In this thesis, I studied the secretory regulation of the long (slow)-acting hormone, cortisol, by several important osmoregulatory fast (short)-acting hormones in the eel (Anguilla japonica). The interaction of fast and slow acting hormones is a crucial step in the process of osmoregulation in euryhaline fish that have the ability to acclimate to both freshwater (FW) and seawater (SW) during their life cycle. Among euryhaline fish, the eel exhibits exceptional adaptability to diverse salinity environments and considered as an excellent model for osmoregulation studies. As cortisol is considered to be a crucial for adaptation to both SW and FW, I analyzed its secretory regulation by three groups of fast-acting hormones including natriuretic peptides (NPs), adrenomedullins (AMs) and angiotensins (Angs).

Chapter 1. Effects of fast-acting hormones on cortisol secretion from eel interrenal tissue

In Chapter 1 of this thesis, I analyzed the diversified NPs present in the eel on cortisol secretion using an in vitro incubation system of steroidogenic interrenal cells from both FW and SW acclimated eels. A previous in vivo experiment from this laboratory showed that ANP increased plasma cortisol concentration in SW eels and CNP (later known as CNP1) increased it in FW eels. Only three NPs (ANP, VNP and CNP1) were identified at that time in teleost fish, but three new NPs have been identified recently in the eel. Thus I examined the effect of all 6 NPs on cortisol secretion in the newly established in vitro system in order to analyze in more detail the mechanisms of action of NPs on the interrenal. Further, I examined the newly discovered AM family and diversified Ang peptides to test their direct effect on interrenal cells.

Although steroidogenic cells are clustered as a compact tissue in tetrapods and some teleosts, the interrenal cells in the eel are scattered along the long head kidney (>10 cm in length). In order to determine the tissue that should be used for the in vitro experiments, I first localized the steroidogenic and chromaffin cells in the eel head kidney by RT-PCR using steroid biosynthetic enzyme (P450scc) and tyrosine hydroxylase (TH) as markers, respectively. I found that the interrenal cells exist only in the anterior part (~2 cm) of the head kidney, but chromaffin cells are distributed more widely along the head kidney. Furthermore, I examined their tissue distribution in the head kidney by immunohistochemistry using the marker enzymes, P450scc and TH. The results indicated that both cell types exist in the close vicinity in the wall of the cardinal vein forming several clusters scattered in the hematopoietic tissue. I also found that the genes of NP receptors (NPR-A and NPR-B) are expressed in the same interrenal region of the head kidney. I also established a radioimmunoassay for cortisol myself to measure large amount of samples.

Using the in vitro incubation system of eel interrenal cells, I first examine the action of the six NPs identified in the eel (ANP, BNP, VNP, CNP1, CNP3 and CNP4) on cortisol secretion. However, none of the six eel NPs given alone produced any significant stimulation of cortisol secretion in either FW or SW eels. These results were inconsistent with the stimulatory effect in the previous in vivo studies. However, when NPs where incubated together with ACTH. ANP and VNP (but not BNP and CNPs) enhanced the stimulatory effect of ACTH on cortisol secretion in SW fish interrenal preparations. By contrast, CNP1 and CNP4 (but not ANP, BNP, VNP and CNP3) enhanced the ACTH action in FW fish preparations. Thus, I eventually confirmed the previous in vivo experiments showing that the different NPs are involved in cortisol secretion in different salinity environments, and thus play important role in the euryhalinity of the eel for adaptation to both FW and SW. In addition, these results demonstrate the importance of the modulation of the steroigogenic action of ACTH, indicating that this fast-acting hormone may be fundamental for osmoregulatory control of cortisol.

I also examined the steroidogenic action of the newly discovered AMs (AM1, AM2 and AM5) and Angs (Ang II, Ang III and Ang IV) in the eel interrenal tissue. For Ang II, I used teleost [Asn1]-Ang II (N-Ang II) and mammalian [Asp1]-Ang II (D-Ang II) as the N-terminal Asn of N-Ang II is known to be converted to Asn by asparaginase in the eel blood. Although AM has a steroidogenic action in mammals, none of the eel AMs caused any effects on cortisol release from either FW or SW eel interrenal preparations. By contrast, Ang II and Ang III given alone to the incubation medium increased cortisol secretion in SW eel preparations. Furthermore, the heterologous D-Ang caused a greater increase than homologous N-Ang I. Ang III induced a slight increase in cortisol secretion in FW eel preparation while N-Ang II and Ang IV had no effect in either FW or SW eel preparations. Ang II acts directly on the interrenal cells to stimulate cortisol secretion. Interestingly, heterologous D-Ang II is more potent than homologous eel N-Ang II. Since previous studies from this lab showed that N-Ang II is more potent than D-Ang II for the vasopressor effect and Ang III is almost ineffective in the eel, a new receptor for Ang that has not been identified to date could exist in the interrenal tissue.

Chapter 2. Effect of NPs on ACTH secretion from the pituitary gland of the eel

From the data in Chapter 1, I realized that ACTH may be playing a central role in cortisol secretion. Considering the differing results from in vitro and in vivo, it is possible that the circulating ACTH may be increased by NPs for cortisol secretion. Supporting this possibility is the previoud data that hypophysectomized eels did not produce cortisol secretion after SW transfer (Hirano 1969). Based on these data, I hypothesized that NPs not only modulate the ACTH action but also stimulate ACTH secretion from the eel pituitary gland.

In Chapter 2 of this thesis, I initially established the ELISA method for ACTH measurement and an in vitro tissue culture system of the eel pituitary gland to measure ACTH secretion from the pituitary. As the steroidogenic action of ANP and CNP1 were demonstrated in SW and FW eel interrenal tissues, I tested the effects of ANP and CNP1 on ACTH secretion in SW and FW eel pituitaries, respectively. As a result, CNP1 significantly increased ACTH secretion in the pituitaries of FW adapted eels, while the ANP effect tended to be stimulatory but non-significant. Altogether, these data suggest a link between NPs and ACTH at the pituitary for cortisol secretion.

Conclusions

Based on the results reported in this thesis, I present a new and more complex model for regulation of cortisol secretion by several fast-acting hormones. Furthermore, it is apparent that the major hormone for cortisol synthesis and secretion is ACTH and thus its regulation becomes of vital importance as the effects of NPs are only visible in the presence of this hormone. In addition, as shown in Chapter 2, NPs seem to act on the pituitary and modulate ACTH secretion, which consequently will influences cortisol secretion in vivo. This data provides a new regulatory step of fast-acting hormones to the hypothalamo-pituitary-interrenal (HPI) axis, also demonstrating the interaction between fast-acting hormones for cortisol secretion (Fig. 1). Further, not only the local action in the interrenal tissue, but the regulation of fast-acting hormones in other glands of the HPI axis like the pituitary become of interest for a wider understanding of the process of osmoregulation that euryhaline teleosts undergo during their life cycle.

Figure 1. Model for the regulation of the hypothalamo-pituitary-interrenal (HPI) axis for cortisol secretion by natriuretic peptides (NPs), adrenomedullins (AMs) and angiotensin (Angs). Previous model based on in vivo results on the effects of ANP and CNP1 in the eel (Li and Takei 2003) and in in the flounder by D-Ang II (Balment et al. 1990)

本論文の基本構成は、Abstract, General Introduction、Chapter 1、 Chapter 2、およびGeneral Discussionの5部からなる。本論文の特色は、浸透圧調節に関わるホルモンのうち、速効性ホルモンであるナトリウム利尿ペプチドが、遅効性ホルモンであるコルチゾルの分泌を促進することにより、短期の調節だけでなく長期の調節にも関与していることを明らかにした点である。すなわち、短期の適応と長期の適応に関わる2つのホルモンの作用協関とその作用機構を、淡水と海水双方によく適応するウナギを用いて初めて明らかにしたことにある。また、本研究の新規性は、(1)ナトリウム利尿ペプチドは単独でコルチゾル分泌を促進しないが、脳下垂体から分泌される副腎皮質刺激ホルモン(ACTH)のコルチゾル分泌促進作用を増強すること、および(2)NPはコルチゾル分泌器官である間腎腺に作用するだけではなく、脳下垂体にも作用してACTHの分泌を促進することを脊椎動物で初めて明らかにしたことにある。

ナトリウム利用ペプチド(natriuretic peptide, NP)ファミリーは真骨魚類で多様化しており、心臓ホルモンであるANP, BNP, VNPと血管内皮細胞に広く存在する4種のCNP(CNP1~4)からなる。ベントゥラ氏は修士課程でこのホルモンファミリーの進化に興味を持ち、原始的な魚類であるチョウザメですでにNPファミリーが多様化していること、およびウナギにはCNP2以外の5種のNPが存在することを明らかにした。そこで博士課程では、この多様化したNPファミリーの作用に興味を拡げた。NPのうちANPは、ウナギを海水に移行させるとすぐに分泌され、すぐに血漿から消失する典型的な速効性ホルモンである。しかし、ウナギは海水に適応するために浸透圧調節器官である鰓、腸、腎臓などを水を吸収して塩分を排出する海水型につくり変えることが重要で、それには遺伝子発現を介した長期作用型ホルモンであるコルチゾルが働いている。これまで速効性ホルモンと遅効性ホルモンの相互作用に関してほとんど研究が行われていなかったが、重要な海水適応ホルモンであるANPとコルチゾルの複雑な相互作用が本研究により明らかになった。

本研究室において、以前海水に適応しているウナギの血液中にANPを投与すると血漿コルチゾル濃度が上昇すること、および淡水ウナギではCNP1がコルチゾルを上昇させることが明らかにされていた。そこでChapter 1において、これらのin vivoにおけるNPの作用が間腎腺への直接作用かどうかを調べるため、まずウナギ間腎腺の培養系を確立した。ウナギの間腎腺は哺乳類の副腎皮質のようにまとまった器官ではないため、中心大静脈を囲む結合組織中に分散して存在する間腎腺を集めることから研究が始まった。まず様々なNPを培養液に投与したが、予想に反してコルチゾルの分泌は見られなかった。そこで、主要なコルチゾル分泌ホルモンであるACTHと共にANPやVNPなどの心臓型NPを海水ウナギから採取した間腎腺に投与すると、ACTHを単独投与した時と比較してコルチゾルの分泌が促進された。いっぽう淡水ウナギから採取した間腎腺では、CNP1とCNP4がACTHのコルチゾル分泌作用を増強した。この結果は、生体内では血液中に常にACTHが存在するため、以前に行われたin vivoの結果と矛盾しない。コルチゾル分泌に関するACTHとNPの相互作用を明らかにしたのは哺乳類を含めて脊椎動物で初めてである。

Chapter 2では,コルチゾルの分泌を支配する視床下部-脳下垂体-間腎腺系へのNPファミリーの作用をさらに詳しく調べるため、ウナギ脳下垂体の器官培養系とウナギACTHのELIAS系を確立し、淡水と海水それぞれに適応したウナギの脳下垂体にANPとCNP1を投与したところ、塩分環境に関わらずANPとCNP1双方がACTHの分泌を促進した。その結果、NPファミリーは間腎腺でACTHの作用を増強するだけではなく、脳下垂体からACTHの分泌を促進してコルチゾルの分泌に関わることが明らかになった。すなわち、速効性の海水適応ホルモンであるNPファミリーは、視床下部―脳下垂体-間腎腺系の複数の部位に作用することにより、遅効性の海水適応ホルモンであるコルチゾルの分泌を促進して長期にわたり海水適応に関わっていることが明らかになった。

なお、本論文の実験は日下部助教の指導のもと全て論文提出者本人が行い分析したものであり、本論文の全ての研究において論文提出者の寄与が十分であると判断する。

したがって、博士(理学)の学位を授与できると認める。