JOURNAL OF EXPERIMENTAL ZOOLOGY 284:407–413 (1999)

© 1999 WILEY-LISS, INC.

Natriuretic Peptide Binding Sites in the Brain of theAtlantic Hagfish, Myxine glutinosa

JOHN A. DONALD,1* TES TOOP,1 AND DAVID H. EVANS2,3

1School of Biological and Chemical Sciences, Deakin University, Geelong,Victoria, Australia

2Department of Zoology, University of Florida, Gainesville, Florida 326113Mount Desert Island Biological Laboratory, Salisbury Cove, Maine 04672

ABSTRACT We have previously used immunohistochemistry to show that the brain of thehagfish, Myxine glutinosa, contains a rich distribution of natriuretic peptide-immunoreactive ele-ments with the densest distribution occurring in the telencephalon and the diencephalon. In thisstudy, the distribution of 125I-rat ANP and 125I-porcine CNP binding sites was determined in thebrain of M. glutinosa. The binding pattern of 125I-rat ANP and 125I-porcine CNP showed similari-ties; however, some differences were observed in the olfactory bulb and the caudal brain regions.Specific 125I-rat ANP and 125I-porcine CNP binding was observed in the olfactory bulb, outer layersof the pallium, and in regions of the diencephalon. Very little specific binding was observed in thehabenula and the primordium hippocampi. In the diencephalon, a distinct zone of specific 125I-rANP binding separated a region of moderate binding in the lateral regions of the diencephalonfrom the thalamic and hypothalamic nuclei. Moderate levels of specific 125I-rANP binding wereobserved in the mesencephalon and medulla oblongata; little or no 125I-porcine CNP binding wasobserved in these regions. The data, in combination with previous immunohistochemical studies,show that the natriuretic peptide system of the hagfish brain is well-developed and suggest thatnatriuretic peptides have a long evolutionary history as neurotransmitters and/or neuromodulatorsin the vertebrate brain. J. Exp. Zool. 284:407–413, 1999. © 1999 Wiley-Liss, Inc.

In mammals, natriuretic peptides (NPs) are afamily of peptide hormones which occur in the brainand periphery and are strongly implicated in thereduction of hypervolemia. To date, four types ofmammalian NP have been isolated: atrial NP(ANP), brain natriuretic peptide (BNP), C-typenatriuretic peptide (CNP), and urodilatin (Yandle,’94). The effects of NPs are mediated by two ge-netically distinct types of natriuretic peptide re-ceptors (NPR-A and NPR-B) which possess anintracellular guanylate cyclase (GC) domain; bind-ing of NPs to the extracellular domain stimulatesthe conversion of guanosine triphosphate to cGMP(Yandle, ’94). NPR-A is primarily activated by atrialnatriuretic peptide (ANP) and brain natriureticpeptide (BNP), whereas NPR-B is primarily acti-vated by C-type natriuretic peptide (CNP). A thirdNPR, called NPR-C, is a unique receptor that bindsall NPs with high affinity but does not have anintracellular GC domain. It is generally consideredthat the NPR-C function as a mechanism for clear-ing NPs from the circulation and tissues by inter-nalization, but it may also function through anon-cGMP signal transduction pathway (Anand-Srivastava and Trachte, ’93; Yandle, ’94).

The distribution of NPs in the central nervoussystem of mammals has been determined with im-munohistochemistry (IHC) and mRNA expression(e.g., see Langub et al., ’95). The presence of NPsin the diencephalon strongly supports a role forNPs in neuroendocrine regulation of osmotic andcardiovascular homeostasis (Gutkowska et al.,’97). However, NPs are extensively expressed inother areas of the brain, which indicates a rolefor NPs in a range of brain functions (Langub etal., ’95). The distribution of NPs and the NPR inthe mammalian brain overlap to some extent(ANP, Quirion et al., ’84; BNP, Konrad et al., ’90;CNP, Brown and Zuo, ’93).

In spite of various NPs having been isolatedfrom either the heart or brain of non-mammalianvertebrates, the function of NPs in lower verte-brates has yet to be unequivocally determined.However, linkage to the control of ion and fluidhomeostasis is emerging (Evans and Takei, ’92;

*Correspondence to: Dr. J.A. Donald, School of Biological andChemical Sciences, Deakin University, Geelong, Victoria, Australia,3217. E-mail: jdonald@deakin.edu.au

Received 17 June 1998; Accepted 6 January 1999.

408 J.A. DONALD ET AL.

Takei and Balment, ’93; Hagiwara et al., ’95). Moststudies in lower vertebrates have focussed on therole of cardiac NPs, but, as in mammals, NPs arewidely distributed in the brain of representativespecies of cyclostome (Donald et al., ’92), elasmo-branch (Donald et al., ’92), and teleost fish (Donaldand Evans, ’92) suggesting that the peptides func-tion as neuromodulators and/or neurotransmit-ters, in addition to their effects in the periphery(Evans and Takei, ’92). Few studies have exam-ined the distribution of NPR in the brains of lowervertebrates, and to date, the distribution of NPRin the brain has only been examined in two am-phibians (Xenopus laevis, Kloas and Hanke, ’92;Rana ridibunda, Tong et al., ’89) and the lung-fish, Protopterus annectens (Vallarino et al., ’96).

Previously, we have used IHC to show an ex-tensive distribution of NP-immunoreactive (IR)structures in the brain of the hagfish, Myxineglutinosa (Donald et al., ’92). These data demon-strated that the presence of NPs in the brain is avery primitive vertebrate characteristic, since itis hypothesised that hagfish branched from thevertebrate line over 500 million years ago (Foreyand Janvier, ’93). In the present study, the distri-bution of NP binding sites in the brain of M.glutinosa has been determined using autoradiog-raphy to correlate the distribution of immunore-active NP with putative sites of action. Both125I-rat ANP and 125I-porcine CNP were used asligands since homologous M. glutinosa peptideshave not been isolated and sequenced.

MATERIALS AND METHODS

AnimalsAtlantic hagfish Myxine glutinosa (25–50 g) of

either sex were trapped in the Gulf of Maine bycollectors at the Huntsman Marine Laboratory(St. Johns, NB, Canada) and maintained in a re-circulating sea water (12–15°C) aquarium. Fourspecimens were used for the study.

AutoradiographyFor autoradiography, brains were freeze-mounted

in Tissue Tek (Miles Inc., Elkhart, IN) in a micro-tome cryostat (Minotome, IEC, Needham Heights,MA), and 20 µm frontal sections were cut andmounted on gelatin-chromium aluminium coatedslides and dried overnight under vacuum at 4°C.The sections were used immediately, or were storedin sealed boxes at –20°C until used.

Preincubation of brain sections was performedat room temperature (22–24°C) for 15 min in 50

mM tris-(hydroxymethyl)aminomethane (Tris)HCl buffer (pH 7.4), 50 mM NaCl, 5 mM MgCl2,0.1% bovine serum albumin (BSA), and 0.05% bac-itracin. This was followed by incubation for 60 minin the same buffer supplemented with 4 µg/ml ofleupeptin, 2 µg/ml chymostatin, 2 µg/ml pepstatin,10–6 M phenylmethylsulfonylfluoride (PMFS), andeither rat 125I-ANP (termed 125I-rANP here) or por-cine 125I-Tyr0-CNP (termed 125I-pCNP here). Dis-placement of specific binding for 125I-rANP wasexamined in adjacent sections in the presence of1 µM rat 3-28 ANP (rANP). Displacement of spe-cific binding for 125I-pCNP was examined in con-secutive sections in the presence of 1 µM porcineCNP. After incubation the slides were washed (2× 10 min at 4°C) in preincubation buffer, fixed for20 min in 4% formaldehyde in 0.1 M phosphatebuffer (pH 7.4, 4°C), washed in 0.1 M phosphatebuffer (pH 7.4, 4°C), and then in distilled water (1min) and dehydrated in alcohol and dried overnightat 60°C. The sections were then apposed to Hyper-film-βmax for 5–21 days (depending on specific ac-tivity) at room temperature. The film was developedin Kodak GBX developer for 4 min, washed in wa-ter for 2 min, and fixed in Kodak GBX fixer for 5min. X-ray films were photographed on a whitelight-box using a Wild dissecting microscope andWild Leitz MPS 46 Photoautomat camera. Brainanatomy was determined according to the descrip-tions of Jensen (’30).

MaterialsRat (3-[125I]iodotyrosol28) atrial natriuretic peptide

(1800–2000 Ci/mmol) and Hyperfilm-βmax werepurchased from Amersham (Arlington Heights, IL).Porcine 125I-Tyr0-CNP (1400–1600 Ci/mmol) was ob-tained from Peninsula (Belmont, CA). Rat ANP (3-28), porcine CNP, and C-ANF (rat des[Gln18, Ser19,Gly20, Leu21, Gly22]ANP-(4-23)-NH2) were obtainedfrom Bachem (Torrance, CA). All other chemicalswere reagent grade and were purchased fromSigma (St. Louis, MO).

Figs. 1–8. Photomicrographs of M. glutinosa brain sec-tions exposed to X-ray film showing the distribution of 125I-rANP binding sites. Specific binding can be determined bycomparison of total 125I-rANP binding (Figs. 1, 3, 5, 7) andnon-specific binding (Figs. 2, 4, 6, 8); non-specific binding isdetermined by incubation of sections with 125I-rANP plus 1µM unlabelled rANP. All figures are frontal sections and onlyFigs. 2, 4, 6, and 8 (non-specific binding) are labelled. Scalebar on Fig. 8 is 500 µm and is the same for each figure.

Figs. 1 and 2. Section through the olfactory bulb. Specific125I-rANP is observed in the outer regions of the olfactorybulb (O).

NATRIURETIC PEPTIDE BINDING SITES IN HAGFISH BRAIN 409

Figs. 3 and 4. Section through the hemisphere. Specific125I-rANP binding is observed in the pallial layers (L) andventral region of the hemisphere (area basilis telencephali,A), but little specific 125I-rANP is present in the primordiumhippocampi (P).

Figs. 5 and 6. Section through the diencephalon. Specific125I-rANP was observed in the lateral regions of the dien-

cephalon, but little specific binding was present in the regionof the thalamic (T) and hypothalamic (H) nuclei. Little spe-cific binding was observed in the habenula (Ha).

Figs. 7 and 8. Section through the mesencephalon. Spe-cific 125I-rANP binding was observed in the dorsal region ofthe tectum (Te) and laterally in the hindbrain in the regionof the trigeminal nuclei (V).

410 J.A. DONALD ET AL.

RESULTSSpecific binding was determined as the differ-

ence in binding between sections incubated witheither 200 pM 125I-rANP or 125I-pCNP alone andadjacent sections incubated with radiolabelledligand and 1 µM of unlabelled ligand.

A dense distribution of 125I-rANP binding siteswas present in the olfactory bulb and pallial re-gion of the telencephalon, particularly in the outerlayers (Figs. 1–4). Further caudal, dense bindingwas again observed in the lateral pallial layersand moderate levels of binding were observed inthe basal region of the telencephalon; very littlespecific binding was observed in the habenula andthe primordium hippocampi (Figs. 3, 4). In thediencephalon, a distinct zone of specific 125I-rANPbinding separated a region of moderate bindingin the lateral regions of the diencephalon fromthe thalamic and hypothalamic nuclei; low levelsof specific 125I-rANP binding were observed in thethalamic and hypothalamic regions of the dien-cephalon (Figs. 5, 6). Little specific binding wasobserved in the habenula (Figs. 5, 6). In the mes-encephalon and medulla oblongata, moderate todense levels of 125I-rANP binding were observedin tectum and the region of the trigeminal nucleirespectively (Figs. 7, 8).

In contrast to 125I-rANP binding, 125I-pCNP bind-ing occurred predominantly in the olfactory bulb,telencephalon, and diencephalon. Specific 125I-pCNPbinding was present in the olfactory bulb (Figs. 9,10), and 125I-pCNP binding in central region of thebulb was denser than 125I-rANP (c.f. Figs. 1 and 9).Specific 125I-pCNP binding was present in the outerpallial layers of the telencephalon, but little if anyspecific binding was present in the primordium hip-pocampus and the ventral regions of the telencepha-lon (Figs. 11, 12). More caudally, diffuse specificbinding was present in the thalamic and hypotha-lamic regions of the diencephalon, with a zone ofdenser binding being present in the area of the hy-pothalamus (Figs. 13, 14). Little if any specific 125I-pCNP binding was observed in the mesencephalonand medulla oblongata.

A comparison of the distribution of 125I-rANPand 125I-pCNP binding sites with the location ofnatriuretic peptide-immunoreactive (NP-IR) struc-tures (Donald et al., ’92) in the brain of the hag-fish, Myxine glutinosa, is shown in Table 1.

DISCUSSION

The vertebrate NP system is a complex pep-tidergic system comprised of peripheral and cen-

tral peptides and receptors which are implicatedin the coordinated control of salt and water bal-ance (Takei and Balment, ’93; Gutkowska et al.,’97). A number of studies have described a broaddistribution of NP and NPR in the mammalianbrain, including the key osmoregulatory centres,and there is emerging evidence of their involve-ment in the control of salt and fluid balance(Gutkowska et al., ’97). In contrast, the role ofNPs in the brain of non-mammalian vertebratesis unknown. Anatomical studies in lower verte-brates have shown that NPs have a broad distri-bution in the brain of cyclostomes [Rienecke, ’87;Donald et al., ’92), elasmobranchs (Vallarino etal., ’90; Donald et al., ’92), teleosts (Donald andEvans, ’92), dipnoans (Vallarino et al., ’96), andamphibians (Feuilloley, ’93; Netchitailo, ’87). How-ever, NP binding sites have only been demon-strated in dipnoans (Vallarino et al., ’90) andamphibians (Tong et al., ’89; Kloas and Hanke,’92). The general conclusion from these studies isthat NPs and NP-binding sites are located inbrain regions such as the diencephalon and thehypophysis which, by homology with the mam-malian brain, are important in the central con-trol of osmoregulation. It is, therefore, interestingto examine the brain NP system of hagfishes,since their requirement for osmoregulation is re-duced because they are isoosmotic with seawater(Evans, ’93).

Previous IHC studies have shown an extensivedistribution of NP-IR in the brain of M. glutinosa,using an antibody that cross-reacts with ANP andCNP (Donald et al., ’92). Immunoreactive-peri-karya and fibers were observed in the pallium,the primordium hippocampi, the pars ventralisthalami, pars dorsalis thalami, the nucleus dif-fusus hypothalami, the nucleus profundus, thenucleus tuberculi posteriosis, and the nucleus ven-tralis tegmenti. The current study has demon-strated for the first time the presence of ANP-and CNP-binding sites in the brain of M. glutinosaand provides further evidence that the NP sys-tem of the agnathan brain is well developed. Thus,the evolution of brain NPs and their receptors oc-curred early in the vertebrate radiation and prob-ably before the migration of early vertebrates intofresh water. The distribution of 125I-rANP and 125I-pCNP binding sites in the brain of M. glutinosawas similar, with the most abundant binding oc-curring in the telencephalon and diencephalon.The major difference occurred in the caudal re-gions of the brain where little 125I-pCNP bindingwas observed in contrast to a moderate distribu-

NATRIURETIC PEPTIDE BINDING SITES IN HAGFISH BRAIN 411

Figs. 9–14. Photomicrographs of M. glutinosa brain sec-tions exposed to X-ray film showing the distribution of 125I-pCNP binding sites. Specific binding can be determined bycomparison of total 125I-pCNP binding (Figs. 9, 11, 13) andnon-specific binding (Figs. 10, 12, 14); non-specific binding isdetermined by incubation of sections with 125I-pCNP plus 1µM unlabelled pCNP. All figures are frontal sections and onlyFigs. 10, 12, and 14 (non-specific binding) are labelled. Scalebar on Fig. 14 is 500 µm and is the same for each figure.

Figs. 9 and 10. Section through the olfactory bulb (O).Specific 125I-pCNP is observed in the olfactory bulb (O) withthe densest binding in dorsal regions.

Figs. 11 and 12. Section through the hemisphere. Spe-cific 125I-pCNP binding is observed in the pallial layers (L),but little specific 125I-pCNP is present in the primordiumhippocampi (P) and the habenula (Ha).

Figs. 13 and 14. Section through the caudal diencepha-lon. Moderate levels of specific 125I-pCNP were present in thethalamic (T) and hypothalamic zones (H) of the diencepha-lon, with a zone of denser binding being present in the areaof the hypothalamus (H).

412 J.A. DONALD ET AL.

tion of 125I-rANP binding. The distribution of NP-IR and NP-binding sites showed concurrence insome areas, but in other areas there was “mis-matching.” A good correlation was observed in thepallial layers of the telencephalon and the rostraldiencephalon, but few binding sites were presentin the primordium hippocampi and the caudal re-gions of the diencephalon and mesencephalonwhere moderate NP-IR was observed (Donald etal., ’92). The “mismatch” between the location ofreceptors and the corresponding ligand has beenreported for other neuropeptide systems in thecentral nervous system (Kuhar et al., ’86).

In earlier studies, we have demonstrated thepresence of multiple types of NPR in the periph-eral tissues of M. glutinosa (Toop et al., ’95a,b).The gills, kidney, and aortae bind 125I-rANP, butonly the gills showed 125I-pCNP binding. The 125I-rANP and 125I-pCNP binding in the gills was dis-placed by unlabelled ANP, CNP, and the NPR-Cspecific ligand C-ANF (Maack, ’92). However, inthe kidney and aortae, the 125I-rANP binding wasonly displaced by ANP which suggests that theNPR in these tissues are NPR-A like. Interest-ingly, both ANP and CNP stimulated guanylatecyclase activity in the kidneys and the gills, withANP being more potent than CNP. Similarly, inthe ventral aortae, rat ANP is more potent thanporcine CNP in mediating relaxation of vascularsmooth muscle (Evans, ’91; Evans et al., ’93).Therefore, in the peripheral tissues that have beenexamined, only the gills appear to possess twotypes of binding site, a putative NPR-A-like bind-ing site and a binding site similar to NPR-C;the kidneys and aortae appear to contain onlythe NPR-A-like binding site. In the present

TABLE 1. A comparison of the distribution of 125I-rANP and 125I-pCNP binding sites with the location of natriureticpeptide-immunoreactive (NP-IR) structures (Donald et al., ’92) in the brain of the hagfish, Myxine glutinosa1

Brain region 125I-rANP Binding 125I-pCNP Binding NP-IR

Olfactory bulbs ++ ++ +, ∆lateral lateral and central

Telencephalon: pallium ++ ++ +, ∆outer layers outer layers

Primordium hippocampi – – ++, ∆Habenula – – +, ∆Thalamus + + ++, ∆Hypothalamus + ++ ++, ∆Mesencephalon: tectum ++ – +Mesencephalon: ventral + – ++, ∆Medullary horns ++ – +Medulla + – +1The distribution of binding sites and NP-IR structures has only been shown for broad areas of the brain. The density of ligand binding andof NP-IR structures has been indicated qualitatively as either (+) or (++), in which (+) represents low to medium density and (++) representsmedium to high density; the ∆ symbol represents the presence of NP-IR cell bodies in the brain.

study, the observation that 125I-rANP and 125I-pCNP show differences in binding suggest thatmore than one NPR is present in the brain. Ifthe predominant receptor in the brain wasNPR-C-like, both ligands would be expected tobind to this receptor with similar affinity (Maack,’92). A full understanding of the hagfish NP sys-tem will only be achieved by molecular character-ization of the NPs and receptors.

The presence of a well-developed NP system inthe brain and the periphery of M. glutinosa is fas-cinating when considered in the functional con-text of NPs as fluid volume regulators in highervertebrates. The fact that hagfishes are osmo-conformers and are almost iso-osmotic with thesurrounding sea water and presumably do notencounter major ionic and volume perturbations(Evans, ’93) raises the question: what is the bio-logical role of NPs as hormones and neuropep-tides in hagfishes? Clearly, analysis of theevolutionary history of the biology of NPs inagnathans (and protochordates) is critical inunderstanding the function of NPs in vertebratesin general. Northcutt (’96) has argued that thebrain of hagfishes is highly derived from that ofancestral craniates due to their invasion of thebenthic environment in which chemosensory andtactile stimuli are critical for feeding and repro-duction. Thus, the hemispheres have become en-larged in comparison to the lampreys (Northcutt,’96). This region of the brain is involved in olfac-tion and is an important sensory modality in hag-fish; the pallial layers of the hagfish brain arebelieved to receive projections from the olfactorybulbs. The telencephalon of hagfish is heavily in-vested with NP-IR neurons and NP binding

NATRIURETIC PEPTIDE BINDING SITES IN HAGFISH BRAIN 413

sites. In addition, previous studies have showna substantial distribution of NPs and NPR inthe telencephalon of lungfish (Vallarino et al.,’96), amphibians (Tong et al., ’89; Kloas andHanke, ’92), birds (Tavolaro et al., ’93), andmammals (see Gutkowska et al., ’97). Thus, arole for NPs in olfaction may occur throughoutthe vertebrates. Interestingly, cGMP, the secondmessenger in NP signalling, is an important cyclicnucleotide in olfactory signal transduction and isfound in high levels in the rat olfactory bulb(Hopkins et al., ’96).

LITERATURE CITEDAnand-Srivastava M, Trachte GJ. 1993. Atrial natriuretic fac-

tor receptors and signal transduction mechanisms. Pharma-col Rev 45:455–497.

Brown J, Zuo Z. 1993. C-type natriuretic peptide and atrialnatriuretic peptide receptors of rat brain. Am J Physiol264:R513–R523.

Donald JA, Evans DH. 1992. Immunohistochemical local-isation of natriuretic peptides in the heart and brain of thegulf toadfish Opsanus beta. Cell Tissue Res 269:151–158.

Donald JA, Vomachka AJ, Evans DH. 1992. Immunohis-tochemical localisation of natriuretic peptides in theheart and brain of the spiny dogfish, Squalus acanthias,and the Atlantic hagfish, Myxine glutinosa. Cell TissueRes 270:535–545.

Evans DH. 1991. Rat atriopeptin dilates vascular smoothmuscle of the ventral aorta from the shark (Squalusacanthias) and the hagfish (Myxine glutinosa). J Exp Biol157:551–555.

Evans DH. 1993. Osmotic and ionic regulation. In: Evans DH,editor. The physiology of fishes. Boca Raton, FL: CRC Press.p 315–341.

Evans DH, Donald JA, Stidham JD. 1993. C-type natriureticpeptides are not particularly potent dilators of hagfish(Myxine glutinosa) vascular smooth muscle. Bull Mt DesertIsland Biol Lab 32:106.

Evans DH, Takei Y. 1992. A putative role for natriuretic pep-tides in fish osmoregulation. News Physiol Sci 7:15–19.

Forey P, Janvier P. 1993. Agnathans and the origin of jawedvertebrates. Nature 361:129–134.

Gutkowska J, Antunes-Rodrigues J, McCann SM. 1997. Atrialnatriuretic peptide in brain and pituitary gland. PhysiolRev 77:465–515.

Hagiwara H, Hirose S, Takei Y. 1995. Natriuretic peptidesand their receptors. Zool Sci 12:141–149.

Hopkins DA, Steinbusch HWM, Markerinkvanittersum M,Devente J. 1996. Nitric oxide synthase, cGMP, and NO-me-diated cGMP production in the olfactory bulb of the rat. JComp Neurol 375:641–658.

Jensen J. 1930. The brain of Myxine glutinosa. J Comp Neurol49:359–507.

Kloas W, Hanke W. 1992. Localisation of binding sites foratrial natriuretic factor and angiotensin II in the centralnervous system of the clawed toad, Xenopus laevis. Cell Tis-sue Res 267:365–373.

Konrad EM, Thibault G, Pelletier S, Genest J, Cantin M.1990. Brain natriuretic peptide binding sites in rats: in vitroautoradiographic study. Am J Physiol 259:E246–E255.

Kuhar MJ, DeSouza EB, Unnerstall JR. 1986. Neurotrans-mitter receptor mapping by autoradiography and othermethods. Ann Rev Neurosci 9:27–59.

Langub MC, Watson RE, Herman JP. 1995. Distribution ofnatriuretic peptide precursors mRNAs in the rat brain. JComp Neurol 356:183–199.

Maack T. 1992. Receptors of atrial natriuretic factor. Ann RevPhysiol 54:11–27.

Quirion RM, Dalpe M, DeLean A, Gutkowska J, Cantin M,Genest J. 1984. Atrial natriuretic factor (ANF) binding sitesin brain and related structures. Peptides 5:1167–1172.

Takei Y, Balment RJ. 1993. Natriuretic factors in non-mam-malian vertebrates. In: Brown JA, Balment RJ, Rankin JC,editors. New insights in vertebrate kidney function. Cam-bridge: Cambridge University Press. p 351–385.

Tavolaro R, Canonaco M, Franzoni MF. 1993. The neuro-anatomic binding pattern of [125I]atrial natriuretic factorin the Japanese quail brain. Neurosci Lett 151:192–195.

Tong Y, Netchitailo P, Leboulenger F, Vaudry H, Pelletier G.1989. Localization of atrial natriuretic factor (ANF) bind-ing sites in the central nervous system of the frog. J CompNeurol 281:384–396.

Toop T, Donald JA, Evans DH. 1995a. Localisation andcharacteristics of natriuretic peptide receptors in thegills of the Atlantic hagfish, Myxine glutinosa. J ExpBiol 198:117–126.

Toop T, Donald JA, Evans DH. 1995b. Natriuretic peptidereceptors in the kidney and the ventral and dorsal aortaeof the Atlantic hagfish, Myxine glutinosa (Agnatha). J ExpBiol 198:1875–1882.

Vallarino M, Goula D, Trabucchi M, Masini MA, ChartrelN, Vaudry H. 1996. Immunocytochemical localisation ofatrial natriuretic factor and autoradiographic distributionof atrial natriuretic factor binding sites in the brain of theAfrican lungfish, Protopterus annectens. J Comp Neurol375:345–362.

Yandle TG. 1994. Biochemistry of natriuretic peptides. J In-tern Med 235:561–576.