tissues White Leghorn hens (18 months of age; kg body weight) laying 3-6 eggs in a sequence (clutch) with a 1-day pause between sequences for more than 2 weeks before experiment were used. Hens which had not been laid an egg for least 1 week before experiments were used as nonlaying hens. The birds used were obtained from a flock of birds kept in individual cages under 14 h ( h) light/day with feed and water provided ad libitum. The laying hens were killed by decapitation at 1000 h without regard to the time of ovulation (30 birds in each determination) and the nonlaying hens were killed at the same hours of a day (30 birds in each determination) for Experiment 1. In the nonlaying hens necropsied, the weight of the ovary and oviduct was less than 10 and 19 g, respectively, and the plasma concentration of progesterone (P), estradio1-17ƒà (E2) and testosterone (T) was less than 101 pg/ml (P), 77 pg/ml (E2), and 78 pg/ml (T), respectively. In Experiment 2, the nonlaying hens (7 birds per determination in each group) received a single iv injection of cvip (5 lug/0.5 ml/bird) or vehicle (0.5 ml of saline/ bird) at 1000 h and were killed 5, 10, 30 and 60 min after the injection. Trunk blood was collected and saved for radioimmunoassay (RIA) of chicken PRL (see below). From all of these birds, the anterior lobe of pituitary (AP; 7-10 mg/bird) was excised, weighed and pooled in each group. Preparation of membrane fraction Membrane fraction was prepared at 4 C according to the method of Wanke and Rorstad [16] with slight modifications. The pituitaries were homogenized in 25 vol/wt TEM buffer (25 mm Tris-HCl, 2 mm EDTA, 2 mm MgC12, ph 7.4) in a Potter- Elvehjem glass teflon homogenizer. The homogenate was centrifuged at 700 X g for 10 min, the supernatant was obtained. The precipitate was homogenized and recentrifuged. The supernatants were combined and centrifuged at 13,000 X g for 30 min. The resultant sediments were washed twice with TS buffer (25 mm Tris-HCl, 0.25 M sucrose, ph 7.4). The washed pellet was suspended in 2 v /w TS buffer and used as a membrane fraction. Radioiodination of cvip The cvip was labeled with Na-125I using the iodogen method [18, 19]. The reaction mixture was placed on a 1 ~ 100 cm Sephadex G-25 (super fine type) column equilibrated with 1% BSA in 0.1 M acetic acid solution as described by Ogawa et al. [20] with slight modifications and eluted with 0.1 M acetic acid solution containing 0.1% BSA. Two radioactive peaks prior to free 1251 peak were obtained and the two peaks were clearly separated. The first peak bound well to the membrane fraction, and the percentage of nonspecific binding to total binding was less 10%, but the second peak bound poorly. The specific activity of 125I-cVIP, expressed as monoiodo cvip, was calculated to be 14,800-25,900 GBq/mmol assuming a complete recovery of the peptide. The labeled VIP was used within 40 days after iodination. cvip binding assay The specific binding of 125I-cVIP was measured by the use of the method of Wanke and Rorstad [16] with modifications. The diluent used was TMB buffer (25 mm Tris HC1, 2 mm MgCl2, 0.2 mm bacitracin, ph 7.4) containing 0.1% BSA and 1 mm PMSF. Aliquots of the membrane fraction (30 pg protein/tube; two tubes per assay) were incubated at 30C for 1 h with 125I-cVIP in the absence (total binding) or presence (nonspecific binding) of unlabeled cvip in a total volume of 200 Al. After the incubation, 2 ml of ice-cold TMB buffer was added. Bound and free radioligands were separated by rapid filtration under vacuum through glass microfiber filters (GF/B type; Whatman, Clifton, NJ, USA) that were presoaked overnight in 0.5% polyethylenimine to reduce the value of back ground [21]. The filters were washed twice

3 VIP RECEPTOR IN HEN PITUITARY 215 with 2 ml TMB buffer and measured for the radioactivity by a gamma counter (Cobra Model B5002; Packard Instrument Co., Meriden, CT, USA) with the counting efficiency of 65-82%. Specific binding was obtained by subtracting the nonspecific binding from the total binding, and expressed as moles/mg protein or per mg tissue. The equilibrium dissociation constant (Kd; indicating the degree of the binding affinity) and the maximum binding capacity (Bmax) were determined by the method of Scatchard [22]. Preliminary experiments examined the relationship of the specific 125I-cVIP binding to incubation period ( min) and to the protein concentration (5-40 Aug/tube) in the membrane fraction. The specific 125I-cVIP binding was found to increase during the first 60 min of the incubation, but a longer period of up to 240 min resulted in much less alteration. The specific 125I-cVIP binding increased linearly with the increase in the protein concentration from 5-40,ƒÊg/ tube. Assay for chicken PRL Immunoreactive chicken prolactin (cprl) in the serum was measured Dy a homoiogous MA system for cprl using the same procedure as reported earlier for chicken LH [23]. The cprl preparation (AFP4444B) for radioiodination, cprl reference preparation (AFP 10328B) and antisera against cprl (AFP : Rabbit) were kindly provided by Dr. A. F. Parlow, Pituitary Hormones and Antisera Center, Harbor-UCLA Medical Center, (Torrance, CA, USA). The coefficient of variation within and between assays was 9 and 12%, respectively, from five assays. unlabeled ligand to estimate the dissociation rate constant (k_1). Student's t test [27] was used to assess the significance of difference between two means. Probability (P) values of less than 0.05 were considered significant. Results Kinetic analysis of 125I-cVIP binding (Experiment 1) The specific 125I-cVIP in the membrane fraction of anterior pituitary of laying hens reached a steady state at 60 min and was stable for up to 240 min (Fig. 1). The decrease of the specific 125I-cVIP binding occurred upon addition of a large excess unlabeled cvip (Fig. 1). The binding of 125I-cVIP to the hen anterior pituitary was reversible and time dependent. The association rate constant (K+1) was ± nm-1 min (n=3). Specific 125I-cVIP binding was reversed (t1/2 = 38.8 } 1.5 min, n=3) by the addition of a large excess of unlabeled cvip. The rate constant for dissociation (K-1) determined from the pseudo-first-order equation was } min-1 (n=3). The kinetic dissociation constant (Kd) for 125I-cVIP calculated from the ratio K-1/K+1 was } (n=3) nm. Other assay Protein concentrations of the membrane fractions were determined by the method of Lowry et al. [24] using BSA as a standard. Plasma concentrations of P, E2 and T were measured by the use of a routine RIA [25]. The coefficient of variation within and between assays was 8 and 11% for P, 11 and 13% for E2, and 10 and 13% for T, respectively, as determined by six assays. Statistical analyses Kinetic data were analyzed by the method of Bylund and Yamamura [26], using pseudo-firstorder conditions to estimate the association rate constant (k +1) and addition of a large excess of Fig. 1. Time-course of the association (0) and dissociation (0) of 125I-cVIP in the membrane fraction of anterior pituitary of laying hens. Samples (30 ƒêg protein/ tube) were incubated at 30C for various times (min) with 125I-cVIP (0.1 mm) in the absence or presence of a 100-fold molar excess or unlabeled cvip and the specific 125I-cVIP binding was measured (see Materials and Methods). The specific 125I-cVIP binding decreased on addition of 1 ƒêm unlabeled cvip (arrow). Each point represents the mean } SEM of three separate pooled samples. Standard errors not shown fall within the point as drawn.

4 216 GONZALES et al. Binding specificity (Experiment 1) The 125I-cVIP binding in the membrane fraction of anterior pituitary of laying hens was drastically reduced by the presence of a 100-fold molar excess of unlabeled cvip or mvip, but little by the presence of an equivalent molar concentration of unlabeled GRF, PHI, Secretin and Glucagon (Fig. 2). GRF, PHI and Secretin except for Glucagon reduced the binding to about 60% when a 10,000- fold molar excess was used. Binding affinity and capacity (Experiment 1) The specific 125I-cVIP binding in the membrane fraction of anterior pituitary of laying hens increased when increasing amounts of 125I-cVIP were added, i.e., when in the amount of free 125I-cVIP was increased, and was saturable at about 0.7 nm (Fig. 3). Scatchard analysis revealed a linear relationship between the amount of specific 125I-cVIP binding and the ratio (B/F) of specific 125I-cVIP binding to free 125I-cVIP (Fig. 3), indicating a single class of binding sites. The value of Kd, Bmax and correlation coefficient (y ) between B/F and specific 125I-cVIP binding (calculated from the data in Fig. 3) were 0.08 nm (Kd), 577 fmol/mg protein Fig. 2. Competition for the 125I-cVIP binding in the membrane fraction of anterior pituitary of laying hens. Samples (30,ƒÊg protein/tube) were incubated at 30 C for 2 h with 0.1 nm 125I-cVIP in the absence (control) or presence of various fold molar excess of unlabeled competitors, and the 125I-cVIP binding was measured (see Materials and Methods). The value of the 125I-cVIP binding in the control was 262 fmol/mg protein. Each point represents the mean of two separate pooled samples. The competitors tested were: cvip ( œ). mvip ( ), GRF ( ), PHI ( ) Secretin ( ), and Glucagon ( ). (Bmax) and (ƒá ), respectively. Table 1 lists the average of Kd and Bmax (expressed as fmol/mg protein and fmol/mg tissue) obtained from five separate pooled samples. The Kd was not significantly different between the laying and nonlaying hens. However, the Bmax was greater in the laying hen than in the nonlaying hen. Changes following in vivo administration of cvip (Experiment 2) The amount of specific 125I-cVIP binding in the membrane fraction of anterior pituitary of Fig. 3. Saturation curves (a) and Scatchard plots (b) of specific 125I-cVIP binding in the membrane fraction of anterior pituitary of laying hens. Samples (30,ƒÊg protein/tube) were incubated at 30 C for 2 h with various concentrations in the absence or presence of a 100-fold molar excess of unlabeled cvip, and 125I-cVIP binding was measured (see Materials and Methods). Each point represents the mean of duplicate on one pooled sample. œ- œ specific binding; -, nonspecific binding. B = bound; F = free.

5 VIP RECEPTOR IN HEN PITUITARY 217 Table 1. Equilibrium dissociation constant (Kd) and maximum binding capacity (Bmax) of the specific 125I-cVIP binding component in the membrane fraction of the anterior pituitary of the laying and nonlaying hens (Experiment 1) nonlaying hens showed a decrease 5 and 10 min after the iv injection of cvip (Fig. 4). No significant change was observed at any time after the injection of vehicle. The binding of 125I-cVIP was recovered to the level of the control 30 and 60 min after the injection of cvip. The cprl concentration in the blood serum of the hens increased 5 min after the iv injection of cvip (Fig. 4). Discussion The membrane fraction of the anterior pituitary of the hen was found to contain a VIP binding component showed reversible binding (Fig. 1), binding specificity (Fig. 2), high affinity and limited capacity (Fig. 3). Since these properties are characteristics of a receptor, the results suggest the presence of VIP receptors in the hen anterior pituitary. The Kd value (0.091 } nm) calculated from kinetic analysis was in close agreement with the Kd value (0.092 } nm) obtained by Scatchard analysis of saturation studies. The Scatchard analysis revealed that the binding component had a single class of binding site. This is different from the finding reported on the turkey [17] and rat [16] anterior pituitary VIP receptor in which the receptor possesses two distinct binding sites of low and high affinity obtained from competitive binding studies and also different from the receptor in the mammalian uterus [29], pineal [30], kidney [31] and blood vessels [32, 33]. However, VIP receptors in the brain synaptic membrane of the rat have a single binding site with an appar- Fig. 4. The specific 125I-cVIP binding in the membrane fraction of anterior pituitary (a), and serum cprl concentrations (b) after an iv injection of cvip (5,ƒÊg/ 0.5 ml saline/bird). Samples (30,ƒÊg protein/tube) obtained from nonlaying hens after the iv injection of cvip ( œ) or saline (control) were incubated at 30 C for 2 h with 0.7 nm 125I-cVIP in the absence or presence of a 100-fold molar excess of unlabeled cvip, and the specific 125I-cVIP binding was measured (see Materials and Methods). The shaded area represents the range of vehicle control values. Each point represents the mean ± SE of four separate pooled samples. *, **, Significantly different from "0 min" at 5 and 1% level, respectively.

6 218 GONZALES et al. ent Kd of 9.8 nm [20] obtained from competitive binding studies. These findings may suggest that VIP receptors are different in their binding properties between different tissues of different animals. The Bmax values obtained in the present study are in a similar order (expressed as fmol/mg protein) to that reported on the high affinity binding site of VIP receptor in various VIP-responsive tissues of mammals [29-33] including the rat anterior pituitary [16, 28]. The Kd value obtained in the present study was not different between the anterior pituitaries of laying and nonlaying hens, but the Bmax was in the laying hens greater than in the nonlaying hens (Table 1). The difference may be related to the difference in the ovarian function, probably in the action of ovarian steroid hormones, because progesterone [34, 35], estrogen [36], and androgen [37] receptors are known to be present in the hen A marked decrease in the VIP receptor binding in the anterior pituitary with a concomitant increase in the blood concentration of PRL was found after the administration of cvip in vivo (Fig. 4). The decrease in the VIP receptor binding may be the results from a down regulation of the receptor. The incidence of down regulation or internalization of VIP receptor has been observed in the rat anterior pituitary [28] and in the human adenocarcinoma cell line [38]. The PRL release from the anterior pituitary in vivo and in vitro by the action of VIP has been found in turkeys [10, 12-15] and pigeons [11] as well as in rats [8, 9]. It may be likely that VIP per se plays a direct action through its receptors in the anterior pituitary of the hen and regulates the release of PRL. Acknowledgment The authors with to express their appreciation to Kazuhiro Matsui and Akiko Hirano for technical assistance. References 1. Roberts GW, Woodhams PL, Bryant MG, Crow TJ, Bloom SR, Polak JM. Vasoactive intestinal polypeptide in the rat brain: evidence for a major pathway linking the amygdala and hypothalamus via the stria terminals. Histochemistry 1980; 65: Yamada S, Mikami SI. Immunohistochemical localization of vasoactive intestinal polypeptide (VIP) containing neurons in the hypothalamus of the Japanese quail, C Coturnix coturnix. Cell Tissue Res 1982; 226: Macnamee MC, Sharp PJ. Evidence that vasoactive intestinal polypeptide is a physiological prolactin-releasing factor in the bantam hen. Gen Comp Endocrinol 1986; 62: Peczely P, Kiss JZ. Immunoreactivity to vasoactive intestinal polypeptide (VIP) and thyrotropin releasing hormone (TRH) in hypothalamic neurons of the domesticated pigeon columbia livia alterations following lactation and exposure to cold. Cell Tissue Res 1988; 251: Arnout MA, Garthwaite TL, Martinson DR, Hagen TC. Vasoactive intestinal polypeptide is synthesized in anterior pituitary tissue. Endocrinology 1986: 119: Nisson A. Structure of the vasoactive intestinal octacosapeptide from chicken intestine. The amino acid sequence. FEBS Letters 1975; 60: Said SI, Mutti V. Polypeptide with broad biological activity isolation from small intestine. Science 1970; 169: Vijayan E, Samson WK, Said SI, McCann SM. Vasoactive intestinal peptide: Evidence for a hypothalamic site of action to release growth hormone luteinizing hormone, and prolactin in conscious ovariectomized rats. Endocrinology 1979; 104: Kato Y, Iwasaki Y, Iwasaki J, Abe H, Yanaihara N, Imura H. Prolactin release by vasoactive intestinal polypeptide in rats. Endocrinology 1978; 103: Fehrer SC, Silsby JL, El Halawani ME. Capacity of various neuropeptides to induce prolactine (PRL) or luteinizing hormone (LH) release by dissociated turkey hen anterior pituitary cells. Poultry Sci 1987; 66 (Suppl 1): Lea RW, Vowles DM. Vasoactive intestinal polypeptide stimulates prolactin release in vivo in the ring dove (Streptopelia risoria). Experientia 1986; 42: Opel H, Proudman JA. Stimulation of prolactin release in turkeys by vasoactive intestinal peptide. Proc Soc Exp Biol Med 1988; 187: Knapp TR, Fehrer SC, Silsby JL, Porter TE, Behnke EJ, El Halawani ME. Gonadal steroid modulation of basal and vasoactive intestinal

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