VIP and PACAP stimulate TSH release from the bullfrog pituitary

peptides 28 (2007) 1784–1789

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VIP and PACAP stimulate TSH release from the bullfrog pituitary Reiko Okada a,b,*, Kazutoshi Yamamoto a, Yoichi Ito a, Hiroshi Mochida c, Marie-Christine Tonon d,e, Alain Fournier f, Je´roˆme Leprince d,e, Hubert Vaudry d,e, Sakae Kikuyama a,c a

Department of Biology, School of Education, Waseda University, Tokyo 169-8050, Japan Department of Regulation Biology, Faculty of Sciences, Saitama University, Saitama 338-8570, Japan c Department of Biology, Faculty of Science, Shizuoka University, Shizuoka 422-8529, Japan d Inserm U413, Laboratory of Cellular and Molecular Neuroendocrinology, European Institute for Peptide Research (IFRMP 23), University of Rouen, 76821 Mont-Saint-Aignan, France e International Associated Laboratory Samuel de Champlain f INRS-Institut Armand-Frappier, Montre´al, Canada b

article info

abstract

Article history:

We have recently shown that corticotropin-releasing hormone (CRH) is a major thyrotropin

Received 15 January 2007

(TSH)-releasing factor in amphibians, but we have also found that, besides CRH, other

Received in revised form

hypothalamic substances stimulate TSH secretion in frog. In order to characterize novel TSH

20 March 2007

secretagogues, we have investigated the effect of frog (Rana ridibunda) vasoactive intestinal

Accepted 23 March 2007

polypeptide (VIP) ( fVIP) and pituitary adenylate cyclase-activating polypeptide (PACAP)

Published on line 30 March 2007

( fPACAP38 and PACAP27) on TSH release from bullfrog (Rana catesbeiana) pituitary cells in primary culture. Incubation of pituitary cells for 24 h with graded concentrations of fVIP, 9

to 10

6

M) induced a dose-dependent stimulation of TSH

Keywords:

fPACAP38 and PACAP27 (10

VIP

release with minimum effective doses of 10

PACAP

PACAP27. The PAC1-R/VPAC2-R antagonist PACAP6–38 (10

Frog TSH

suppressed the stimulatory effects of fVIP and fPACAP38 (10

VPAC2-receptor

antagonist (10

PAC1-receptor

PACAP27 (10 VIP (10

6

7

6

PACAP27 (10

M for fVIP and 10 7

and 10 7

8

M for fPACAP38 and

6

M) dose-dependently

M each). Likewise, this

M) dose-dependently attenuated the stimulatory effect of

M). On the other hand, the VPAC1-R/VPAC2-R antagonist [D-pCl-Phe6, Leu17]-

and 10 8

and 10

5

9

5

M) dose-dependently inhibited the stimulatory effect of fVIP (10

M), but did not affect the response to fPACAP38 (10

8

9

M) and

M). These data indicate

that, in amphibians, the activity of thyrotrophs can be regulated by VIP and PACAP acting likely through VPAC2-R and PAC1-R. # 2007 Elsevier Inc. All rights reserved.

1.

Introduction

Although the pituitary thyroid axis plays a major role in the control of metamorphosis [31], the neuroendocrine mechanisms regulating thyrotropin (TSH) secretion in amphibians have long remained elusive. Indeed, because of the very low

concentration of TSH and the relative abundance of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the frog pituitary, highly purified TSH preparations could not be obtained in sufficient amounts to develop a radioimmunoassay (RIA) for amphibian TSH. Recently, cloning of the bullfrog (Rana catesbeiana) TSH b-subunit [24] has eventually

* Corresponding author. Tel.: +81 48 858 3420; fax: +81 48 858 3420. E-mail address: [email protected] (R. Okada). 0196-9781/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2007.03.012

peptides 28 (2007) 1784–1789

made it possible to raise antibodies against a C-terminal peptide deduced from the cDNA sequence and to develop a homologous RIA for frog TSH [25]. Using this RIA technique, we have shown that corticotropin-releasing hormone (CRH) is a very potent stimulator of TSH secretion from pituitary cells in both adult and larval bullfrogs while thyrotropin-releasing hormone (TRH) and gonadotropin-releasing hormone (GnRH) exhibit weak TSH-releasing activity for the pituitary cells of adults, but not of larvae [25]. However, while extracts of adult and larval bullfrog hypothalami provoked a robust increase in TSH release, the CRH antagonist a-helical CRH9–41 could only reduce by 40–50% the stimulatory effect of the tissue extracts, indicating that hypothalamic factors other than CRH, TRH and GnRH can actually influence the secretion of TSH [26]. Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two members of a superfamily of structurally related peptides that also include secretin, glucagon, glucagon-like peptide and growth hormone (GH)-releasing hormone [16]. VIP is a 28-amino acid a-amidated peptide, originally isolated from the porcine small intestine by virtue of its vasodilator activity [30], which is also widely expressed in the brain [29]. PACAP is a 38-amino acid a-amidated polypeptide (PACAP38) initially isolated from the ovine hypothalamus, based on its ability to stimulate adenylyl cyclase activity in anterior pituitary cells [20]. PACAP is considered to be a pleiotropic neuropeptide that functions as neurotransmitter, neuromodulator, neurotrophic factor, vasodilator and regulatory factor for pituitary and peripheral hormone release [4,32]. Alternative processing of the PACAP precursor has the potential to generate a 27-amino acid a-amidated peptide (PACAP27) that exhibits 68% sequence identity with VIP [21]. The biological actions of VIP and PACAP are mediated through three types of receptors: the VPAC1 and VPAC2 receptors (VPAC1-R and VPAC2-R) which bind VIP and PACAP with equal affinity, and the PAC1 receptor (PAC1-R) which shows high affinity for PACAP and a much lower affinity for VIP [32]. The primary structures of VIP and PACAP have been strongly preserved during evolution. In particular, frog VIP only differs from porcine VIP by 4 amino acid substitutions [8] while the sequence of frog PACAP38 exhibits a single conservative substitution when compared to the mammalian one [2,7]. The strong evolutionary pressure that has acted to conserve the sequences of VIP and PACAP across vertebrates strongly suggests that these two peptides subserve important functions. Indeed, both VIP and PACAP stimulate the activity of anterior pituitary cells in mammals [28,32], and VIP acts as a prolactin (PRL)-releasing factor in amphibians [14]. In order to elucidate the possible involvement of hypothalamic peptides other than CRH, TRH and GnRH in the control of thyrotroph activity in amphibians, in the present study, we have investigated the possible effects of VIP and PACAP on TSH release from bullfrog pituitary cells.

2.

Materials and methods

2.1.

Animals

Adult bullfrogs, weighing approximately 600 g, were supplied from Ohuchi-AAS (Misato, Saitama, Japan) and kept under

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laboratory conditions for one week in plastic containers filled with tap water under a 12L:12D photoperiod and constant temperature at 23 8C, being fed with bovine liver. All experiments were approved by the Steering Committee for Animal Experimentation at Waseda University.

2.2.

Peptides

Frog (Rana ridibunda) VIP ( fVIP) [8] was synthesized (0.1-mmol scale) by the solid-phase methodology on a Rink amide 4methylbenzhydrylamine resin (Biochem, Meudon, France) using a 433A peptide synthesizer (Applied Biosystems, Courtaboeuf, France) and the standard procedure as previously described [15]. The synthetic peptide was purified by reversed phase high-performance liquid chromatography (RP-HPLC) on a 2.2  25 cm Vydac 218TP1022 C18 column (Alltech, Templemars, France), using a linear gradient (20–40% over 60 min) of acetonitrile/TFA (99.9:0.1, v/v) in water, at a flow rate of 10 ml/min. Analytical RP-HPLC, performed on a 0.46  25 cm Vydac 218TP54 C18 column, showed that the purity of the peptide was greater than 99%. The molecular mass of the peptide was verified by mass spectrometry on a MALDI-TOF Voyager DE-PRO instrument (Applied Biosystems). Frog (R. ridibunda) PACAP38 ( fPACAP38) [2,7] was synthesized by solidphase methodology as previously described [22]. PACAP27 (identical sequences for mammalian and frog peptides), the PAC1-R/VPAC2-R antagonist PACAP6–38 and the VPAC1-R/ VPAC2-R antagonist [D-pCl-Phe6, Leu17]VIP were purchased from Sigma (St. Louis, MO, USA).

2.3.

Pituitary cell culture

In order to test the hypothalamic peptides for their hypophysiotropic activity in vitro, enzymatically dispersed anterior lobe cells were used, mainly because they yield homogenous samples in large number as compared with intact lobes. The dispersed cells from adult bullfrogs were prepared as previously described [23]. Briefly, frogs were decapitated and the distal lobes were rapidly dissected out under sterile conditions. The pituitaries were cut in small pieces and transferred into Medium 199 (M199; Nissui Pharmaceutical, Tokyo, Japan) adjusted to R. catesbeiana osmolality (M199/water, 70:30) containing 0.2% collagenase (248 U/mg; Wako Chemicals, Osaka, Japan) and 0.1% DNase I (Sigma). After mechanical and enzymatic dispersion, the suspension was centrifuged at 100  g for 5 min, and the supernatant was removed. Dispersed cells were then resuspended in M199 containing 0.1% BSA (Fraction V; Sigma). An aliquot of the suspension was used to count the cell number and the density of cells was adjusted to 3.5  105 cells/ ml. Two hundred microliters of the suspension (containing 70,000 cells) were plated in each well of 96-multiwell plates (Asahi Techno Glass, Tokyo, Japan) and incubated at 23 8C in a humidified atmosphere of 95% air–5% CO2. After preincubation for 24 h, the culture medium was replaced with fresh medium containing fVIP, fPACAP38 or PACAP27 in the absence or presence of PACAP6–38 or [D-pCl-Phe6, Leu17]VIP. Incubation was continued for 24 h. After incubation, the medium was collected from each well and centrifuged, and the supernatant was subjected to RIA for bullfrog TSH [25]. The values, given as mean  S.E.M., are expressed as ng/10,000 cells.

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2.4.

peptides 28 (2007) 1784–1789

Statistical analysis

The significance of differences between the values obtained in each experiment was assessed by Kruskal–Wallis test followed by Scheffe’s test. A P value lower than 0.05 was considered significant.

3.

Results

Incubation of cultured pituitary cells for 24 h with graded concentrations of fVIP (10 9 to 10 6 M) provoked a dosedependent increase (340–700% of the control value) in TSH release, the minimum effective concentration being 10 9 M (Fig. 1A). Incubation of cells with graded concentrations of fPACAP38 or PACAP27 (10 9 to 10 6 M) also induced a dosedependent stimulation of TSH release (140–360% of the control value for fPACAP38 and 150–520% of the control value for PACAP27), but the minimum effective concentration was 10 8 M (Fig. 1B and C). Exposure of cultured pituitary cells to the PAC1-R/VPAC2-R antagonist PACAP6–38 (10 7 to 10 5 M), induced a modest, doserelated stimulation (130–190% of the control value) of TSH secretion, with a significant effect at a concentration of 10 6 M (Fig. 2A). Co-incubation of cells with fVIP (10 7 M) and PACAP6– 7 and 10 6 M) provoked a concentration-dependent 38 (10 reduction (60 and 35% of the value for the nonadditive) of the stimulatory action of fVIP on TSH release (Fig. 2B). Similarly, PACAP6–38 (10 7 and 10 6 M) dose-dependently attenuated the stimulatory effect of 10 7 M fPACAP38 on TSH release (85 and 45% of the value for the nonadditive) (Fig. 2C). fPACAP38 at concentrations of 10 6 and 10 5 M suppressed the stimulatory effect of 10 7 M PACAP27 on TSH release (70 and 50% of the value for the nonadditive) (Fig. 2D). Incubation of pituitary cells with the VPAC1-R/VPAC2-R antagonist [D-pCl-Phe6, Leu17]VIP, at concentrations of 10 6 M and 10 5 M, did not modify TSH release (Fig. 3A). Coincubation of cells with fVIP (10 9 M) and [D-pCl-Phe6, Leu17]VIP (10 6 and 10 5 M) provoked a dose-dependent decrease (85 and 55% of the value for the nonadditive) of fVIP (10 9 M)-induced TSH release (Fig. 3B). In contrast, [D-pCl-Phe6, Leu17]VIP did not affect fPACAP38 (10 8 M)-induced TSH release (Fig. 3C) and only produced a substantial reduction (75% of the value for the nonadditive) of PACAP27 (10 8 M)-evoked TSH secretion at a concentration of 10 5 M (Fig. 3D).

4.

Fig. 1 – Effects of fVIP, fPACAP38 and PACAP27 on TSH release from bullfrog pituitary cells in primary culture. Cells were incubated for 24 h with graded concentrations of each peptide and the culture medium was subjected to RIA for bullfrog TSH. The values, given as means W S.E.M., are expressed as ng/10,000 cells (n = 9–12). Blank columns (control) represent the basal amount of TSH release during 24 h of culture. The values with the same superscript do not differ from each other at the 5% level of significance (Scheffe’s test).

Discussion

Recent studies have shown that the neuroendocrine mechanisms regulating amphibian thyrotrophs are clearly different from those operating in mammals. Thus, in frog (R. catesbeiana and Rana pipiens) and toad (Xenopus laevis), TRH exerts only a modest effect on TSH secretion whereas CRH is a very potent TSH-releasing factor [6,10,25,26]. The hypophysiotropic effects of VIP and PACAP have been extensively investigated in mammals [28,32] (for review): VIP triggers the secretion of PRL, GH and LH while PACAP stimulates the secretion of adrenocorticotropic hormone, GH and LH. However, in mammals, neither VIP [5] nor PACAP [9,12,20] have any effect on

thyrotroph activity. The present data, showing that VIP and PACAP stimulate TSH release from bullfrog pituitary cells, thus provide additional evidence for differential regulation of thyrotrophs in amphibians and mammals. In order to test VIP and PACAP for their TSH-releasing activity with pituitary cells of R. catesbeiana, we used the peptides from the frog belonging to the same genus, but different species (R. ridibunda). Considering that the amino acid sequences of both VIP and PACAP are highly conserved across vertebrates, the results obtained in the present experiments seem to reflect the activity close to that of the homologous peptides, which is yet unavailable.

peptides 28 (2007) 1784–1789

Fig. 2 – Effect of the PAC1-R/VPAC2-R antagonist PACAP6–38 on fVIP-, fPACAP38- and PACAP27-induced TSH release from bullfrog pituitary cells in primary culture. Cells were incubated for 24 h with PACAP6–38 (10S7 to 10S5 M) in the absence (A) or in the presence of 10S7 M fVIP (B), 10S7 M fPACAP38 (C) or 10S7 M PACAP27 (D), and the culture medium was subjected to RIA for bullfrog TSH. The values, given as means W S.E.M., are expressed as ng/10,000 cells (n = 6). Blank columns (control) represent the basal amount of TSH release during 24 h of culture. The values with the same superscript do not differ from each other at the 5% level of significance (Scheffe’s test).

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Fig. 3 – Effect of the VPAC1-R/VPAC2-R antagonist [D-pClPhe6, Leu17]VIP on fVIP-, fPACAP38- and PACAP27induced TSH release from bullfrog pituitary cells in primary culture. Cells were incubated for 24 h with [D-pClPhe6, Leu17]VIP (10S6 or 10S5 M) in the absence (A) or presence of 10S9 M fVIP (B), 10S8 M fPACAP38 (C) or 10S8 M PACAP27 (D), and the culture medium was subjected to RIA for bullfrog TSH. The values, given as means W S.E.M., are expressed as ng/10,000 cells (n = 6). Blank columns represent the basal amount of TSH released during 24 h of culture. The values with the same superscript do not differ from each other at the 5% level of significance (Scheffe’s test).

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peptides 28 (2007) 1784–1789

Consistent with the TSH-releasing activity of VIP and PACAP in bullfrog pituitary cells, an early study had shown that PACAP38 increases cytosolic calcium concentration in frog (R. ridibunda) thyrotrophs [11]. The occurrence of the mRNAs encoding all three types of VIP/PACAP receptors has been previously reported in the frog pituitary, i.e., the VIP/PACAP mutual receptors VPAC1-R [1] and VPAC2-R [13] and the PACAPselective receptor PAC1-R [3], but the type of pituitary cells that express each receptor subtype has not been determined. The present data now indicate that thyrotrophs express receptors for VIP and PACAP. The fact that fVIP was more potent than fPACAP38 and PACAP27 to stimulate TSH release strongly suggests the occurrence of VPAC1-R and/or VPAC2-R on frog thyrotrophs. In agreement with this notion, the stimulatory effect of fVIP was dose-dependently attenuated by the VPAC1-R/ VPAC2-R antagonist [D-pCl-Phe6, Leu17]VIP [27]. At the highest concentration tested, this antagonist slightly reduced the TSHreleasing effect of PACAP27 but did not affect that of fPACAP38. This latter observation indicates that the stimulatory effect of fPACAP38, and to some extent PACAP27, are mediated via the PACAP-selective receptor PAC1-R. Concurrently, the fact that the PAC1-R/VPAC2-R antagonist PACAP6–38 suppressed fVIPinduced TSH release reveals the presence of VPAC2-R on frog thyrotrophs inasmuch as frog PAC1-R, like their mammalian counterparts, have very low affinity for VIP [3]. Taken together, the present data strongly suggest that frog thyrotrophs express both VPAC2-R and PAC1-R. The quantitative distribution and chromatographic characterization of immunoreactive PACAP in the frog brain have been reported by Matsuda et al. [19]. According to them, the concentration of immunoreactive PACAP is highest in the diencephalon, the predominant form of this peptide being PACAP38. Immunohistochemical studies have shown that, in both adult [33] and larval frog [18], PACAP-immunoreactive neurons are located in hypothalamic hypophysiotropic nuclei that project to the external zone of the median eminence. Although there is no information regarding the distribution of VIP-producing neurons in the brain of adult amphibians, the presence of VIP-immunoreactive cell bodies and fibers has been demonstrated in the hypothalamus and median eminence of tadpoles [17]. These neuroanatomical data provide strong evidence that VIP and PACAP may act as physiological regulators of TSH secretion in amphibians, notably during metamorphosis. In conclusion, the present report has shown that VIP and PACAP stimulate in vitro the release of TSH from frog pituitary cells in primary culture through activation of VPAC2-R and PAC1-R. The occurrence of VIP and PACAP fibers in the external zone of the median eminence indicates that, in frog, the two neuropeptides may actually act as TSH-releasing factors. Since, in mammals, thyrotrophs are not affected by VIP or PACAP, the present data provide additional evidence that the neuroendocrine control of TSH markedly differs between amphibians and mammals.

Acknowledgments This work was supported by JSPS (18570063 and 18001847), Grant from Inamori Foundation, Inserm (U413), the European

Institute for Peptide Research (IFRMP 23) and the ‘‘conseil re´gional de Haute-Normandie’’.

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