Corrigendum Natriuretic peptide binding sites in the gills of the pouched lamprey Geotria australis T. Toop, D. Grozdanovski and I. C. Potter 10.1242/jeb.018200 The authors would like to correct an error in J. Exp. Biol. 201, 1799-1808. In this paper, supporting evidence for the presence of an NPR-C/D type receptor used a partial sequence of putative lamprey (Geotria australis) NPR-C/D obtained by sequencing a PCR-generated fragment that was subsequently cloned for sequencing. In subsequent work on rainbow trout (Oncorhynchus mykiss) in her laboratory, Dr Toop and colleagues have obtained the identical cDNA sequence several times from rainbow trout gill but have been unable to repeat the amplification with lamprey cDNA. In addition, comparisons with the now available expressed sequence tag (GenBank accession number DY697355) from the Atlantic salmon, Salmo salar, a close relative of the trout, show 90% identity with the sequence in question. This information leads them to conclude that the published sequence is not from lamprey but from rainbow trout, tissue of which was present in the laboratory at the time. They conclude that, owing to the sensitive nature of the PCR reaction, the reagents or the lamprey mRNA/cDNA were contaminated with trout nucleic acid, which was amplified instead of the lamprey cDNA. In the paper, the cDNA sequence and deduced amino acid sequence are presented in Figs·7 and 8, detailed in the Results section entitled ‘Molecular Cloning’ on p. 1805 and discussed in the fourth paragraph of the Discussion on pp. 1806-1807. The authors apologise to readers for this error. Publisher’s note: we would like to reassure readers that expert opinion has confirmed that this error does not affect any of the other experiments presented in the paper or the conclusions drawn from this study. The Journal of Experimental Biology 201, 1799–1808 (1998) 1799 Printed in Great Britain © The Company of Biologists Limited 1998 JEB1476 NATRIURETIC PEPTIDE BINDING SITES IN THE GILLS OF THE POUCHED LAMPREY GEOTRIA AUSTRALIS T. TOOP1,*, D. GROZDANOVSKI1 AND I. C. POTTER2 1School of Biological and Chemical Sciences, Deakin University, Geelong, Victoria 3228, Australia and 2School of Biological and Environmental Sciences, Murdoch University, Perth, Western Australia 6150, Australia *e-mail: [email protected] Accepted 5 March; published on WWW 12 May 1998 Summary Iodinated atrial natriuretic peptide (ANP) binding sites 65 kDa under non-reducing conditions and at 85 and were examined in the gills and ventral aorta of the adult 65 kDa under reducing conditions. Guanylate cyclase upstream-migrating lamprey Geotria australis using tissue assays demonstrated that, while no NP-stimulated GC section autoradiography, in vitro competition analysis and activity occurred in adult lampreys, NP-stimulated affinity cross-linking, while guanylate cyclase assays were enhancement of cyclic GMP accumulation was found in performed on gill membranes of both adult and juvenile juveniles in fresh water and more particularly in salt water. lampreys. A partial natriuretic peptide (NP) receptor RT-PCR amplified a 471 base pair fragment with 68 % sequence was amplified using reverse transcription/ amino acid sequence homology to the eel natriuretic polymerase chain reaction (RT-PCR). The results peptide receptor D (NPR-D). This study suggests that NP indicated that there was specific NP binding to the aortic binding sites in the adult gill and aorta are of an NPR-C/D endothelium and to pillar cell regions in the axial plate and type, whereas an additional GC-coupled site exists in secondary lamellae. In competition studies, 50 % of NP juveniles. binding was abolished by 4 nmol l−1 rat ANP, 35 nmol l−1 porcine C-type NP (CNP) and 45 nmol l−1 C-ANF (a truncated ANP). Affinity cross-linking followed by Key words: lamprey, Geotria australis, Agnatha, natriuretic peptide, SDS–PAGE demonstrated two binding sites at 205 and natriuretic peptide receptor. Introduction Early research on the natriuretic peptide (NP) system in guanosine 3′,5′-cyclic monophosphate (cGMP) intracellular mammals gave rise to the view that the main function of NPs, second messenger system, and the ‘clearance’ receptor (NPR- atrial natriuretic peptide (ANP), brain natriuretic peptide C, a homodimer of a 65 kDa protein), which is not coupled to (BNP) and C-type natriuretic peptide (CNP), was to control guanylate cyclase (GC) activity (Rosensweig and Seidman, salt and water balance via primary and secondary effects on 1991). NPR-C was originally thus named because it was the cardiovascular and renal systems (Brenner et al. 1990). The believed to modulate the circulating concentrations of NPs by nomenclature of this peptide family reflects its ability to removing them from the blood (Maack, 1992). However, stimulate renal salt excretion, a feature which, in combination subsequent research has linked NPR-C with other second with diuretic and vasodilatatory effects, reduces the systemic messenger systems (Anand-Srivastava and Trachte, 1993). At blood volume and hence blood pressure. However, the most least two GC-linked receptors have been identified, namely recent research on the mammalian system indicates that the NPR-A and NPR-B. Although NPR-A binds ANP functions of the above peptide family are wide-ranging and preferentially, it will also bind BNP and to a lesser extent CNP, complex, and include roles in the function of the central whereas NPR-B binds CNP almost exclusively. nervous system, vascular remodelling and antiproliferation The discovery that a mammalian peptide system was (see, for example, Anand-Srivastava and Trachte, 1993; implicated in salt and water balance suggested that NPs might Espiner et al. 1995). play a role in fish osmoregulation (Evans, 1990). Ensuing In common with other peptide hormone systems, NPs studies quickly identified NP immunoreactivity in the heart, require a plasma-membrane-bound receptor on target cells to plasma and brain of both agnathan and gnathostome fishes and mediate their effects via intracellular second messenger demonstrated vasodilatatory and various iono- and osmo- systems. There are two NP membrane-bound receptor (NPR) regulatory effects (Evans and Takei, 1992). The precise actions types, namely the guanylate-cyclase-coupled receptors of NPs in fish still await clarification (for general reviews, see (molecular mass approximately 130 kDa) that activate the Takei and Balment, 1993; Hagiwara et al. 1995). The 1800 T. TOOP, D. GROZDANOVSKI AND I. C. POTTER traditional view that NPs act as saltwater hormones, because demonstrate NP binding characteristics in these tissues because the circulating NP concentrations increase during saltwater the native NP is not available. In addition, we have examined adaptation (Evans and Takei, 1992), may need to be NP stimulation of guanylate cyclase activity and the apparent reappraised in the light of recent data indicating that an molecular mass of putative lamprey NPRs in the gills using the upregulation of gill NP receptors occurs in fresh water heterologous NPs. We have also used polymerase chain (Katafuchi et al. 1994; Takashima et al. 1995; Sakaguchi et al. reaction (PCR) on lamprey gill cDNA with primers designed 1996). Such findings become particularly relevant as there is to conserved regions of NPR-C and NPR-D to identify a partial now evidence that NPR-C may be linked with second sequence of a lamprey NPR. messenger systems, as opposed to being a ‘silent’ clearance receptor present only to modulate circulating concentrations of NPs (Maack, 1992; Anand-Srivastava and Trachte, 1993). Materials and methods However, there is no doubt that the gills are a location for the Animal maintenance clearance of NPs from the blood. Olson and Duff (1993) Adult pouched lampreys Geotrila australis Gray were demonstrated that 60 % of injected ANP is removed from the trapped in eel traps on the Derwent River, Tasmania, as they circulation in a single pass through the gills of the rainbow were migrating upstream, following the completion of the trout Oncorhynchus mykiss; furthermore, when the gills were marine trophic phase. They were held in running stream water treated with an NPR-C inhibitor, the extracted proportion fell at the Salmon Ponds Hatchery, Plenty, Tasmania, before either to 18 %. The influence of receptor regulation on NP function dissection at the site or shipment to Deakin University, in fish has only just begun to be explored, although binding Geelong, Australia, where they were held at 7 °C in aerated sites have been identified in the gills, kidneys, heart and recirculating fresh water before dissection. Juvenile (young vasculature of all fishes examined (Donald et al. 1994, 1997; adult) lampreys were obtained just prior to their downstream Duff and Olson, 1992; Kloas et al. 1988; Toop et al. 1995a,b). migration to the sea by electrofishing in the Donnelly River, Several NPRs have been cloned and sequenced from the Western Australia. They were transported to Murdoch Japanese eel Anguilla japonica: NPR-B (Katafuchi et al. University, Perth. All fish were held in appropriate aerated or 1994), NPR-C (Takashimi et al. 1995) and a novel NPR, NPR- running water for at least a week prior to experiments. Half of D, which is related to NPR-C (Kashiwagi et al. 1995). the juveniles were progressively acclimated to full-strength sea Recently, a fragment of the gill NPR-C has been cloned from water (35 ‰) over 3 weeks and maintained in full-strength sea the dogfish Squalus acanthias (Donald et al. 1997). water for at least a further week before use. All animals were Lampreys, together with the hagfishes, are the sole survivors anaesthetised in either MS222 (0.1 %) or benzocaine (0.01 %) of the jawless (agnathan) stage in vertebrate evolution before being killed by either decapitation or spinal cord (Hardisty, 1979). Lampreys, such as Geotria australis, severance caudad to the brain. The gills and ventral aorta were undergo an anadromous migration, i.e.