Arginine vasotocin is the major adrenocorticotropic hormone-releasing factor in the bullfrog Rana catesbeiana

Arginine vasotocin is the major adrenocorticotropic hormone-releasing factor in the bullfrog Rana catesbeiana

General and Comparative Endocrinology 237 (2016) 121–130 Contents lists available at ScienceDirect General and Comparative Endocrinology journal hom...

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General and Comparative Endocrinology 237 (2016) 121–130

Contents lists available at ScienceDirect

General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen

Research paper

Arginine vasotocin is the major adrenocorticotropic hormone-releasing factor in the bullfrog Rana catesbeiana Reiko Okada a,⇑,1, Kazutoshi Yamamoto b,1, Itaru Hasunuma c, Jota Asahina a, Sakae Kikuyama b a

Department of Biological Science, Faculty of Science, Shizuoka University, Shizuoka 422-8529, Japan Department of Biology, Faculty of Education and Integrated Sciences, Center for Advanced Biomedical Sciences, Waseda University, Tokyo 162-8480, Japan c Department of Biology, Faculty of Science, Toho University, Chiba 274-8510, Japan b

a r t i c l e

i n f o

Article history: Received 16 June 2016 Revised 6 August 2016 Accepted 24 August 2016 Available online 26 August 2016 Keywords: AVT CRF ACTH Corticotrope AVT receptors Bullfrog

a b s t r a c t In a previous study, we showed that corticotropin-releasing factor (CRF) is the major thyroid-stimulating hormone (TSH)-releasing factor in the bullfrog (Rana catesbeiana) hypothalamus. Our findings prompted us to ascertain whether CRF or arginine vasotocin (AVT), a known adrenocorticotropic hormone (ACTH) secretagogue in several vertebrates, is the main stimulator of the release of ACTH from the bullfrog pituitary. Both the frog CRF and AVT stimulated the release of immunoassayable ACTH from dispersed anterior pituitary cells in vitro in a concentration-dependent manner. AVT, however, exhibited far more potent ACTH-releasing activity than CRF. Although CRF by itself weakly stimulated ACTH release, it acted synergistically with AVT to enhance the release of ACTH markedly. Mesotocin and AVT-related peptides such as hydrin 1 and hydrin 2 showed relatively weak ACTH-releasing activity. Subsequently, cDNAs encoding the bullfrog AVT V1a-type and V1b-type receptors were molecularly cloned. Reverse transcriptase-PCR using specific primers revealed that the anterior lobe of the pituitary predominantly expressed AVT V1b-type receptor mRNA but scarcely expressed AVT V1a-type receptor mRNA. Abundant signals for V1b-type receptor mRNA in the corticotropes were also detected by in situ hybridization. The results obtained by the experiments with the bullfrog pituitary indicate that AVT acts as the main ACTH-releasing factor through the AVT V1b-type receptor and that CRF acts synergistically with AVT to enhance the release of ACTH. Ó 2016 Elsevier Inc. All rights reserved.

1. Introduction In some non-mammalian vertebrates, corticotropin-releasing factor (CRF) stimulates the release of thyroid-stimulating hormone (TSH) from the pituitary (De Groef et al., 2006). In fact, we have reported that both frog and ovine CRFs potently enhance the release of TSH from pituitary cells of the bullfrog Rana catesbeiana using a homologous radioimmunoassay for bullfrog TSH (Ito et al., 2004; Okada et al., 2005, 2004). Moreover, it has been demonstrated that the TSH-releasing activity of bullfrog hypothalamic extract is considerably decreased in the presence of a CRF receptor antagonist, a-helical CRF9–41 (Ito et al., 2004), and that CRF acts through the type 2 CRF receptor (CRFR2) expressed in thyrotropes (Okada et al., 2009, 2007). These findings suggest that CRF acts as the main TSH-releasing factor, contributing to the regulation of the hypothalamus–pituitary–thyroid axis in amphibians. ⇑ Corresponding author at: Department of Biological Science, Faculty of Science, Shizuoka University, 836 Oya, Suruga-ku, Shizuoka 422-8529, Japan. E-mail address: [email protected] (R. Okada). 1 Both these authors contributed equally to this work. http://dx.doi.org/10.1016/j.ygcen.2016.08.014 0016-6480/Ó 2016 Elsevier Inc. All rights reserved.

CRF was first purified from the ovine hypothalamus on the basis of its adrenocorticotropic hormone (ACTH)-releasing activity (Vale et al., 1981). Subsequently, arginine vasopressin (AVP) was shown to act synergistically with CRF on corticotropes to enhance the release of ACTH in the rat (Gillies et al., 1982; Rivier and Vale, 1983). This phenomenon has been reported in other mammalian species, including humans (DeBold et al., 1984; Favrod-Coune et al., 1993; Salata et al., 1988), non-human primates (Goncharova and Lapin, 2002), horses (Evans et al., 1993), sheep (Familari et al., 1989; Hassan et al., 2003; McFarlane et al., 1995), and mice (Lolait et al., 2007; Müller et al., 2000). However, AVP is generally less potent in stimulating ACTH release than CRF, except in sheep (Familari et al., 1989; Hassan et al., 2003). In some species of submammalian vertebrates, the stimulatory effects of arginine vasotocin (AVT, an AVP ortholog) on the release of ACTH have also been reported. AVT has been shown to stimulate the release of ACTH from the rainbow trout pituitary to a lesser extent than rat CRF and to potentiate the ACTH-releasing activity of rat CRF (Baker et al., 1996). In goldfish, both ovine CRF and AVT stimulate the release of ACTH from dispersed pituitary cells (Fryer et al., 1985), but their action is additive and not synergistic.

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According to Tonon et al. (1986), ovine CRF and AVP exhibit almost equipotent ability to induce the release of ACTH from the marsh frog pituitary, although a synergistic action between these two peptides has not been recognized. In the duck, ovine CRF and AVT synergistically stimulate the release of ACTH, AVT being more potent than CRF in this respect (Castro et al., 1986). In the chicken, a synergistic action of CRF and AVT on corticotropes has also been shown (Madison et al., 2008; Mikhailova et al., 2007), although the results regarding the dominance of the effectiveness between these two peptides are conflicting. Thus, in the submammalian vertebrates, the responsiveness of corticotropes to CRF and AVT seems to vary according to the species. To clarify the roles of CRF and AVT in controlling the release of ACTH from the pituitary of the bullfrog, in which CRF is the major TSH-releasing factor, we first developed a homologous timeresolved fluoroimmunoassay (TR-FIA) system for bullfrog ACTH. Using this system, we analyzed the stimulatory effects of AVT and homologous CRF, either separately or in combination, on the release of ACTH from the bullfrog pituitary. We also investigated the effects of mesotocin (MT) and the hydrins vasotocinyl-GlyLys-Arg (hydrin 1) and vasotocinyl-Gly (hydrin 2), which are present in the amphibian neurohypophysis (Acher et al., 1997), on the release of ACTH. Additionally, we cloned cDNAs encoding the bullfrog AVT V1a-type and V1b-type receptors, and introduced them to examine the expression of mRNAs for these two receptors in corticotropes. 2. Materials and methods 2.1. Animals Adult bullfrogs, R. catesbeiana, harvested from the fields in the vicinity of Tokyo, Japan, were supplied by Oh-uchi Aquatic Animal Supply (Saitama, Japan). They were housed under laboratory conditions and fed bovine liver twice per week. All animal experiments were performed in accordance with the Guide for Care and Use of Laboratory Animals of Shizuoka, Waseda, and Toho Universities. 2.2. Peptides and antibodies Bullfrog CRF (fCRF) was synthesized according to the amino acid sequence deduced from the cDNA sequence as described previously (Ito et al., 2004). Ovine CRF (oCRF; Peptide Institute, Osaka, Japan), AVT (Peptide Institute), MT (Alpha Diagnostic International, San Antonio, TX, USA), hydrin 1 (vasotocinyl-Gly-Lys-Arg; Bachem, Bubendorf, Switzerland), and hydrin 2 (vasotocinyl-Gly; Bachem) were purchased from commercial suppliers. Bullfrog ACTH (fACTH: SYSMEHFRWGKPVGKKRRPIKVFPTDAEEESSESFPLEL) and a-melanocyte-stimulating hormone (a-MSH: SYSMEHFRWGKPVNH2) were synthesized according to the amino acid sequence deduced from the nucleotide sequence of bullfrog proopiomelanocortin (POMC) cDNA (Aida et al., 1999). Antiserum to fACTH was produced in a mature female rabbit using the lymph node injection technique (Goudie et al., 1966). For immunization, 100 lg of synthetic fACTH conjugated with bovine serum albumin (BSA) according to the carbodiimide method (Previero et al., 1973) was dissolved in 100 lL saline, emulsified with an equal volume of Freund’s complete adjuvant (Gibco, Detroit, MI, USA), and injected into the popliteal lymph nodes under pentobarbital (Nembutal) anesthesia. Starting two weeks after the first injection, four subcutaneous injections (50 lg fACTH, each) were administered into the dorsal surface of the animal every two weeks. Blood was collected from the marginal ear vein every week, starting after the second set of injections, and tested for antibody titer. Two weeks after

the last injection, the rabbit was bled from the carotid artery. The serum was separated by centrifugation and stored at 80 °C. 2.3. TR-FIA Synthetic fACTH was labeled by europium (Eu) using the DELFIA Eu-labeling kit (Perkin-Elmer, Boston, MA, USA). The labeling was performed at 25 °C for 16 h in 50 lL of 0.1 M carbonate buffer (pH 8.5), containing 50 lg fACTH and 10 lg Eu-labeling reagent (Perkin-Elmer). The Eu-labeled peptide was separated from free Eu by gel filtration chromatography with Sephadex G-25 (GE Healthcare, Buckinghamshire, UK). The TR-FIA of fACTH was performed using the double-antibody method (Kaneko and Hasegawa, 2007). A 96-well microtiter plate (Nunc, Roskilde, Denmark) was coated with coating buffer [0.05 M K2HPO4, 0.15 M NaCl, 0.05 % (w/v) sodium azide] containing goat IgG against the rabbit IgG (50 lg/mL) at room temperature for 16 h. After removal of the goat IgG solution, anti-fACTH diluted to 1:16,000 by assay buffer [0.05 M Tris, 0.15 M NaCl, 0.5% (w/v) BSA, 0.05% (w/v) c-globulins, 20 mM diethylenetriaminepentaacetic acid, 0.05% (w/v) sodium azide, 0.15% (w/v) phenol red, 0.01% (v/v) Tween 40, pH 7.8] was bound to the plate at room temperature for 16 h and then washed six times with wash buffer [1% (v/v) Tween 20, 0.05 M Tris-buffered saline (pH 7.8)]. After 100 lL of fACTH reference standard or test samples that were serially diluted with assay buffer and 100 lL of Eu-labeled fACTH (1:200) were added to each well of the plate, a competitive antigen-antibody reaction proceeded at room temperature for 2 h. After washing eight times with the wash buffer, 100 lL DELFIA Enhancement Solution (Perkin-Elmer) was added to each well and the plate was shaken for 5 min to dissociate Eu from the antibodyantigen complex on the well surface. The fluorescence intensity of the dissociated Eu was measured using the multilabel reader ARVO X3 (Perkin-Elmer). The intensity in the well containing the labeled ACTH and antiserum but no unlabeled ligand was designated 100%, and the counts in the other wells were expressed as a fraction of this fluorescence intensity. To check the specificity of the TR-FIA, several dilutions of the anterior pituitary extract [one piece in 1 mL phosphate-buffered saline (PBS)], medium in which anterior pituitary cells were cultured for 12 h (see below), and a-MSH were subjected to this assay system. 2.4. Pituitary cell culture Cells from the anterior lobe of the pituitary were dispersed in Medium 199 (M199; Nissui Pharmaceutical, Tokyo, Japan) diluted to 70% for amphibian cells containing collagenase (Wako Pure Chemicals, Osaka, Japan) according to the procedures previously described (Oguchi et al., 1996; Okada et al., 2004). The cell suspension was adjusted to a density of 3.5  105 cells/mL in 70% M199. Aliquots (200 lL) of the medium containing 7.0  104 cells were plated in each well of a 96-multiwell plate (Nunc) and incubated at 23 °C in a humidified atmosphere of 95% air–5% CO2. After preincubation for 24 h, the medium was replaced with 70% M199 containing the desired test substance. After incubation for 0, 3, 6, or 12 h, the medium was collected from each well and centrifuged at 100g for 5 min. The resulting supernatant was stored at 20 °C until use. 2.5. Molecular cloning of bullfrog (f) AVT V1a- and V1b-type receptor cDNAs Sequences of all PCR primers used in this study are listed in Table 1. Total RNA was extracted from bullfrog brains using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. Contaminating genomic DNA in the total

R. Okada et al. / General and Comparative Endocrinology 237 (2016) 121–130 Table 1 Primers used for cDNA cloning, RT-PCR, and in situ hybridization. Name

Sequence (50 to 30 )

fV1a-ORFS fV1a-ORFA fV1a-S1 fV1a-S2 fV1a-A1 fV1a-A2 V1b-S V1b-A fV1b-S fV1b-A Adaptor-(dT)17 primer 30 -RACE adaptor primer 50 -RACE abridged anchor primer Abridged universal amplification primer fV1a-PCRS fV1a-PCRA fV1b-PCRS fV1b-PCRA fb-actin-PCRS fb-actin-PCRA fV1a-ISHS fV1a-ISHA fV1b-ISHS fV1b-ISHA fPOMC-ISHS fPOMC-ISHA

TATAGCGATCGCCATGGGCTTCTCTAAACTGGG TATAGTTTAAACTCAGATTTGCAGGGGCAGGA TCATCTTGCTGACCTGCTATGGC TCACCGTGTCCGCTTTGTTAGC GGCAAACATCCCAAACACTTGAAG CTTGACGATGCTGCTCTCAT ACYGTGAAGATGACCTTTTGT AGRAGCATGGWGATGGTGAA TGTGGTCTGTATGGGATGAAAATGC CATCTGAACGCTGAAGAAGGGTGTC GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT GGCCACGCGTCGACTAGTAC GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG GGCCACGCGTCGACTAGTAC GGTGGTGGCTTTTTTCCAGGTG CGCAAACATCCCAAACACTTGAAG TGTGCCCGTTGGAATCCTTG CCCTGGAAATAGTTTGTATGCTGC TCTTCCAGCCATCCTTCTTGGG TACCTCCAGACAGCACAGTGTTGG TGGATAATGCCACCAGTGAG GTAGCAAATAAAGCCATAGCAGG CCAGGATGCATCTCTTTATTG ATCTGGAGCATTTTCATCCC GTGCTGGGAAAGCAATAAGT AATGGGGAAACTTTCTGAGG

RT = reverse transcriptase; RACE = rapid amplification of cDNA ends.

RNA sample was digested with deoxyribonuclease I (Takara Bio, Shiga, Japan). One microgram of the total RNA was reverse transcribed using Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and oligo-deoxythymidine (dT)12–18 primer. The cDNA containing the entire coding sequence for fAVT V1a-type receptor was amplified from the brain cDNA with primers fV1a-ORFS and fV1a-ORFA, designed according to the sequence of bullfrog AVT receptor mRNA (GenBank accession number AY277924) (Acharjee et al., 2004). The sense primer contained a SgfI site and translation initiation signal sequence, and the antisense primer contained a PmeI site and translation stop codon sequence. PCR amplification was performed with KOD Plus DNA polymerase (Toyobo, Osaka, Japan). PCR consisted of 35 cycles of 10 s at 98 °C, 30 s at 60 °C, and 90 s at 68 °C carried out in a 50 lL reaction mixture. The amplified product was subcloned into the pF9A Flexi CMV vector (Promega, Madison, WI, USA) and sequenced. The unknown sequences including 50 - and 30 -untranslated regions of the fAVT V1a-type receptor cDNA were analyzed by 50 - and 30 -rapid amplification of cDNA ends (RACE) methods, respectively. For 50 -RACE, the total RNA obtained from the brain was reverse transcribed with fV1a-A1 primer, and single-strand cDNA was subsequently obtained following the application of RNase H (Takara Bio). Poly(C) was added to the 30 terminal of the single-strand cDNA by terminal deoxynucleotidyl transferase (Takara Bio), and the resultant poly(C)-tailed single-strand cDNA was used as a template in the first-round PCR with the 50 -RACE abridged anchor primer and fV1a-A1 primer. The second-round PCR was performed with an abridged universal amplification primer and fV1a-A2 primer, using the diluted first-round PCR reaction solution as a template. The first- and second-round PCR consisted of 30 cycles of 30 s at 94 °C, 30 s at 60 °C, and 1 min at 72 °C in a 20 lL reaction mixture. For 30 -RACE, the first-strand cDNA for 30 -RACE was synthesized using the adaptor-(dT)17 primer, and the first-round PCR was performed with the fV1a-S1 primer and 30 -RACE adaptor primer. The second-round PCR was

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performed with the fV1a-S2 primer and 30 -RACE adaptor primer using the diluted first-round PCR reaction solution as a template. The first- and second-round PCR consisted of 30 cycles of 30 s at 94 °C, 30 s at 60 °C, and 2 min at 72 °C in a 20 lL reaction mixture. The RACE products of the expected sizes were excised after agarose gel electrophoresis, purified, subcloned into pT7Blue T-vector (Novagen, Madison, WI, USA), and sequenced. A cDNA fragment of fAVT V1b-type receptor was obtained by reverse-transcriptase (RT)-PCR using primers V1b-S and V1b-A designed through a BLAST alignment search on the Xenopus tropicalis genome database (http://genome.jgi-psf.org/Xentr4/Xentr4. home.html) (Hasunuma et al., 2007). Total RNA was extracted from bullfrog anterior pituitary, treated with deoxyribonuclease I (Takara Bio), and reverse transcribed using Superscript III Reverse Transcriptase (Invitrogen) and oligo dT12–18 primer as described above. PCR amplification was performed with Ex Taq DNA polymerase (Takara Bio) under the following cycling conditions: denaturation at 94 °C for 5 min, followed by 40 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. The PCR products of the expected size were excised after agarose gel electrophoresis, purified, subcloned into pT7Blue T-vector (Novagen), and sequenced. To determine the full-length sequence of fAVT V1b-type receptor cDNA, 50 - and 30 -RACE methods were conducted as described above for the fAVT V1a-type receptor cDNA. For 50 -RACE, anterior pituitary total RNA was reverse transcribed with V1b-A primer and added poly(C) tail. The cDNA was used as a template for the first-round PCR with a 50 -RACE abridged anchor primer and V1b-A primer, followed by second-round PCR with abridged universal amplification primer and gene-specific fV1b-A primer. A first-strand cDNA for 30 -RACE was synthesized using adaptor(dT)17 primer, and the first-round PCR was performed with V1b-S and 30 -RACE adaptor primers. For the second-round PCR, fV1b-S and 30 -RACE adaptor primers were used. 2.6. RT-PCR Total RNAs were extracted from various organs, including the anterior and neurointermediate lobes of the pituitary, kidney, interrenal gland, urinary bladder, liver, heart, and oviduct, and were reverse transcribed as described above. Specific primer pairs for V1a-type receptor (fV1a-PCR), V1b-type receptor (fV1b-PCR), and b-actin (fb-actin-PCR) were used in PCRs to amplify 118-, 140-, and 126-bp products, respectively. The PCRs consisted of 1 min at 94 °C followed by 30 (V1a- and V1b-type receptors) or 24 (b-actin) cycles of 30 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C in 10 lL reaction mixtures. The amplified products were visualized on a 3% agarose gel containing ethidium bromide. Authenticity of the products was confirmed by sequencing. 2.7. Dual-label in situ hybridization Partial bullfrog POMC, AVT V1a-, and V1b-type receptor cDNA fragments were amplified by RT-PCR with specific sense and antisense primer pairs of fPOMC-ISH, fV1a-ISH, and fV1b-ISH, respectively. PCR amplification was performed in 50 lL reaction mixtures with EmeraldAmp MAX PCR Master Mix (Takara Bio) under the following conditions: 30 cycles of 10 s at 98 °C, 30 s at 60 °C, and 1 min at 72 °C. The cDNA fragments (460-bp, corresponding to POMC nucleotide positions 145–604; 680-bp, V1a-type receptor 176–855; and 680-bp, V1b-type receptor 381–1060) were subcloned into pSTBlue-1 vector (Novagen) and subjected to PCR to obtain cDNA templates for in vitro transcription. Fluorescein-labeled probes for detection of POMC mRNA and digoxigenin-labeled probes for detection of V1a- and V1b-type receptor mRNAs were synthesized using a fluorescein or digoxigenin RNA labeling kit (Roche Diagnostics, Basel, Switzerland).

R. Okada et al. / General and Comparative Endocrinology 237 (2016) 121–130

2.8. Statistical analysis The significance of differences between the values obtained in each experiment was assessed by Tukey’s test. To estimate the effects of two factors, the data were subjected to two-way ANOVA. If two-way ANOVA revealed the effects were significant, the data were reanalyzed by the procedures described above. A p value of less than 0.05 was considered significant. 3. Results 3.1. Development of TR-FIA for fACTH Using a specific antiserum against fACTH, we developed a homologous TR-FIA system for fACTH. The sensitivity of this TR-FIA, defined as the amount of ACTH that significantly decreased the counts by 2SD from the 100% value (means ± SEM, n = 10), was 18.7 ± 1.2 pg/100 lL assay buffer. The inter- and intra-assay coefficients of variation were 4.2% and 8.0%, respectively. The slopes of inhibition produced by several dilutions of the anterior pituitary extract and the medium in which pituitary cells were cultured for 12 h was parallel to the standard curve of fACTH (Fig. 1). It was confirmed that the fACTH antiserum did not cross-react with a-MSH in this TR-FIA system (Fig. 1).

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% Bound (B/B0 × 100)

Before frogs were sacrificed, they were anesthetized with 0.1% tricaine methanesulfonate (Sigma-Aldrich, St. Louis, MO, USA) and perfused through the heart with an ice-cold solution of 0.65% NaCl, followed by ice-cold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). The pituitary glands with adjacent tissue were removed, postfixed in the same fixative at 4 °C overnight, immersed into 20% sucrose in PB until they sank, and then embedded in OCT compound (Sakura Finetek Japan, Tokyo, Japan). Sections of 10 lm thickness were rinsed in PBS, digested with 1 lg/mL proteinase K in PBS at 37 °C for 15 min, and soaked in 4% PFA to arrest proteolysis. After washing with PBS, the sections were immersed into 2 mg/mL glycine–PBS for 10 min, and then washed in 2 saline sodium citrate buffer (SSC). The sections were prehybridized for 1 h at 50 °C in prehybridization buffer (50% formamide, 2 SSC, 1 Denhardt’s solution, 0.5 mg/mL yeast transfer RNA, 0.5 mg/mL heparin sodium, and 0.1% sodium pyrophosphate). Hybridization was performed at 50 °C for 16 h with a mixture of 1 lg/mL fluorescein-labeled POMC probes and digoxigeninlabeled V1a- or V1b-type receptor probes diluted with hybridization buffer (prehybridization buffer supplemented with 10% dextran sulfate). Following hybridization, the sections were sequentially washed with four changes of 2 SSC at room temperature for 10 min each, washed in 2 SSC–50% formamide at 50 °C for 1 h, washed in 1 SSC–50% formamide at 50 °C for 1 h, and then rinsed in 2 SSC. After post-hybridization steps, slides were incubated with mouse monoclonal anti-fluorescein antibody (Roche Diagnostics) diluted 1:500 with buffer 1 (100 mM TrisHCl, 150 mM NaCl, pH 7.5) and alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche Diagnostics) diluted 1:500 with buffer 1. Sections were washed three times with buffer 1 and incubated with Alexa488-labeled anti-mouse IgG antibody (Invitrogen) at room temperature for 1 h. The sections were then washed three times with buffer 1, immersed in buffer 2 (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl2, pH 9.5), and incubated with a solution of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphste at room temperature for 48 h in the dark for detection of V1a- or V1b-type receptor mRNA signals. Fluorescence of Alexa488 for detection of POMC mRNA signals was detected with an Olympus BX60 microscope equipped with the BX-epifluorescence attachment (Olympus, Tokyo, Japan).

α-MSH

100

8 4 2 1 16 8 4 2 1

80 60 40 20 0 0.01 0.1

1

10 100

ng protein/well Fig. 1. Displacement of Eu-labeled fACTH with serial dilutions of fACTH1–39 standard, extract of the adult bullfrog anterior pituitary (one piece equivalent in 1 mL), the medium in which pituitary cells (7.0  104 cells/200 lL) had been cultured for 6 h, and a-MSH. The linearity and parallelism were verified by Bliss’s method of parallel line assay (Bliss, 1952). All points are averages of two determinations.

3.2. Effects of various peptides on the release of ACTH from the anterior pituitary cells The amounts of fACTH released from the dispersed anterior lobe cells into the culture media in the presence of 107 M AVT or 107 M fCRF were monitored for 12 h. As shown in Fig. 2, AVT markedly enhanced the release of ACTH, and the effect of the secretagogue became conspicuous within 3 h after its addition to the cells. Although the stimulatory effect of fCRF on the release of ACTH also became conspicuous by 3 h of incubation, it was far less potent than that of AVT. AVT at concentrations of 1010–106 M enhanced ACTH release in a concentration-dependent manner, as measured after a 6-h incubation. A much higher concentration of fCRF (108 M) than AVT was required to enhance ACTH release

ACTH release (ng from 104 cells)

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2.0 Control 10−7 M AVT 10−7 M fCRF

1.5

c

B″

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a

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a

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0.5 b

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12

Fig. 2. Changes in the release of ACTH from dispersed anterior lobe cells of the bullfrog pituitary in the presence of 107 M AVT or fCRF during a 12 h culture. Medium containing no test substance was used as the control. Data represent the means ± SEM (n = 7). Values with different lowercase and uppercase letters are significantly different at the 5% level among the values for different additives with the same incubation time and for different incubation times with the same additives, respectively (two-way ANOVA followed by Tukey’s test).

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R. Okada et al. / General and Comparative Endocrinology 237 (2016) 121–130

1.5 cd cd c

1.0 b

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a

e

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0 −10 −9 −8 −7 −6 −10 −9 −8 −7 −6 Control 10 10 10 10 10 10 10 10 10 10 AVT (M) fCRF (M)

from the pituitary cells (Fig. 3). Even when the fCRF concentration was elevated to 106 M, the amount of ACTH released into the medium was only twice the control value, whereas the same concentration of AVT enhanced the release of ACTH approximately 15 times higher than the control value after a 6 h incubation (Fig. 3). As for oCRF, it demonstrated a trend of ACTH-releasing activity slightly above the control level during a 6 h culture at concentrations of 109–106 M (data not shown). To examine the interaction between AVT and fCRF in stimulating the release of ACTH, the anterior pituitary cells were incubated in medium containing a fixed concentration of fCRF (107 M) and various concentrations of AVT (1012–1010 M), either individually or in combination. As shown clearly in Fig. 4, the effects of fCRF and AVT were not additive but synergistic. The values (percent increase over the control value) for the effects of CRF and AVT in

1.5

(971) (837) (678)

1.0 (581) (433)

f

B

ACTH release (ng from 104 cells)

ACTH release (ng from 104 cells)

d

Fig. 3. Effect of AVT or fCRF on the release of ACTH from dispersed anterior lobe cells of the bullfrog pituitary during a 6 h incubation. Medium containing no test substance was used as the control. Data represent the means ± SEM (n = 7). Values with different letters are significantly different from each other at the 5% level (Tukey’s test).

ACTH release (ng from 104 cells)

ACTH release (ng from 104 cells)

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0.6 0.4 0.2

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0 10−7 10−6 10−5 Control Hydrin 1 (M)

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Hydrin 2 (M)

0.3 c c

0.2 b

0.1

0

a a

a

Control 10−10 10−9 10−8 10−7 10−6 MT (M)

Fig. 5. Effects of hydrin 1 and 2 (A) and MT (B) on the release of ACTH from dispersed anterior lobe cells of the bullfrog pituitary during a 6 h incubation. Medium containing no test substance was used as control. Data represent the means ± SEM (n = 7). Values with the different letters are significantly different from each other at the 5% level (Tukey’s test).

combination were significantly higher than those for the sum of the effects of CRF and AVT alone. Hydrin 1 and 2 also stimulated the release of ACTH from the pituitary cells during the 6 h incubation, but their effects were far less potent than that of AVT (Fig. 5A). Likewise, the ACTH-releasing activity of MT was low, only 5–15% of that of AVT (Fig. 5B).

f

ef

de

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3.3. Molecular cloning of cDNAs encoding fAVT V1a- and V1b-type receptor

(233)

0.5 (83)

bc

b a

0 Control

10−12 10−11 10−10 10−7 10−12 10−11 10−10 fCRF (M) AVT (M) fCRF (10−7 M) + AVT (M)

Fig. 4. Effect of AVT and/or 107 M fCRF on the release of ACTH from dispersed anterior lobe cells of the bullfrog pituitary during a 6 h incubation. Medium containing no test substance was used as the control. Data represent the means ± SEM (n = 7). Values with different letters are significantly different from each other at the 5% level (Tukey’s test). Numbers in parentheses indicate the percent increase over the control value. Note that at any comparable AVT concentration, the value for the combination of CRF and AVT is higher than the sum of the value for CRF and AVT.

To clarify the function of AVT with respect to the ACTH release at the receptor level, cDNAs encoding fAVT V1a- and V1b-type receptors were cloned from bullfrog brain and anterior pituitary, respectively, using the 50 - and 30 -RACE method. The fAVT V1atype receptor cDNA included 1260 bp of a coding region, which encoded 419 amino acid residues (Fig. 6). When compared with the sequence of the fAVT V1a-type receptor cDNA reported by Acharjee et al. (2004), there were some inconsistencies in the nucleotide sequences: 26 bases in the 50 -untranslated region, 5 bases in the coding region, and 5 bases in the 30 -untranslated region. The difference in nucleotides in the coding region caused replacement of three amino acid residues. The fAVT V1b-type

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Fig. 6. Nucleotide and deduced amino acid sequences of the bullfrog AVT V1a-type receptor (GenBank Accession number LC155110). An asterisk indicates the termination codon. Seven putative transmembrane domains are underlined (1–7, SOSUI engine version 1.11, http://harrier.nagahama-j-bio.ac.jp/sosui/) (Hirokawa et al., 1998). Circled residues are putative N-glycosylation sites. Triangles indicate potential phosphorylation sites. The Asp in the second transmembrane domain and a tripeptide (Asp-Arg-Tyr) in the surface of the second intracellular loop, which are important for receptor activation, are shadowed. Nucleotides or amino acid residues that were not consistent with the corresponding nucleotides or amino acid residues of the vasotocin receptor cloned from the brain of the bullfrog inhabiting South Korea (Acharjee et al., 2004) are boxed in black.

receptor cDNA consisted of 1176 bp of a coding region, which encoded a protein of 391 amino acid residues (Fig. 7). The AVT V1b-type receptor of the bullfrog showed 78%, 64%, 62%, 58%,

and 55% amino acid identity with AVT/AVP V1b-type receptors of X. tropicalis (GenBank accession number ALG00073) (Yun et al., 2015), Japanese red-bellied newt (BAF38756) (Hasunuma et al.,

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Fig. 7. Nucleotide and deduced amino acid sequences of the bullfrog AVT V1b-type receptor (GenBank Accession number LC149903). An asterisk indicates the termination codon. Seven putative transmembrane domains are underlined (1–7, SOSUI engine version 1.11, http://harrier.nagahama-j-bio.ac.jp/sosui/) (Hirokawa et al., 1998). Circled residues are putative N-glycosylation sites. Triangles indicate potential phosphorylation sites. The Asp in the second transmembrane domain and a tripeptide (Asp-Arg-Tyr) in the surface of the third transmembrane domain, which are important for receptor activation, are shadowed.

2007), Western painted turtle (XP_005306529), rat (AAC52235) (Lolait et al., 1995), and chicken (NP_001026669) (Cornett et al., 2003), respectively. The predicted amino acid sequences of V1a- and V1b-type receptors exhibited the typical features of a G protein-coupled receptor: seven transmembrane domains, a few putative N-linked glycosylation sites, and several potential phosphorylation sites in the intracellular loop and C-terminal intracellular domain (Figs. 6 and 7). A relatively low sequence identity was measured between the fV1b-type receptor and

fV1a-type receptor (43%) or MT receptor (43%, AAQ22365) (Acharjee et al., 2004). 3.4. Expression of fAVT V1a- and V1b-type receptor mRNAs in the anterior pituitary We examined the expression of AVT V1a- and V1b-type receptor mRNAs in the anterior lobe of the pituitary by RT-PCR using specific primers. For comparison, the neurointermediate lobe of

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4. Discussion

Fig. 8. Expression of AVT V1a- and V1b-type receptor mRNAs in the anterior lobe (AL) of the pituitary of the bullfrog. The expression in the neurointermediate lobe (NIL) of the pituitary, kidney, interrenal gland (IG), urinary bladder (UB), liver, heart, and oviduct were also examined for reference. PCR products were electrophoresed on an agarose gel and stained with ethidium bromide. b-actin mRNA was used as an internal control.

the pituitary, kidney, interrenal gland, urinary bladder, liver, heart, and oviduct were analyzed according to the same procedure. The V1a-type receptor mRNA expression in the anterior lobe was detected only slightly, whereas its expression was clearly observed in the kidney, interrenal gland, and urinary bladder. In contrast, V1b-type receptor mRNA was prominently expressed in the anterior pituitary and at relatively low levels in the kidney, interrenal gland, and urinary bladder (Fig. 8). Subsequently, distribution of V1a- and V1b-type receptor mRNAs in the anterior lobe of the pituitary was investigated by in situ hybridization, using a digoxigenin-labeled antisense probe for mRNAs of fAVT V1a- or fV1b-type receptors. The positive signals for V1a-type receptor mRNA were not observed in any cells of the anterior pituitary (data not shown). On the contrary, abundant signals for V1b-type receptor mRNA were observed mainly in the ventro-rostral region of the anterior lobe (Fig. 9A). In situ hybridization with a fluorescein-labeled antisense probe for POMC mRNA revealed V1b-type receptor mRNA signals expressed in the corticotropes (Fig. 9C and D). Control sections incubated with a digoxigeninlabeled sense probe for V1b-type receptor mRNA showed only background signals (Fig. 9B).

To determine the main ACTH secretagogue in the bullfrog, we developed a TR-FIA system employing ACTH synthesized according to the amino acid sequence deduced from the nucleotide sequence of the bullfrog POMC cDNA that was previously cloned by our group (Aida et al., 1999). This is the first homologous assay system for amphibian ACTH. A direct assay of ACTH, instead of measuring corticoids, is of particular importance for studying the effect of AVT on the release of ACTH from the pituitary, because not only ACTH, which stimulates the release of corticosteroids from the amphibian interrenal gland (Maser et al., 1982; Morra et al., 1990), but also AVT stimulates the release of corticoids acting directly on the interrenal gland (Iwamuro et al., 1991; Larcher et al., 1989). Using the TR-FIA system, we showed that the ACTH-releasing activity of AVT was far more potent than that of fCRF at every concentration examined. It is notable, however, that fCRF by itself moderately enhances the release of ACTH in a concentrationdependent manner. As mentioned in the Introduction, in most mammals, the effect of CRF in stimulating the release of ACTH is greater compared with that of AVP, and in non-mammalian vertebrates, the relative effectiveness of these two peptides varies according to the species. In the present experiments, we examined the effects of AVT and CRF only on the release of ACTH. It is, however, of interest to examine the ability of these two peptides in terms of stimulation of the synthesis of ACTH precursor protein (POMC). We also tested the ACTH-releasing activity of MT, hydrin 1, and hydrin 2. MT showed very weak ACTH-releasing activity, considering that its minimum effective concentration is approximately 108 M, whereas 1012 M AVT is enough to stimulate the release of ACTH. Hydrins are non-amidated AVT-related peptides that were isolated from the neurohypophysis of anuran amphibians. Vasotocinyl-Gly-Lys-Arg (hydrin 1) and vasotocinyl-Gly-Lys (hydrin 10 ) were found in the neurohypophysis of Xenopus by Acher’s group (Rouillé et al., 1989) and our group (Iwamuro et al., 1993), respectively. The presence of vasotocinyl-Gly (hydrin

Fig. 9. Expression of AVT V1b-type receptor and POMC mRNAs in the anterior pituitary of the bullfrog as demonstrated by in situ hybridization. A digoxigenin-labeled antisense probe for V1b-type receptor mRNA was hybridized in a pituitary section (A). To ascertain the specificity of the probe binding, an adjacent section was reacted with a digoxigenin-labeled sense probe for V1b-type receptor mRNA (B). Panel C is the magnified image of the framed area in panel A. A fluorescein-labeled antisense probe for POMC mRNA was also hybridized in the same section used for hybridization with V1b-type receptor mRNA. The area comparable to panel C is shown in panel D. Arrows in panels C and D point to the representative cells that express both V1b-type receptor mRNA and POMC mRNA. ME, median eminence; AL, anterior lobe of the pituitary; IL, intermediate lobe of the pituitary; PL, posterior lobe of the pituitary. Bars represent 200 (A and B) or 20 (C and D) lm.

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2) in the neurohypophysis of Bufonidae and Ranidae has also been reported by the former group (Rouillé et al., 1989). We previously isolated two peptides that exert a potent aldosterone-releasing activity from the bullfrog neurointermediate lobes and the active substances were revealed to be AVT and hydrin 2 (Iwamuro et al., 1991). The corticoid-releasing activity of AVT and hydrin 2 were nearly equipotent. Hydrin 1 and hydrin 10 also possess a considerable steroid-releasing activity in Xenopus adrenal gland in vitro (Iwamuro et al., 1993). In the present study, however, both hydrin 1 and hydrin 2 exhibited very weak activity in stimulating the release of ACTH from the bullfrog anterior pituitary, with the minimum effective concentration being 105 M. Thus, we conclude that in the bullfrog, hydrin 2 is not involved in the release of ACTH release from the pituitary and that AVT, which is transported from the hypothalamus through the median eminence to the anterior pituitary, acts as the main ACTH-releasing factor. It has been reported that AVT-immunoreactive neurons terminate around the portal vessels in the median eminence of the bullfrog (Carr and Norris, 1990). To determine the receptor for AVT that mediates its ACTH-releasing activity, we cloned fAVT V1a- and V1b-type receptor cDNAs. The fAVT V1a-type receptor cDNA had already been cloned (Acharjee et al., 2004) from the brain of a bullfrog collected in South Korea. Comparing the cDNA that they obtained from the bullfrog brain with the one obtained in the present study, there was a slight inconsistency in the nucleotide and amino acid sequences, which may have arisen from a geographical variation in the bullfrogs in South Korea and Japan. The primary structure of the bullfrog AVT V1b-type receptor showed a considerably high sequence similarity with those of other vertebrates, whereas a relatively low sequence similarity was observed between fAVT V1b-type receptor and fAVT V1a-type receptor or MT receptor. Analysis of mRNA expression of these two AVT receptors in the anterior pituitary using RT-PCR revealed that mRNA for AVT V1btype receptor was markedly expressed, whereas AVT V1a-type receptor mRNA was only slightly expressed. It was noted that among the organs examined in this experiment, expression of mRNA for AVT V1a-type receptor is conspicuous in the kidney, interrenal gland, and urinary bladder. Acharjee et al. (2004) conducted in situ hybridization using the pituitary of the same frog species, and showed that V1a-type receptor mRNA is expressed in both the anterior and neurointermediate lobes of the bullfrog pituitary. Unlike their observation, our in situ hybridization experiment did not yield positive signals for V1a-type receptor mRNA in the area of the pituitary. The definite reason for this discrepancy is not clear. In situ hybridization using an antisense probe for AVT V1b-type receptor mRNA revealed its expression specifically in cells in the rostral region of the anterior pituitary, and that the V1b-type receptor mRNA-expressing cells almost coincide with those expressing POMC mRNA. Thus, we concluded that in the bullfrog, ACTH cells are equipped with the AVT V1b-type receptors, through which AVT exerts its ACTH-releasing activity. This suggests that AVT in the bullfrog stimulates corticotropes through the AVT V1b-type receptor as AVP in mammals (Lolait et al., 1995) and AVT in birds (Jurkevich et al., 2008) do through AVP V1b- and AVT V1b-type (designated as VT2 in avians) receptors, respectively. As mentioned above, our RT-PCR study revealed that both AVT V1a- and V1b-type receptor mRNAs are expressed abundantly in the interrenal gland of the bullfrog. Thus, the direct stimulatory effect of AVT on the release of corticoids from the frog interrenal gland as reported by Larcher et al. (1989) and Iwamuro et al. (1991) is considered to be mediated through V1a- and/or V1b-type receptors. In the present study, we used CRF synthesized according to the sequence of fCRF that was deduced from a cDNA encoding the

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bullfrog CRF precursor (Ito et al., 2004). The amino acid sequence of the fCRF is identical to the sequence of CRF isolated from the brain of the European green frog Rana esculenta (Okada et al., 2005). It is worth mentioning that notwithstanding the use of the homologous CRF, it exerted a far less potent effect on the bullfrog corticotropes to release ACTH than AVT. Another phenomenon observed in the present study is that fCRF, which possesses a relatively weak ACTH-releasing activity by itself, markedly augmented the ACTH-releasing activity of AVT. When both peptides were added simultaneously into the pituitary cell culture medium, the amount of ACTH released by the combination of AVT and CRF was far greater than the sum of the amounts released by AVT and CRF individually. The cellular mechanism underlying the synergistic action of AVT and CRF is not fully understood. In avian species, it was reported that AVT V1b-type receptor and CRF receptor are coexpressed in corticotropes (Mikhailova et al., 2007) and that type 1 CRF receptor (CRFR1) mRNA is dominantly expressed in corticotropes (De Groef et al., 2003). It is of interest to note that mammalian AVP V1b-type receptor and CRFR1 (Young et al., 2007), and avian AVT and CRF receptors (Mikhailova et al., 2007) form heterodimers in corticotropes. The latter investigators infer that this may enhance the ability of the CRF receptor to activate downstream signal transduction. In the bullfrog, we demonstrated the expression of CRFR2 mRNA in thyrotropes, but could not detect CRFR1 mRNA in the corticotropes using in situ RT-PCR (Okada et al., 2009), although the expression of mRNAs for both CRFR1 and CRFR2 in the anterior lobe of the pituitary has been detected by RT-PCR (Ito et al., 2006). To demonstrate the coexpression of AVT V1b-type receptor and CRFR1 mRNAs in the corticotropes of the bullfrog pituitary, some manipulation such as subjecting the frogs to stressful conditions may be necessary. Further studies are needed to elucidate the mechanism of the synergistic action of CRF and AVT to enhance the release of ACTH from corticotropes in the bullfrog at their receptor levels. In conclusion, AVT is the major ACTH-releasing factor in the bullfrog. AVT acts on corticotropes through AVT V1b-type receptor. CRF possesses relatively weak ACTH-releasing activity by itself, but it potentiates the activity of AVT when both peptides act simultaneously. The fact that in the bullfrog, CRF is the major TSHreleasing factor (Okada et al., 2009, 2004), whereas AVT is the major ACTH-releasing factor indicates that shifts in the role of hypothalamic factors among the vertebrate species have occurred during their evolution. Acknowledgments We are grateful to Professor Y. Hasegawa and Ms. C. Miyauchi (Kitasato University) for their valuable advice in performing TR-FIA. We thank Professors S. Tanaka and M. Suzuki (Shizuoka University) for their valuable advice during the experiments. Thanks are also extended to Dr. H. Mochida (Tanpaku Seisei Co.) for his generous technical assistance. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan to R.O. and S.K. (16K074020) and to I.H. (15K07135). References Acharjee, S., Do-Rego, J.L., Oh, D.Y., Moon, J.S., Ahn, R.S., Lee, K., Bai, D.G., Vaudry, H., Kwon, H.B., Seong, J.Y., 2004. Molecular cloning, pharmacological characterization, and histochemical distribution of frog vasotocin and mesotocin receptors. J. Mol. Endocrinol. 33, 293–313. Acher, R., Chauvet, J., Rouillé, Y., 1997. Adaptive evolution of water homeostasis regulation in amphibians: vasotocin and hydrins. Biol. Cell 89, 283–291. Aida, T., Iwamuro, S., Miura, S., Kikuyama, S., 1999. Changes of pituitary proopiomelanocortin mRNA levels during metamorphosis of the bullfrog larvae. Zool. Sci. 16, 255–260.

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