Pituitary adenylate cyclase-activating polypeptide induces somatolactin release from cultured goldfish pituitary cells

Peptides 30 (2009) 1260–1266

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Pituitary adenylate cyclase-activating polypeptide induces somatolactin release from cultured goldfish pituitary cells Morio Azuma a, Mio Tanaka a, Yumika Nejigaki a, Minoru Uchiyama a, Akiyoshi Takahashi b, Seiji Shioda c, Kouhei Matsuda a,* a b c

Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190-Gofuku, Toyama 930-8555, Japan School of Marine Biosciences, Kitasato University, Ofunato, Iwate 022-0101, Japan Department of Anatomy, Showa University School of Medicine, Shinagawa-ku, Tokyo 142-8555, Japan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 12 February 2009 Received in revised form 17 March 2009 Accepted 17 March 2009 Available online 26 March 2009

In the goldfish pituitary, nerve fibers containing pituitary adenylate cyclase-activating polypeptide (PACAP) are located in close proximity to somatolactin (SL)-producing cells, and PACAP enhances SL release from cultured pituitary cells. However, there is little information about the mechanism of PACAPinduced SL release. In order to elucidate this issue, we used the cell immunoblot method. Treatment with PACAP at 108 and 107 M, but not with vasoactive intestinal polypeptide (VIP) at the same concentrations, increased the immunoblot area for SL-like immunoreactivity from dispersed pituitary cells, and PACAP-induced SL release was blocked by treatment with the PACAP selective receptor (PAC1R) antagonist, PACAP(6–38), at 106 M, but not with the PACAP/VIP receptor antagonist, VIP(6–28). PACAPinduced SL release was also attenuated by treatment with the calmodulin inhibitor, calmidazolium at 106 M. This led us to explore the signal transduction mechanism up to SL release, and we examined whether PACAP-induced SL release is mediated by the adenylate cyclase (AC)/cAMP/protein kinase A (PKA)- or the phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP3)/protein kinase C (PKC)-signaling pathway. PACAP-induced SL release was attenuated by treatment with the AC inhibitor, MDL-12330A, at 105 M or with the PKA inhibitor, H-89, at 105 M. PACAP-induced SL release was suppressed by treatment with the PLC inhibitor, U-73122, at 3  106 M or with the PKC inhibitor, GF109203X, at 106 M. These results suggest that PACAP can potentially function as a hypophysiotropic factor mediating SL release via the PAC1R and subsequently through perhaps the AC/cAMP/PKA- and the PLC/ IP3/PKC-signaling pathways in goldfish pituitary cells. ß 2009 Elsevier Inc. All rights reserved.

Keywords: PACAP Goldfish Pituitary Somatolactin Cell immunoblot Hypophysiotropic factor PAC1R Signal transduction

1. Introduction Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally isolated from the ovine hypothalamus during an attempt to isolate a novel hypophysiotropic neuropeptide that could activate adenylate cyclase in cultured rat pituitary cells [28]. Phylogenetically, PACAP occurs throughout the chordate phylum, and its primary structure is highly conserved among vertebrates [47]. PACAP and its receptors including the PACAP selective receptor, PAC1R, and the PACAP/vasoactive intestinal polypeptide (VIP) receptor, VPACR, are widely distributed in the central nervous system and peripheral tissues, and are involved in many physiological processes, such as cell proliferation and differentiation, cell death, neuroendocrine function, neurotransmission and neuroprotection in mammals [3,22,41,47]. Several reports have

* Corresponding author. Tel.: +81 76 445 6638; fax: +81 76 445 6549. E-mail address: [email protected] (K. Matsuda). 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.03.011

also described the distribution and physiological function of PACAP in teleost fish [29,41,52]. Neuronal cell bodies with PACAP-like immunoreactivity are distributed mainly in the diencephalon, and their fibers project into the adenohypophysis of teleosts [26,30,50,52]. PACAP is able to stimulate growth hormone (GH) and gonadotropin release from cultured pituitary cells of teleosts, such as the European eel and salmon, in vitro [30,35]. PAC1R and VPACR have also been cloned and characterized from the goldfish pituitary and brain [9,19,25,50]. Recent studies have indicated that PACAP-containing nerve fibers or endings innervate prolactin (PRL)- and somatolactin (SL)-producing cells in addition to GHand gonadotropin-producing cells in the stargazer pituitary [23,24], that PACAP also stimulates PRL and SL release from cultured pituitary cells in the goldfish [25], and that PACAP enhances SL-a and -b gene expression in the grass carp [13,14]. In the grass carp, PACAP-induced SL-a and -b gene expression is mediated by the PAC1R system and subsequently through two intracellular signal transduction pathways, the adenylate cyclase (AC)/cAMP/protein kinase (PKA)-signaling pathway and the

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phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP3)/protein kinase C (PKC)-signaling pathway, suggesting that, in this species, PAC1R is also coupled with the Gq protein in addition to the Gs protein, as in the case of the goldfish [13,14]. An understanding of the evolutionary background of the regulation of adenohypophysial hormone release by PACAP could provide insight into the role of PACAP in the hypothalamopituitary region throughout the teleost fish. The goldfish has been widely used as an animal model to investigate the effect of PACAP on GH, gonadotropin and PRL release from the pituitary [7,25,39,49,50]. In this species, PACAP-containing nerve fibers or endings also seem to innervate adenohypophysial cells including SL-producing cells, in addition to GH-, gonadotropin- and PRLproducing cells, as is the case in other species [24]. However, there is little information about the effect of PACAP on SL release from the pituitary in goldfish. It is known that SL, recently identified in the pars intermedia of bony and cartilaginous fish comprising two subtypes, SL-a and -b [16,32,56], is involved in the regulation of steroidogenesis, water-mineral balance and body pigmentation, although functional analysis of SL in fish is still under way [15,21,36]. The aim of the present study was to examine the functional relationship between PACAP and SL in the teleost pituitary using the goldfish, a fish model commonly employed for studies of the neuroendocrine actions of PACAP. PACAP is known to modulate the AC/cAMP/PKA-signaling pathway or the PLC/IP3/PKC-signaling pathway in teleost pituitary cells as described above. Given that a similar mechanism might occur in relation to the regulation of SL release from pituitary cells, we examined the effect of PACAP on SL release in dispersed and cultured pituitary cells using the cell immunoblot method [25]. 2. Materials and methods 2.1. Animals Goldfish (Carassius auratus, body weight [BW] 10–25 g) of both sexes were purchased from a commercial supplier, and kept for 2 weeks under controlled light/dark conditions (12L/12D) with the water temperature maintained at 20–24 8C. The fish were fed once per day at 12:00 noon with a uniform granular diet (containing 32% protein, 5% dietary fat, 2% dietary fiber, 6% minerals and 8% water, Tetragold, Tetra GmbH, Melle, Germany) until used in experiments. All animal experiments were conducted in accordance with the University of Toyama guidelines for the care and use of animals. 2.2. Chemicals In order to examine the effect of PACAP on SL release from cultured pituitary cells, the following chemicals were used. Human/ovine/rat PACAP (Peptide Institute Inc., Osaka, Japan), bovine/canine/human/rat VIP (Peptide Institute), the PACAP selective receptor (PAC1R) antagonist PACAP(6–38) (Peptide Institute) and the PACAP/VIP receptor (VPACR) antagonist VIP(6–28) (Sigma–Aldrich Co., St. Louis, MO). All were purchased commercially, dissolved in distilled water at 0.1 mM for storage at 80 8C, and diluted with 0.6% NaCl and 0.02% Na2CO3 solutions (saline) before use. In order to examine the signal transduction pathways for PACAP-induced SL release, the calmodulin inhibitor calmidazolium (calmidazolium chloride) (1-[bis(4-chlorophenyl)methyl]3-[2,4-dichloro-b-(2,4-dichlorobenzyloxy)phenethyl]imidazolium chloride; Calbiochem, La Jolla, CA), the AC inhibitor MDL-12330A (cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine hydrochloride; Calbiochem), the PKA inhibitor H-89 (N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide dihydrochlor-

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ide; Calbiochem), the PLC inhibitor U-73122 (1-[6-((17b-3methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole2,5-dione; Calbiochem) and the PKC inhibitor GF109203X (bisindolylmaleimide I) (2-[1-(3-dimethylaminopropyl)-1H-indol-3yl]-3-81h-indo-3-yl)-maleimide; Calbiochem) were also purchased commercially, dissolved in dimethyl sulfoxide at 10 mM for storage, and diluted with saline before use. 2.3. Primary cell culture and cell immunoblot In order to examine the effect of PACAP on SL release from goldfish pituitary cells, the cell immunoblot method was carried out as described previously [4,10,42,43] with modifications for use on isolated goldfish pituitary cells [25]. This method allowed for the semiquantitative measurement of SL release from individual cells [25]. In brief, goldfish were anesthetized with MS-222 (3aminobenzoic acid ethyl ester; Sigma–Aldrich), decapitated, and the whole pituitaries were rapidly dissected out under sterile conditions. The pituitaries were cut into small pieces and transferred into Leibovitz’s L-15 medium (Gibco Invitrogen Co., Grand Island, NY) containing 25 mM HEPES pH 7.4, 3 mM EDTA and 2500 U/ml collagenase (type I; Worthington Biochemical Co., Lakewood, NJ). After mechanical and enzymatic dispersion, the suspension was centrifuged at 200  g for 10 min, and the supernatant was removed. Dispersed cells were then resuspended in Leibovitz’s L-15 medium containing 25 mM HEPES pH 7.4, 10% fetal bovine serum (Sigma–Aldrich), 100 mg/ml streptomycin (MBC, Tokyo, Japan) and 100 U/ml penicillin (MBC) at 27 8C for 24 h to avoid contamination and degradation of the neurohypophysial cells. The number of cells in the suspension was then calculated and the density of the cells was adjusted to 105 cells/ml. Five hundred microliters of the suspension was plated in the cassette chamber that was located beneath the PVDF membrane (Hybond-P, polyvinylidene difluoride transfer membrane, Amersham Biosciences, Bucks, UK) and incubated at room temperature in a humidified atmosphere with filtered-clean air. After preincubation for 3 h, PACAP or VIP were added to the culture medium at concentrations of 109, 108 and 107 M or an equivalent volume of saline (control), and the incubation was continued for a further 3 h. After incubation, the PVDF membrane was collected from each cassette chamber and for immunostaining of SL in the PVDF membrane, we used rabbit primary antiserum against salmon SL (anti-SL serum, diluted 1:8000, [37]). Primary structure of salmon SL has 44–72% homology to those of goldfish SL-a and -b (directly submitted by Jiang and Wong, 2008; GenBank accession no EU580712 and U72940) and anti-SL serum can cross-react with goldfish SL-a and -b. Previous preabsorption tests of anti-SL serum resulted in no specific staining; the specificity of this antiserum has been well demonstrated in teleost pituitaries and checked by immunoblotting analysis and radioimmunoassay [23–25,37]. In brief, each membrane was treated with 10% bovine serum albumin (Sigma–Aldrich) in 0.1 M Tris–HCl, pH 7.4, 0.1 M NaCl and 0.1% Tween-20 (TBS-T) for 1.5 h, washed with TBS-T, and then stained with anti-SL serum (diluted 1:8000) at room temperature overnight. Following a 1-h incubation with biotinylated swine antirabbit immunoglobulin G (diluted 1:100, Vector Laboratories Inc., Burlingame, CA), each membrane was treated with avidinbiotinylated peroxidase complex (ABC) (Vector Laboratories) for 1.5 h and then incubated with 3,30 -diaminobenzidine-4HCl (DAB, Sigma–Aldrich) and 0.03% H2O2 in 15 ml 0.05 M Tris–HCl buffer (pH 7.6) containing 150 mM NaCl. Immunostained membranes were observed under a light microscope (BH40, Olympus, Tokyo, Japan), and digital images as a gray scale were recorded with a digital camera (CoolPix 995, Nikon, Tokyo, Japan). The density, total area and number of the immunoblots were measured using the Scion Image freeware program (Scion Corporation, ML).

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2.4. Effect of PACAP receptor antagonists on PACAP-induced SL release In order to examine the effect of the PACAP receptor antagonists on PACAP-induced SL release from goldfish pituitary cells, the cell immunoblot method was carried out as described above. After preincubation of dispersed pituitary cells for 3 h, PACAP at 108 M and/or PACAP(6–38) at 106 M or VIP(6–28) at 106 M was added to the culture medium or an equivalent volume of saline (control) was added, and the incubation was continued for a further 3 h. The incubation doses of PACAP(6–38) and VIP(6–28) had been determined in previous studies using goldfish and rainbow trout, and it had been demonstrated that, in these species, these compounds antagonized each respective agonist [31,49,50]. After incubation, each PVDF membrane was collected from each

cassette chamber and immunostained with the anti-SL serum as described above. 2.5. Effect of calmodulin inhibitor on PACAP-induced SL release In order to examine the effect of the calmodulin inhibitor on PACAP-induced SL release from goldfish pituitary cells, the cell immunoblot method was carried out as described above. After preincubation of dispersed pituitary cells for 3 h, PACAP at 108 M and/or calmidazolium at 106 M were added to the culture medium or an equivalent volume of vehicle (same concentration of dimethyl sulfoxide diluted with saline) was added, and the incubation was continued for a further 3 h. The incubation doses of calmidazolium had been determined in previous studies using

Fig. 1. Effect of PACAP and VIP on SL release from cultured goldfish pituitary cells (A,B), and effect of PACAP receptor antagonists on PACAP-induced SL release (C,D). Each column and bar represents the mean  SEM, respectively, and numbers in parentheses in each panel indicate the number of immunoblots in each experimental or control group. Significance of differences between saline- and PACAP-incubated groups (A) was evaluated by one-way ANOVA with the Bonferroni test (*p < 0.05; **p < 0.01). Significance of differences between groups (C,D) was evaluated by two-way ANOVA with the Bonferroni test (**p < 0.01). A photomicrograph in each column indicates representative cell immunoblots in each experimental or control group. Scale bar in the photomicrographs is 200 mm.

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Fig. 2. Effect of calmodulin inhibitor on PACAP-induced SL release from cultured goldfish pituitary cells. Each column and bar represents the mean  SEM, respectively, and numbers in parentheses in each panel indicate the number of immunoblots in each experimental or control group. Significance of differences between groups was evaluated by two-way ANOVA with the Bonferroni test (**p < 0.01). A photomicrograph in each column indicates representative cell immunoblots in each experimental or control group. Scale bar in the photomicrographs is 200 mm.

rainbow trout, and it had been demonstrated that, in this species, the reagent specifically inhibits the calmodulin-signaling pathway [2]. After incubation, each PVDF membrane was collected from each cassette chamber and immunostained with the anti-SL serum as described above. 2.6. Effect of AC, PKA, PLC and PKC inhibitors on PACAP-induced SL release In order to examine the effect of the AC, PKA, PLC and PKC inhibitors on PACAP-induced SL release from goldfish pituitary cells, the cell immunoblot method was carried out as described above. After preincubation of dispersed pituitary cells for 3 h, PACAP at 108 M and/or MDL-12330A at 105 M, H-89 at 105 M, U-73122 at 3  106 M or GF109203X at 106 M was added to the culture medium or an equivalent volume of vehicle (same concentration of dimethyl sulfoxide diluted with saline) was added, and the incubation was continued for a further 3 h. The incubation doses of MDL-12330A, H-89, U-73122 and GF109203X had been determined in previous studies using fish including goldfish, grass carp and tilapia, and it had been demonstrated that, in these species, these reagents specifically inhibit each respective target enzyme [7,18,46,49,51]. After incubation, each PVDF membrane was collected from each cassette chamber and immunostained with the anti-SL serum as described above. 2.7. Data analysis All results are expressed as the mean  SEM. Statistical analysis was performed using one-way or two-way ANOVA with Bonferroni’s method. Statistical significance was determined at the 5% level.

Fig. 3. Effect of AC (A) and PKA (B) inhibitors on PACAP-induced SL release from cultured goldfish pituitary cells. Each column and bar represents the mean  SEM, respectively, and numbers in parentheses in each panel indicate the number of immunoblots in each experimental or control group. Significance of differences between groups was evaluated by two-way ANOVA with the Bonferroni test (**p < 0.01). A photomicrograph in each column indicates representative cell immunoblots in each experimental or control group. Scale bar in the photomicrographs is 200 mm.

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3. Results 3.1. Effect of PACAP and VIP on SL release from cultured pituitary cells, and effect of PACAP receptor antagonists on PACAP-induced SL release Incubation of pituitary cells for 3 h with graded concentrations of PACAP at doses of 109, 108 and 107 M provoked a dosedependent increase in the immunoblot area for SL-like immunoreactivity. Treatment of cells with 108 and 107 M PACAP induced a significant increase in the immunoblot area for SL-like immunoreactivity (Fig. 1A). On the other hand, there was no evident change in the immunoblot area for SL-like immunoreactivity after treatment with 109, 108 and 107 M VIP (Fig. 1B). Treatment with PACAP alone at 108 M for 3 h enhanced the immunoblot area for SL-like immunoreactivity in comparison with treatment with saline, or with PACAP(6–38) alone at 106 M. PACAP(6–38) at 106 M, which had no effect by itself, suppressed the stimulatory action of PACAP (Fig. 1C). The interaction between PACAP and PACAP(6–38) was shown to be significant by two-way ANOVA with Bonferroni’s method (F and p values, 14.26 and 0.0002, respectively). VIP(6–28) at 106 M did not affect the response to PACAP at 108 M (Fig. 1D). There was no interaction between PACAP and VIP(6–28). 3.2. Effect of calmodulin inhibitor on PACAP-induced release in cultured pituitary cells The stimulatory effect of PACAP on SL release was blocked by treatment with calmidazolium at 106 M (Fig. 2). Interaction between the effects of PACAP and calmidazolium was shown to be significant by two-way ANOVA with Bonferroni’s method (d.f., F and p values, 1, 8.60 and 0.0034, respectively). 3.3. Effect of AC and PKA inhibitors on PACAP-induced SL release in cultured pituitary cells The stimulatory effect of PACAP on SL release was blocked by treatment with MDL-12330A at 105 M (Fig. 3A). Interaction between effects of PACAP and MDL-12330A was shown to be significant by two-way ANOVA with Bonferroni’s method (F and p values, 5.90 and 0.015, respectively). PACAP-induced increase in the immunoblot area for SL-like immunoreactivity was also abolished by treatment with H-89 at 105 M (Fig. 3B). Interaction between the effects of PACAP and H-89 was shown to be significant by two-way ANOVA with Bonferroni’s method (d.f., F and p values, 1, 6.65 and 0.01, respectively). 3.4. Effect of PLC and PKC inhibitors on PACAP-induced SL release from cultured pituitary cells The stimulatory effect of PACAP on SL release was blocked by treatment with U-73122 at 3  106 M (Fig. 4A). Interaction between the effects of PACAP and U-73122 was shown to be significant by two-way ANOVA with Bonferroni’s method (F and p values, 5.11 and 0.024, respectively). The stimulatory effect of PACAP on SL release was also abolished by treatment with GF109203X at 106 M (Fig. 4B). Interaction between the effects of PACAP and GF109203X was shown to be significant by two-way ANOVA with Bonferroni’s method (d.f., F and p values, 1, 8.56 and 0.0035, respectively). 4. Discussion This is the first report to describe in detail the stimulatory effect of PACAP on SL release from cultured goldfish pituitary cells in vitro. Recent studies have indicated that the PACAP gene in teleosts

Fig. 4. Effect of PLC (A) and PKC (B) inhibitors on PACAP-induced SL release from cultured goldfish pituitary cells. Each column and bar represents the mean  SEM, respectively, and numbers in parentheses in each panel indicate the number of immunoblots in each experimental or control group. Significance of differences between groups was evaluated by two-way ANOVA with the Bonferroni test (**p < 0.01). A photomicrograph in each column indicates representative cell immunoblots in each experimental or control group. Scale bar in the photomicrographs is 200 mm.

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encodes PACAP and PACAP-related peptide [20,45], but not GHreleasing hormone [1,11,27,34,35,40]. PACAP is able to stimulate GH release from cultured pituitary cells of salmon [35], European eel [30], goldfish [39,50], carp [53], grass carp [44,51] and turbot [38]. In the present study, we used a cell immunoblot to examine the effect of PACAP on SL release from the goldfish pituitary. This method has been used to examine the regulation of pituitary hormone secretion [4,10,42,43], and has been shown to be suitable for demonstrating hormone release from individual cells isolated from rat pituitary. As no such immunoassay system has been developed for measuring adenohypophysial hormone levels in the goldfish pituitary, we have previously modified this method to examine whether PACAP stimulates the release of PRL and SL from dispersed and cultured pituitary cells [25]. An anti-SL serum used in the present study crossreacts with SL-a and -b. Our preliminary study found that the mean level of SL-b mRNA expression was approximately three times that of SL-a mRNA expression in cultured goldfish pituitary cells using a real-time PCR method (Azuma and Matsuda, unpublished data). Whether PACAP induces SL-a and -b release from cultured pituitary cells remains to be determined. Previous immunohistochemical studies of teleost pituitaries have positioned the main populations of PACAP-containing neuronal cell bodies in the diencephalon, with nerve fibers in various areas of the brain and nerve endings in the adenohypophysis; PACAP-positive nerve fibers innervate GH-producing cells in the pituitary of the goldfish and European eel [30,50]. Our previous studies have also indicated that, in some teleosts, PACAPcontaining nerve fibers or endings originating from the hypothalamus are present throughout the neurohypophysis and even the adenohypophysis, and specifically in close proximity to PRL- and SL-producing cells in the adenohypophysis, and that PAC1R-like immunoreactivity is present along with PRL- and SL-like immunoreactivities in goldfish pituitary cells, suggesting that these cells are modulated by PACAP [23–25]. Our physiological and pharmacological findings in the present study indicate that PACAPinduced SL release from cultured pituitary cells is mediated by the PACAP-selective receptor, PAC1R, because the effect of PACAP was abolished by treatment with PACAP(6–38), but not with VIP(6–28), and VIP elicited no evident changes in SL release. Our previous study of the expression levels of PAC1R and VPACR mRNAs in the goldfish pituitary indicated that the mean level of PAC1R mRNA expression was approximately twice that of VPACR mRNA [25]. Although a previous report has indicated that VIP affects PRL secretion in tilapia [17], in good agreement with our present findings, PACAP stimulates the expression of SL-a and -b mRNAs and subsequent SL production in cultured pituitary cells from grass carp via the PAC1R, but not via the VPACR [14]. In fish, the actions of PACAP on release of GH and gonadotropin from pituitary cells are related to its ability to modulate the AC/ cAMP/PKA-signaling pathway and thereby increase the intracellular Ca2+ concentration [Ca2+]i [5,7,8,39,48,51,53,54]. Salmon gonadotropin-releasing hormone (sGnRH) and chicken GnRH II stimulate gonadotropin secretion via mainly the PLC/IP3/PKC- and throughout subsequent Ca2+-signaling pathways from the goldfish and masu salmon pituitaries [6,33], and sGnRH also induces PRL release via the same pathway from the cultured tilapia pituitary cells [46]. Enhancement of [Ca2+]i is thought to be accomplished through increased extracellular Ca2+ entry via voltage-sensitive Ca2+ channels and mobilization of Ca2+ from intracellular stores [39,49,52]. In our previous study using goldfish, PACAP stimulated an increase of [Ca2+]i in isolated pituitary cells showing SL-like immunoreactivity [25], and treatment with the Ca2+ ionophore, ionomycin, at 106–105 M induced SL release from the cultured pituitary cells (Azuma and Matsuda, unpublished data). In the present study, we observed that treatment with the calmodulin inhibitor, calmidazolium, attenuated PACAP-induced SL release

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from cultured goldfish pituitary cells, suggesting that an increase of [Ca2+]i mediates SL release from cultured goldfish pituitary cells. In the present study, we found that treatment with the AC and PKA inhibitors, MDL-12330A and H-89, abolished PACAP-induced SL release, suggesting that a similar signaling mechanism for GH release operates. Consistent with these results, it has been shown that PACAP stimulates SL-a and -b mRNA expression via the AC/ cAMP/PKA-signaling pathway in cultured pituitary cells from grass carp [13]. In this species, the PLC/IP3/PKC- and subsequently through the Ca2+-signaling pathway also mediates PACAP-induced SL-a and -b mRNA expression [13]. The present study clearly indicated that, in cultured goldfish pituitary cells, PACAP-induced SL release is blocked by treatment with the PLC and PKC inhibitors U-73122 and GF109203X, suggesting that the PLC/IP3/PKCsignaling pathway also mediates SL release in goldfish pituitary. PACAP signaling pathway is increased or activated by means of phosphorylation of the extracellular signal-regulated kinase type (ERK) of the mitogen-activated protein kinase (MAPK) in the brain of rodents, and PACAP-induced actions are mediated via the AC/ cAMP/PKA- and the PLC/IP3/PKC-signaling pathways, converging at the ERK cascade in cultured tilapia and grass carp pituitaries [12,55]. Further investigations to clarify the regulatory mechanism of SL release by PACAP are warranted. In conclusion, our investigation indicates that PACAP can potentially act as a hypophysiotropic factor, not only for GH, gonadotropin and PRL, but also for SL, in the goldfish pituitary. On the basis of the present data, it seems likely that PACAP-induced SL release is mediated by the PAC1R system, and subsequently through perhaps the AC/cAMP/PKA- and PLC/IP3/PKC-signaling pathways in cultured goldfish pituitary cells. Whether these signal transductions are co-localized in the same SL-producing cells remains to be determined, and further investigations to clarify the mechanism of SL regulation by PACAP are warranted. Acknowledgments This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (K.M. and A.T.), and by research grants from the Faculty of Science, University of Toyama (K.M.) and from the Toyama Marine Biotechnology Association (K.M.). References [1] Adams BA, Lescheid DW, Vickers ED, Crim LW, Sherwood NM. Pituitary adenylate cyclase-activating polypeptide in sturgeon, whitefish, grayling, flounder and halibut: cDNA sequence, exon skipping and evolution. Regul Pept 2002;109:27–37. [2] Ahmed KH, Pelster B, Krumschnabel G. Signaling pathways involved in hypertonicity- and acidification-induced activation of Na+/H+ exchange in trout hepatocytes. J Exp Biol 2006;209:3101–13. [3] Arimura A. Perspectives on pituitary adenylate cyclase activating polypeptide (PACAP) in the neuroendocrine, endocrine, and nervous systems. Jpn J Physiol 1998;48:301–31. [4] Arita J, Kojima Y, Kimura F. Measurement of the secretion of a small peptide at the single cell level by the cell immunoblot assay: thyroidectomy increases the number of substance P-secreting anterior pituitary cells. Endocrinology 1993;132:2682–8. [5] Canosa LF, Stacey N, Peter RE. Changes in brain mRNA levels of gonadotropinreleasing hormone, pituitary adenylate cyclase activating polypeptide, and somatostatin during ovulatory luteinizing hormone and growth hormone surges in goldfish. Am J Physiol Regul Integr Comp Physiol 2008;295:R1815–21. [6] Chang JP, Johnson JD, Sawisky GR, Grey CL, Mitchell G, Booth M, et al. Signal transduction in multifactorial neuroendocrine control of gonadotropin secretion and synthesis in teleosts-studies on the goldfish model. Gen Comp Endocrinol 2009;161:42–52. [7] Chang JP, Wirachowsky NR, Kwong P, Johnson JD. PACAP stimulation of gonadotropin-II secretion in goldfish pituitary cells: mechanisms of action and interaction with gonadotropin releasing hormone signaling. J Neuroendocrinol 2001;13:540–50. [8] Chang JP, Wong CJH, Davis PJ, Soetaert B, Fedorow C, Sawisky G. Role of Ca2+ stores in dopamine- and PACAP-evoked growth hormone release in goldfish. Mol Cell Endocrinol 2003;206:63–74.

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