Hormonal control of Ca homeostasis in lower vertebrates: Considering the evolution

金沢大学環日本海域環境研究センター生物多様性研究部門Early vertebrates may have had genes that would later produce hormones concerning Ca homeostasis in advanced vertebrates. These genes may possibly have expressed the respective product for the local control of Ca in some tissues, which must be a paracrine control. When vertebrates acquired the jaw, however, delicate but systemic Ca control system may have been needed to cope with fluctuations in blood Ca levels resulting from the digestion of food. Furthermore, when vertebrates were transferred from freshwater to seawater or to land, new control systems must have been required. Therefore, such vertebrates might have coped with two ways in their evolution. One is the increase of the number of local production sites of hormones, for the delicate control of Ca in various tissues, which must be the paracrine control. The other is the creation of new glands for the systemic control of Ca homeostasis by making paracrine cells independent from tissues. This style must be an endocrine control. Therefore, in modern vertebrates, in various tissues, calcitonin, stanniocalcin, parathyroid hormone-related protein, and maybe also prolactin, or at least substances immunoreactive to antibodies against these hormones, are expressed locally as paracrine tissues. At the same time, these hormones or similar molecules are also produced in special endocrine glands such as the ultimobranchial glands, Stannius corpuscles, the parathyroid glands and the pituitary gland, respectively. This supposition is summarized in Fig. 1

Alpha-glucosidase-like activity detected in a siboglinid polychaete, Oligobrachia mashikoi

元金沢大学大学院自然科学研究科生命科学専攻 / キッセイ薬品工業株式会社 開発研究部　薬理研究所金沢大学環日本海域環境研究センター生物多様性研究部門Siboglinid worms live on carbohydrates produced by symbiotic bacteria. In this study, α-glucosidase-like activity was detected in the surface of the body and in the trophosome of Oligobrachia mashikoi. The enzyme exhibiting this activity was partially purified by consecutively applying the crude enzyme extract to Con-A-Sepharose and Sephadex-200 HR columns. The enzyme sample thus obtained gave a single activity peak at a position corresponding to 550 kDa in the Sephadex-200 HR gel filtration column. The enzyme was active in the range of pH 6.0-8.0, with a maximum activity at around pH 6.5. It specifically hydrolyzed maltose, and was inhibited by voglibose and miglitol. Moreover, a glucose transporter 2-like protein was detected by immunohistochemical and Western-blotting analyses using anti-rat GLUT2 polyclonal antibody. These results raise the question how this unique species lives. © 2008 Zoological Society of Japan

爬虫類のカルシトニン: その一次構造と生理活性の解明

Polymerase Chain Reaction (PCR)法を用いて3目4種の爬虫類(メガネカイマン、アオダイショウ、カナヘビ及びクサガメ)のゲノムより、カルシトニン遺伝子の増幅を試みた。その結果、ニワトリのカルシトニン遺伝子の塩基配列の一部をプライマーとして用いた時のみ、それらの爬虫類でヒトのカルシトニン遺伝子と同じ大きさである約150塩基対のDNA断片の増幅が見られた。それらを大腸菌に組み込み、クローニングした結果、それらの増幅されたDNAの塩基配列はニワトリのカルシトニン遺伝子の塩基配列と非常に良く似ていることが明らかになった。 ニワトリのカルシトニン遺伝子と増幅されたDNA断片の類似性は、クサガメにおいては100%、メガネカイマンでは99%、カナヘビでは96%、アオダイショウでは93%であった。この類似の順序は爬虫類内において、これらの動物が系統発生学的にはカメ目、ワニ目、有鱗目のトカゲ亜目、ヘビ亜目の順で分化し、鳥類はワニ目と同じ祖先から分岐してきたという地史的資料と良く一致する。 本研究で増幅されたDNA断片の塩基配列が、爬虫類のカルシトニン遺伝子の一つであると考え、脊椎動物全体のカルシトニンの分子進化を考察すると、原子硬骨魚類が持っていたと思われる基本型のカルシトニンは、進化の過程を通して長期間に渡って保存され、現世の硬骨魚類に広がる一方、爬虫類を経て、鳥類へと受け継がれたと思われる。 以上の結果は、新知見であり、今後、日本動物学会の機関誌であるズオロジカルサイエンス等に投稿予定である。Amplifying of calcitonin gene of 4 species of reptiles (caiman : Gaiman crocodiles, snake : Elaphe climacophora, skink : Takydromus taky-dromoides and turtle : Geoclemys reevisii) was tried using genomic polymerase chain reaction (PCR) method. When primer designed from 2 parts of base sequence of chicken calcitonin gene was applied, about 150 bp of DNA fraction, of which sizu is similar to that of human calcitonin gene, was amplified in each species of those reptiles. After cloning them, it became clear that each base sequence was very similar to that of chicken calcitonin gene. The similarity was 100% in the turtle, 99% in the caiman, 96% in the skink and 93% in the snake. The order of the similarity was coincident to the fact that these species was differentiated from their ancestors in the same order (turtle is the oldest) in reptiles.研究課題/領域番号:06640861, 研究期間(年度):1994–1995出典：「爬虫類のカルシトニン: その一次構造と生理活性の解明」研究成果報告書　課題番号06640861(KAKEN：科学研究費助成事業データベース（国立情報学研究所）)　　　本文データは著者版報告書より作

軟骨魚類の骨硬化ホルモン〈カルントニン〉: 細胞レベルにおける生理作用の解明

金沢大学理学部本研究は平成7年度と8年度に渡って研究された。しかしながら、当初に目的とするところまでは、残念ながら達していない。これは7年度の配分が10月末日に補正予算として遅れて来たこと、その額が予定をした物品の購入にはるかに及ばない為、自分で装置の手作りから始めねばならなかったことによる。さらに、日本海における原油流出という全く不足の事態が発生して漁船が出ず、アカエイ等の軟骨魚類の入手がかなり困難になってしまったことも原因の一つである。現在、本研究で申請した細胞内イオン測定装置(日本分光:CAM230型)に相当する、細胞内のCa濃度の変化を調べる装置を、倒立顕微鏡と落射蛍光装置及び光電子倍増管を組み合わせて完成させた。これはニコンTMD300という倒立顕微鏡に蛍光カルシウム指示薬Indo-1用の落射蛍光装置(EF-S/Ca)を組み込み、細胞を380nmの波長でたたき、そこから出る蛍光をダイクロイックミラーDM455により2つの波長に分け、波長405と485のフィルターを通して細胞内Ca濃度の盛衰を見るという装置である。実際は、倒立顕微鏡のサイドポートへ光電子倍増管(浜松ホトニクスR1477)を2本組み込んだ装置を取り付け、2ペンのレコーダーへシグナルを導き、記録した。すなわちCa濃度が高いときは405nmのピークが高く、低いときは485nmのピークが高くなる。軟骨細胞の培養は試みてはいるが現在、上手く行かない。最近、神経細胞の細胞膜は軟骨細胞が生産する軟骨基質に似た硫酸プロテオグリカンからなるという報告がある。従って、上記の装置の有効性を試すため、軟体動物のアメフラシの腹部神経節より過去に、申請者らがカルシトニンに反応する事を見いだしたR9と名付けられている細胞で細胞内Ca濃度の変化を検出を試みた。しかしながら、うまく細胞内Caの変化を捕らえらず、その原因をやっきとなって調べている。As we have clarified a primary structure of calcitonin from the stingray before, as a next step, the physiological function of this hormone was tried to study at a cellular level. This project was performed in 1995 and 1996. At present, however, the fruitful results are not yet obtained for some reasons. The main difficulty was to have to build up the instrument for determining cytoplasmic Ca level by ourselves, because financial support was fairly small when compared to the amount requested. Nevertheless, we made an instrument for the purpose by combining an inversed microscope (Nikon TMD 300) with an apparatus of epi-fluorescence for Indo-1 (Nikon EF-S/Ca) and 2 photo multipliers (Hamamatsu Photonics R1477). The principle of this instrument is following. When the cell is irradiated by a wave length 380 nm from a mercury lamp, the fluorescence must be released from the cell, because the cell is treated with Indo-1 already.The fluorescence is divided into two wave lengths of 405 nm and 485 nm by a dichroic mirror DM455. If Ca level in the cytoplasm is high, the peak of 405 nm should be high, and the Ca level is low, peak of 485 nm should be high. Such changes in the Ca level is recorded by a two-pen recorder. On the other hand, although we tried to cultivate chondrocyte from branchial cartilage of goldfish in vitro, but good results are not yet obtained. Recently, it was reported that cellular membrane of neurons is composed of the same component as the cartilaginous matrix of cartilage. Therefore, to check the performance of our instrument, we examined the cytoplasmic changes of Ca level in neurons from abdominal ganglion of Aplysia, as we have checked some of neurons can respond to calcitonin before. However, we could not detect Ca changes, when calcitonin was added to the incubation medium. At present, the causes are examined, whether the sensitivity of this instrument was low, or quantity of the administration of calcitonin was two much low.研究課題/領域番号:07554089, 研究期間(年度):1995 – 1996出典：研究課題「軟骨魚類の骨硬化ホルモン〈カルントニン〉: 細胞レベルにおける生理作用の解明」課題番号07554089（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07554089/）を加工して作

Anuran calcitonins are diverse in lower vertebrates

金沢大学環日本海域環境研究センター生物多様性研究部門We investigated the nucleotide sequences of cDNA fragments coding calcitonin from ultimobranchial glands in 2 species of urodelans (1 salamander and 1 newt) and 4 species of anurans (1 toad and 3 frogs) by the reverse transcription polymerase chain reaction (RT-PCR) method and rapid amplification of cDNA ends (RACE)-PCR method. The salamander and newt calcitonins were each 97% and 94% similar to the lungfish and caiman calcitonins that we have already reported, in the amino acid sequences. However, anuran calcitonins were not only dissimilar (63-81%) to the lungfish and caiman calcitonins but also diversified (59-91%) even among anurans. The sequence identity of toad calcitonin was always low (59-66%) among anurans

Expression levels of hormone receptors and bone morphogenic protein in fins of medaka

金沢大学環日本海域環境研究センター生物多様性研究部門In the genus Oryzias, the morphologies of the dorsal and anal fins are typical secondary sex characters. In the Japanese medaka (Oryzias latipes) and Thai medaka (Oryzias minutillus), androgen receptor (AR) expression levels in the dorsal, anal, and pectoral fins were higher in males than in females. Conversely, in both species estrogen receptor (ER) β expression levels in the dorsal and anal fins were higher in females than in males. AR and ERβ expression levels in the dorsal and anal fins of sex-undeterminable individuals of Thai medaka were intermediate between those in normal male and female Thai medaka. There was no difference in the bone morphogenic protein (Bmp) 2b expression level between male and female Japanese medaka. In contrast, the Bmp2b expression level in the dorsal fin of sex-undeterminable individuals was lower than in normal male and female Thai medaka. It is thus clear that androgen and estrogen regulate the sex-dependent characters of fin morphology in both Oryzias species. In sex-undeterminable individuals of Thai medaka, the low levels of Bmp2b expression in the dorsal fin are evidence that androgen and estrogen are necessary for adequate expression of Bmp2b in the normal development of at least the dorsal fin. © 2009 Zoological Society of Japan.全文公開20100

Plasma calcium and calcitonin levels at food intake in eels and goldfish

金沢大学環日本海域環境研究センター生物多様性研究部門In order to examine whether calcitonin plays an important role in Ca homeostasis of teleosts, such as suppressing hypercalcemia at food intake, we compared the plasma Ca levels and calcitonin levels in eels fed normally with eels starved for one week (Experiment I), and in goldfish administered with a high Ca-consomme solution into the digestive tract with goldfish given physiological saline solution (Experiment II). In Experiment I, the plasma Ca levels and calcitonin levels in the fed eels were significantly higher than those in the starved eels after one week. In Experiment II, the plasma Ca levels in the high Ca-treated goldfish were significantly higher than those in the saline-treated goldfish after 1 hr and 3 hr. The number of goldfish showing over 500 pg/ml of plasma calcitonin was significantly higher in the high Ca-treated group than in the saline-treated group. From the results of both experiments, we conclude that in these two species, Ca and/or nutriment absorbed via the digestive tract may affect plasma calcitonin levels. However, more experiments are needed to directly demonstrate that calcitonin suppresses hypercalcemia at food intake

Nucleotide sequences of reptile calcitonins: Their high homology to chicken calcitonin

金沢大学環日本海域環境研究センター生物多様性研究部門The calcitonin genes of four species of reptiles (Reeve\u27s turtle, rat snake, grass lizard, and spectacled caiman) were amplified from the genomic DNA, as well as from the mRNA of the ultimobranchial glands of the former three species, by the polymerase chain reaction (PCR) method, and were sequenced. Among several primer sets, only one primer set synthesized from the chicken calcitonin gene was compatible with those of the reptiles. The nucleotide sequences of the reptile calcitonin genes were highly homologous with that of chicken calcitonin (100% for turtle, 99% for caiman, 96% for lizard and 93% for snake). The products amplified from mRNA by the RT-PCR method matched completely those from genomic DNA in the turtle, snake and lizard