Peptides 24 (2003) 17–26 Identification of a vasopressin-like immunoreactive substance in hydra F. Morishita a,∗ , Y. Nitagai a , Y. Furukawa a , O. Matsushima a , T. Takahashi b,1 , M. Hatta b,2 , T. Fujisawa b , S. Tunamoto c , O. Koizumi c a Department of Biological Science, Graduate School of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima 739-8526, Japan b National Institute of Genetics, Mishima 411-8540, Japan c Fukuoka Women’s University, Fukuoka 813-0003, Japan Received 26 April 2002; accepted 17 September 2002 Abstract Vasopressin (VP)-like immunoreactivity has long been known in the hydra nervous system, but has not yet been structurally identified. In this study, using HPLC fractionation and an immunological assay, we have purified two peptides, FPQSFLPRGamide and SFLPRGamide, from Hydra magnipapillata. Both the peptides shared the same C-terminal structure, -PRGamide, with Arg-VP. The nonapeptide proved to be Hym-355, a peptide that stimulates neuronal differentiation in hydra. Detailed evaluation by competitive enzyme-linked immunosorbent assay (ELISA) and double immunostaining using anti-VP and anti-Hym-355 antibodies enabled us to conclude that the two peptides account for a major part of the VP-like immunoreactivity in hydra nerve cells. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Hydra; Vasopressin; Oxytocin; Immunohistochemistry; ELISA; Cnidaria; Neuropeptide; Hormone; Neurotransmitter 1. Introduction The oxytocin (OT)–vasopressin (VP) superfamily of peptides are neurohypophysial hormones that are present throughout vertebrates [9]. In invertebrates, the OT–VP su- perfamily of peptides has been identified in a small group of phylogenetically closely related phyla such as molluscs [2,16,26,37], annelids [23,28] and an arthropod [25]. In cnidaria, which are the oldest metazoans, immuno- histochemical studies revealed that hydra nerve cells con- tain peptides immunoreactive with anti-vasopressin or anti-oxytocin antibodies [5,14,15,19]. If the peptide is actually an OT–VP superfamily peptide, then hydra will be the most primitive animal that has the OT–VP superfamily of peptides [2,8,20]. This would be intriguing information about the origin of the peptide family. However, the chem- ical nature of the vasopressin-like or oxytocin-like peptide in hydra has not yet been determined. Here, we report the purification and structural determina- tion of two Arg-vasopressin-like immunoreactive peptides ∗ Corresponding author. Tel.: +81-824-24-7439; fax: +81-824-24-0734. E-mail address: fumi425@hiroshima-u.ac.jp (F. Morishita). 1 Present address: Developmental Biology Center, University of Califor- nia, Irvine, CA 92697-2275, USA. 2 Present address: Department of Biology, Faculty of Science, Ochano- mizu University, Tokyo 112-8610, Japan. in hydra, FPQSFLPRGamide and SFLPRGamide. These peptides, however, do not represent the OT–VP superfam- ily of peptides, but share the same C-terminal tripeptide, -Pro-Arg-Gly-amide, with Arg-vasopressin (Table 1). The longer of these peptides was Hym-355, which has recently been reported as a neuron-inducing peptide [34]. The im- munohistochemical results strongly suggest that the two peptides identified in the present study account for the previ- ously reported vasopressin-like immunoreactivity of hydra. 2. Materials and methods 2.1. Extraction and purification Hydra magnipapillata were cultured as described pre- viously [33]. The collected animals were frozen in liquid nitrogen and stored at -80 ◦ C until use for extraction of the peptide components. About 2 kg of the animals was pulverized in liquid nitrogen and boiled in 8 l of de-ionized water for 10 min. After homogenization, the extract was acidified by adding acetic acid (final concentration 3% acetic acid) and centrifuged at 16,000 × g for 30 min (4 ◦ C). The pellet was re-extracted with 3% acetic acid. The two supernatants were combined and condensed using a rotary evaporator. To remove the viscous materials, 2.5vol. of 0196-9781/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S0196-9781(02)00272-3