autoradiographic localization and characterization of atrial natriuretic peptide binding sites in...

7/28/2019 Autoradiographic Localization and Characterization of Atrial Natriuretic Peptide Binding Sites in the Rat Central Ner

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The Journal of NeuroscienceJuly 1966, 6(7): 2004-2011

Autoradiographic Localization and Characterization of AtrialNatriuretic Peptide Binding Sites in the Rat Central Nervous Systemand Adrenal GlandThomas R. Gibson, Gary M. Wildey, Scott Manaker, and Christopher C. GlembotskiDepartment of Pharmacology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Atria1 natriuretic peptides (ANP) have recently been identif iedin both heart and CNS. These peptides possess potent natri-uretic, diuretic, and vasorelaxant activities, and are al l appar-ently derived from a single prohormone. Specific ANP bindingsites have been characterized in the adrenal zona glomerulosaand kidney cortex, and one study reported ANP binding sitesin the CNS. However, a detailed examination of the localizationof ANP binding sites throughout the brain has not been report-ed. In this study, quantitative autoradiography was employed toexamine the distribution of ANP receptors in the rat CNS. Thebind ing of (3-Z51-iodotyrosy128) rat ANP-28 to binding sites inthe rat CNS was saturable, specific for ANP-related peptides,and displayed high affinity (& = 600 PM). When the relativeconcentrations of ANP bind ing sites were determined through-out the rat brain, the highest levels of ANP bind ing were local-ized to the circumventricular organs, includ ing the area postre-ma and subfornical organ, and the olfactory apparatus. Moderatelevels of ANP binding sites were present throughout the mid-brain and brain stem, while low levels were found in the fore-brain, diencephalon, basal ganglia, cortex, and cerebellum. Thepresence of ANP binding sites in the subfornical organ and thearea postrema, regions considered to be outside the blood-brainbarrier, suggests that peripheral ANP levels may regulate someaspects of CNS control of salt and water balance. The possiblefunctions of ANP binding sites in other regions of the rat brainare not known, but, like many other peptides, ANP may act asa neurotransmitter or neuromodulator at these loci .Much interest has been generated by the recent discovery ofbioactive peptides within cardiac atria. These peptides, collec-tively referred to as atria1 natriuretic factor (ANF) or peptides(ANP), have been shown to exhibit natriuretic, diuretic, andvasorelaxant activities in several bioassay model systems (Cur-rie et al., 1983; DeBold et al., 198 1). ANP is also suggested tohave an inhib itory effect on adrenal steroidogenesis (Matsuokaet al., 1985). Although many bioactive peptides of varying sizehave been purified from atria1 tissue, al l contain common aminoacid sequences (Atlas et al., 1984; Currie et al., 1984) suggestingReceived Aug. 15, 1985; revised Jan. 9, 1986; accepted Jan. 9, 1986.

This work w as supported by National Institutes of Health Grant NS20396,American Heart Associa tion Grant-in-aid 84 653, with funds contributed in partby the Philadelphia Chapter of the American Heart Association, and Biomed icalResearch Support Grants SO7-05415-23 and -24, and Medical Scientist TrainingProgram G rant 5-T32-GM07170 awarded by the NIH. T.R.G. is a Spe cial In-vestigator ofthe American Heart Associatio n, Southeastern Pennsylvania Chapter,and C.C.G. i s an Established Investigator of the American Heart Association . Wewould like to thank Barry Wolfe, Richard Miselis, and Paul Shields for theircomments during the preparation of this manuscript.Correspondence should be addressed to Thomas R. Gibson, Department ofPharmacology/G3, University of Pennsylvania, 36th and Hamilton Walk, Phila-delphia, PA 19 104.Present address: Department of Medicine, Boston City Hospital, Boston, MA.Copyright 0 1986 Societ y for Neuro scienc e 0270-6474/86/0720 04-08$02.00/O

that these peptides may be shorter fragments of a large, commonprecursor. The isolation and characterization of both rat (Makiet al., 1984; Yamanka et al., 1984) and human (Oikawa et al.,1984) cloned atria1 cDNA encoding for ANP supports the hy-pothesis that all ANP-related peptides isolated to date can begenerated from a common preprohormone.ANP is one of many peptides originally discovered and char-acterized in peripheral organs and subsequently shown to existin the CNS (Iversen, 1983; Krieger, 1983). ANP-related pep-tides have been found in particularly high concentrations in thepons and hypothalamus, using both immunohistochemistry (Ja-cobowitz et al., 1985; Saper et al., 1985) and radioimmunoassay(Tanaka et al., 1984). In addi tion, different forms of ANP-re-lated peptides are found in the rat hypothalamus, atria, andplasma (Glembotski et al., 1985; Mot-ii et al., 1985; Tanaka etal., 1984). Therefore, ANP-related peptides may also be im-portant physiological regulators of fluid balance through cen-trally mediated mechanisms.ANP-related peptides are thought to produce the ir physio-logical effects by interacting with specific surface receptors onappropriate target tissues. Utilizing radioligand binding tech-

niques, high-affini ty (picomolar) bind ing sites for ANP havebeen identified and characterized on membranes derived fromrabbit aorta, rabbit and rat kidney (Napier et al., 1984) andbovine adrenal cortex (Currie et al., 1984). Specific bind ing siteswith lower affin ity (nanomolar) for ANP have also been shownto exist on cultured rat vascular smooth muscle cells (Hirata etal., 1984). That these binding sites described by radioligandbinding techniques are biolog ically relevant is suggested by 2observations: (1) their affinities for ANP-related peptides aresimilar to the reported plasma concentrations of immunoreac-tive ANP (Tanaka et al., 1984) and (2) the abi lity of variousANP analogs to inhib it the binding of labeled ANP to high-affin ity membrane sites parallels the bioactive potencies of theseanalogs (DeLean et al., 1984; Napier et al., 1984).ANP binding sites in the rat brain have been demonstratedusing autoradiography (Quirion et al., 1984). The autoradio-graphic distribution of these bind ing sites was found to be uniquecompared to other peptide receptors, and binding was not in-hibi ted by other peptide hormones or neurotransmitters. Thisreport, however, did not contain quantitative data character-izing these central ANP bind ing sites. In the present report weutilize quantitative autoradiography to localize and characterizethe ANP binding sites in the rat brain and adrenal gland.Materials and MethodsTerminologySinceconflicting erminology asevolved n describingariousANP-relatedpeptides, he following standardabbreviations ill be usedthroughout his paper:ANP-28, atria1natriureticpeptide- or pro-ANP(99-126);AP III, atriopeptinII or pro-ANP( 103-l 26); AP I, atrio-

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The Journal of Neuroscience Localization of Atrial Natriuretic Peptide Receptors 2005peptin I or pro-ANP( 103-l 24); and ANP( 13-28), atria1 natriuret ic pep-tide (13-28) or pro-ANP( 11 -l 26).AnimalsANP receptor leve ls were measured in brain and adrenal tissue takenfrom male Sprague-Dawley rats (1 Xl-200 gm; Charles River BreedingLabs Inc., Wilmington, MA). The rats were housed under a 14:lO hrlight-dark cyc le and were maintained with Purina rat chow and waterad ibitum.Chemicals and peptidesRat ANP-28, AP I, AP III , ANP( 13-28), adrenocorticotropic hormone(ACTH), cu-melanocyte-stimulating hormone ((uMSH), thyrotropin-re-leasing hormone (TRH), o-Ala-o-Leu-enkephalin (DADLE), and &en-dorohin were obtained from Peninsula Laboratories (Belmont, CA) orBachem (Torrance, CA). (3-1251-iodotyrosy12*) rat ANP-28 (lZiI-ANP-28; 1920-2000 Ci/mmol) was purchased from Amersham (ArlingtonHeights, IL). Biochemicals were obtained from Sigma Chemical Co. (St.Louis, MO), and all other compounds were reagent grade.Tissue preparationFor regional localization by quantitative autoradiography, animals weredecapitated and their brain tissue rapidly removed, mounted on cryostatchucks, and frozen in dry ice. Coronal sections (32 wrn) were cut at- 18C and thaw-mounted onto chromate/gelatin-subbed slides. Sec-tions were then stored with desiccant at -20C. Four consecutive sec-tions at 500 pm intervals were mounted, 2 per slide, for total andnonspecific binding determinations.For the biochemical characterization of Y-ANP-28 binding sites, 2methods were employed. First, for analysis by gamma counting, olfac-tory bulbs or adrenal glands were obtained from freshly killed rats andquickly frozen on dry ice. This tissue was then thawed, minced, andhomogenized to form a tissue mash. This mash was then placed into a3 ml plastic syringe and frozen, and sections (32 rm) were cut and thaw-mounted (2 sections per slide). Second, for densitometric studies, ol-fac tory bulb or adrenal tissue was mounted directly onto cryostat chucksand frozen; then, 32 pm sections were cut and thaw-mounted (2-4sections per slide). All sections were stored desiccated at -20C untilneeded.Binding proceduresSlides were warmed to room temperature and preincubated for 10 minin slide jars filled with buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mMMgCl,, 0.1% BSA, pH 7.4) at 25C. After preincubation, sections wereallowed to air-dry for at least 1 hr at 4C. Slides were then incubatedwith 300 ~1 (whole-brain slices) or 200 pl (ol factory bulb and adrenalmash and slices) of 4C buffer containing 5 &ml leupeptin, 10 PMbestatin, and varying concentrations of 12SI-ANP-28. These volumeswere sufficient to cover completely both sections on a slide. Since thebinding of 12+ANP-28 was shown to reach equilibrium after 90 min(not shown), the sections were incubated at 4C for 2 hr; more than95% of the iodinated lisand co-eluted with Y-ANP-28 standard onRP-HPLC (Glembotski-et al., 1985) after the incubation. They werethen washed with 4C buffer 4 times for 15 min each. Preliminaryexperiments established that little specif ic binding was lost by this pro-cedure. and fewer washes after incubation resulted in a greater propor-tion of nonspecific binding. For gamma counting, the sections werewiued of f the slides with Schleicher and Schuell no. 30 glass-fiber filterdisks, placed in tubes, and counted in an LKB Minigamma gammacounter. For autoradiography, the slides were brief ly dipped in 4Cdistilled water to remove buffe r salts and then rapidly dried on a 60-70C slide drier.Concentrations of lz51-ANP-28 employed were 50 PM for studies ofreaional localization and for inhibition analysis . and from 35 to 1000PM for Scatchard analysis. For inhibition analysis, 10 concentrations ofunlabeled ANP-related peptides spanning 5 orders of magnitude wereincluded; other compounds were included at concentrations indicatedin the text. Nonspecific binding was defined as the binding of lZSI-ANP-28 in the presence of 100 nM AP III . At 50 PM 12Y-ANP-28, specificbinding in sections o f tissue mash was 50-70% of total binding (1 OOO-2000 pm).Quantitative densitometryQuantitative autoradiograms of lZSI-ANP-28 binding to tissue sectionswere prepared as described previously (Rainbow et al., 1982). Sections

were apposed to LRB Ultro film (LRB, Inc., Gaithersburg, MD), exposedat room temperature for 5 d, and developed.Densitometric analysis ofautoradiograms was performed as describedpreviously (Rainbow et al., 1982, 1984; Unnerstall et al., 1982). Opticaldensity values were converted into fmol/mg of protein using lZ51stan-dards prepared from brain mash mixed with known quantities of lz51-olMSH (Rainbow et al., 1984; Unnerstall et al., 1982). Values for totaland nonspecific binding of lZ51-ANP-28 for each brain region were ob-tained by averaging 4-8 readings of the 2 sections on each autoradio-gram. Equal numbers of readings were taken bilaterally and averagedtogether. Af ter autoradiography, the sections were stained with cresylviolet to allow the overlaying of the section and autoradiogram forlocalization of the binding to specific brain structures. For Scatchardand inhibition analyses, the plexiform layer of the olfac tory bulb andthe zona glomerulosa of the adrenal gland were analyzed utilizing thetechniques described above.ResultsScatchard analysis of 12I-ANP-28 binding to olfactory bulbmash by gamma countingLinear Scatchard plots were generated by varying the concen-tration ofradiolabeled ligand from 50 to 1000 PM. A Kdof 567 f178 PM (mean of 3 experiments f SEM) was obtained, with aB,,, of 9.8 + 2.0 fmol/mg protein.Inhibition analysis of lz51-ANP-28 binding to olfactorybulb and adrenal mash by gamma countingWhen unlabeled ANP-related peptides were added to the in-cubation mixture, differing potencies of inhibition were ob-tained (Table 1). AP II I and ANP-28, comprising the 24 and28 COOH-terminal amino acids of pro-ANP, respectively , in-hibited the binding of 1251-ANP-28 by 50% with low nanomolarpotency. At peptide concentrations higher than 100 nM, thecompetit ion curves were no longer monophasic, with an ap-parent low-af finity binding component. This low-af finity bind-ing component has been reported previous ly for ANP binding(DeLean, 1984; Napier et al., 1984) and was not examined inthe present study. AP I, which is COOH-terminally shortenedby 2 amino acids ascompared o AP III, inhibited the bindingof Y-ANP-28 to olfactory bulb mashonly at highernanomolarconcentrations.ANP( 13-28)-which doesnot contain both cys-teine residues, and thus does not have the disulfide bond thatis present n longer ANP-related peptides-failed to block thebinding of the radiolabeled igand even at a concentration of 10PM. Inhibition of binding of 12sI-ANP-28 to adrenal mash byANP-related peptideshad a similar profile, although nhibitionby AP I seemedo be more potent. Other peptide hormones-including TRH, DADLE, ACTH, aMSH, insulin, and P-en-dorphin (all at 1 &-were unable to compete for the bindingof lZ51-ANP-28 n olfactory bulb mash. Propranolol, phentol-amine, atropine (all at 10@M), and carbachol(1 mM) were alsoineffective.Densitometric characterization of lZsI-ANP-28 binding toolfactory bulb and adrenal sectionsFrozen sectionsof olfactory bulb and adrenal tissue were in-cubated in the presence of 1251-ANP-28and various con-centrations of unlabeled ANP-related peptides. The resultingautoradiograms were quantified by densitometry for inhibitionanalysis.The IC,, values calculated for olfactory bulb sections(Table 1) correspond losely o resultsobtainedutilizing olfactorybulb mash as describedabove. The IC,,value for ANP-28 inadrenalsectionsof 0.84 f 0.20 nM (not shown)alsoagreeswiththe data obtained using adrenal mash (Table 1). Scatchardanalysis of incubation mixtures containing Xl-1000 PM 251-ANP-28 wasalso performed on sectionsof olfactory bulb, andaKd of 630PM andB,,, of 3 1.9 fmol/mg protein were.obtained.This K,, value is similar to that obtained by Scatchard analysis


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2006 Gibson et al. Vol. 6, No. 7, Jul. 1986

Table 1. Relative potencies of ANP-related peptides in inhibiting1z51-AP-28 binding to rat olfactory bulb and adrenal sections

CompoundGo bf)Mash,olfactory

Tissue,olfactory

Mash,adrenal

AP-28AP II IAP IANP( 13-28)

0.98 + 0.05 0.52 * 0.13 1.89 -t 0.280.96 f 0.32 1.13 + 0.44 2.90 + 1.1416.76 + 6.19 17.54 t 6.21 2.16 + 1.43> 1000 ND >lOOO

Several concentrations of ANP-related peptides were tested for their abilities toinhibit the binding of 50 PM Y-AP-28. Binding was assayed by gamma countingfor mash section s or autoradiography for tissue sections as described in Materialsand Methods. Values are expressed as means f SE for 3-5 experiments. ND, notdetermined.

of olfactory bulb mashdescribedabove, while the higher B,,,value reflects he concentration of binding sites o only a specificlayer of the olfactory bulb.Densitometricquantitation of regional bindingThe distribution of atria1 natriuretic peptide binding sites nindividual nuclei and subregions f rat brain was discrete andheterogeneousFig. 1). Moderately high (1 O-2.0 fmol/mg pro-tein) to very high (2.0-4.0 fmol/mg protein) levels of 1251-ANP-28 binding sites were localized within the circumventricularorgansand the olfactory apparatus Table 2). Moderate levels(0.5-1.0 fmol/mg protein) of ANP binding siteswere presentthroughout the midbrain and brain stem.Low levels


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The Journal of Neuroscience Loca lization of Atrial Natriuretic Peptide Receptors 2007Table 2. Relative concentrations of 1z51-AP-28 binding sites in ratbrain determined by quantitative autoradiography

Structure Y-ANP-28 bound(fmol/mg protein)Olfactory apparatus

Olfactory bulbPlexiform layerGranular layerGlomerular layer

Olfactory nerveAccessory olfactory bulbOlfactory nucleiLateral olfactory tract

CortexFrontal cortexPrimary olfactory cortexCorpus callosum

MedialLateral

Anterior cingulate cortexPosterior cingulate cortexSplenium corpus callosumStriate cortex

Limbic forebrainOlfactory tubercleLateral septum

DorsalIntermediateVentral

Medial septumDiagonal band of BrocaAnterior commissureBed nucleus of the stria terminalisVentral hippocampal commissureHippocampus

CA3Dentate gyrus

AmygdalaCentral nucleusLateral nucleus

Basal gangliaCaudate putamenAccumbens nucleusVentral palladium

Circumventricular organsSubfornical organMedian eminenceArea postrema

HypothalamusPeriventricular nucleusMedial preoptic areaLateral preoptic areaAnterior hypothalamic areaLateral hypothalamic areaSupraoptic nucleusParaventricular nucleusArcuate nucleusVentromedial nucleus

1.97 + 0.110.70 + 0.070.76 dz 0.190.65 k 0.153.13 -t 0.100.36 + 0.041.24 + 0.130.18 + 0.040.16 + 0.020.90 f 0.020.59 * 0.020.40 I!I 0.050.28 t- 0.061.09 + 0.100.52 + 0.08

0.18 + 0.050.25 k 0.060.24 -t 0.070.22 -t 0.070.30 f 0.050.35 IL 0.060.69 f 0.120.28 k 0.060.70 + 0.100.28 + 0.020.29 f 0.030.21 + 0.100.24 + 0.02

0.12 2 0.020.22 + 0.020.22 f 0.04

1.51 + 0.260.52 f 0.112.27 -t 0.55

0.33 f 0.050.32 f 0.040.34 + 0.060.17 + 0.060.25 + 0.020.33 * 0.040.30 t- 0.030.40 + 0.060.30 f 0.04

Table 2. Continued1251-ANP-28 boundStructure (fmol/mg protein)

Dorsomedial nucleus 0.32 + 0.06Posterior nucleus 0.31 + 0.08Mammillary nuclei 0.38 k 0.12ThalamusParaventricular nucleus 0.68 + 0.02Anteroventral nucleus 0.45 + 0.05Anteromedial nucleus 0.32 f 0.02Ventrolateral nucleus 0.43 * 0.03Ventroposterolateral nucleus 0.50 + 0.04Lateral dorsal thalamus 0.34 + 0.06Lateral posterior thalamus 0.24 + 0.05Habenular nucleus 1.05 + 0.14

MidbrainSubstantia nigra 0.36 + 0.09Central gray 0.41 k 0.09Superior colliculus 0.51 k 0.12lnterpeduncular nucleus 0.86 + 0.08

Cranial nerves and nucleiOptic nerve 0.45 + 0.03Optic chiasm 0.40 + 0.05Motor nucleus (5) 0.47 I! 0.13Principal sensory nucleus (5) 0.48 + 0.08Facial nucleus (7) 0.53 + 0.10Vestibular nucleus (8) 0.43 f 0.06Solitary nucleus (10) 0.34 + 0.06Hypoglossal nucleus (12) 0.37 + 0.04

Brain stemPontine nucleus 0.56 + 0.10Inferior colliculus 0.56 2 0.15Dorsal raphe 0.55 + 0.09Medial raphe 0.55 + 0.13Pontine caudal reticular nucleus 0.58 + 0.08Superior olive 0.47 + 0.11Inferior olive 0.44 k 0.05Parvocellular reticular nucleus 0.53 + 0.09Paragigantocellular nucleus 0.55 * 0.10Lateral reticular nucleus 0.35 + 0.04Medullary reticular nucleus 0.40 k 0.07

Cerebellum 0.28 + 0.03Frozen 32 am thick brain sectio ns were labeled as described in Materials andMethods with 50 PM T-AP-28, and apposed against LK B Ultrofdm for 5 d togenerate autoradiograms. Brain sections (32 pm) were cut at 500 pm intervalscorresponding to coronal levels of the atlas of Paxinos and Watson (1982). Seriallycut sections at each level were used for determination of total and nonspe cificbinding. Nonspec ific binding was subtracted from each structure at each level.Values are means + SE from readings taken from 4 rat brains.

levels of ANP-related peptides in the preoptic area and thepontine tegmentum (Saper et al., 1985) sites of low levels ofANP receptors.This disparity in localization betweena peptideand its receptor has been observed previously, and several ex-planations have been proposed Kuhar and Unnerstall, 1985).However, the existence of ANP-related peptides n the AV3Vregion of the preoptic area s especially ntriguing, since ibersfrom the subfornicalorganextend directly to the AV3V (Miselis,1981). The subfornical organ also has afferent connections tothe paraventricular nucleus and lateral area of the hypothala-


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Figure I. Distribution of AP-28 receptors in rat brain and adrenal gland determined by quantitative autoradiography. Frozen 32 pm thick sectionsof rat brain and adrenal gland were labeled, as described in Materials and Methods, with 50 PM 1251-AP-28 and apposed to LKEi Ultrofilm for 5 dto generate autoradiograms. Sections representing total binding are on the left , while sections representing nonspecific binding are on the right. A-D, Coronal level f rom the atlas of Paxinos and Watson (1982). All brain sections are presented at the same magnification. A, Level 2: PL, plexiform


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The Journal of Neuroscience Localization of Atrial Natriuretic Peptide Receptors 2009mus, the bed nucleus of the stria terminalis, and the perifomicalregion (Miselis, 1981; Weiss et al., 1984), al l areas shown topossess neurons that contain ANP immunoreactivity (Saper etal., 1985). In addition, a major projection from the area pos-trema extends to the lateral parabranchial nucleus (Shapiro andMiselis, 1985), which has been shown to contain fibers fromthe AV3V that contain ANP-immunoreactive material (Saperet al., 1985).Thus, it is apparent that numerous connections exist betweenANP receptor-containing and ANP-related peptide-containingstructures. Also, fibers from the subfomical organ extend to theorganum vasculosum of the lamina terminalis (OVLT), a struc-ture postulated to be directly involved in peripheral ANP feed-back control of CNS fluid balance regulation (Saper et al., 1985).Although it is difficult to visualize the OVLT in coronal sections,in several sections we observed moderate to moderately highlevels of ANP bind ing sites in the OVLT region. Thus, theOVLT itself might be included in the group of circumventricularorgans that contain ANP receptors.Another region conta ining high levels of ANP receptors, theolfactory apparatus, has few known functions other than olfac-tion. However, the olfactory bulb contains numerous neuro-peptides and peptide receptors. For example, insulin, luteiniz inghormone-releasing hormone, substance P, Met-enkephalin,TRH, somatostatin, and angiotensin II are al l high ly concen-trated in the olfactory bulb (Bogen et al., 1982; Brownstein etal., 1975, 1976; Havrankova et al., 1978; Jennes and Stumpf,1980; Kreider et al., 1981; Lind et al., 1985). Peptide receptors,such as those for TRH and cholecystokinin, are also present inthe olfactory bulb in high levels (Gaudreau et al., 1983; Manakeret al., 1985; Van Dijk et al., 1984; Zarbin et al., 1983a). Thisconcentration of peptides and peptide receptors may indicatethat the olfactory apparatus utilizes a rich variety of peptidesin the neurochemistry of olfaction, or it could suggest that theolfactory bulb may have other (as yet undiscovered) functions.ANP binding was also found i n the lateral region of the media lhabenular nucleus of the thalamus and the interpeduncular nu-cleus of the midbrain . Interestingly, these 2 nuclei are connectedby the fasciculus retroflexus, which also contains ANP bindingsites (Fig. 1). The function of ANP receptors in these areas isnot known, but the presence of ANP binding in all 3 structuressuggests that the habenular and interpeduncular nuclei may bepart of an integrated network, functional ly as well as structurally.It has been discovered that different forms of ANP-relatedpeptides are present in various tissues. In the atria, ANP-im-munoreactive material has been found predominately in a, lo-15 kilodalton (kDa) form, while smaller forms have been lo-calized to the plasma (3 kDa) and hypothalamus (1.5 kDa)(Glembotski et al., 1985; Morii et al., 1985; Tanaka et al., 1984).Thus, cardiac-derived ANP-related peptides in the plasma (the3 kDa form) may act on the CNS through receptors in thesubfomical organ and area postrema (both circumventricularorgans outside the blood-brain barrier), perhaps similarly to theway that angiotensin II acts on these structures (Edwards andRitter, 1982; Simpson, 1981). In contrast, the different formsof ANP found within the brain (such as the hypothalamus) mayinteract with receptors found in the olfactory apparatus andhindbrain, much like classical neurotransmitters or neuromodu-lators.Given the relative lack of data on the physiological effects ofANP in the CNS, we cannot now rule out the possibility thatt

these bind ing sites in the CNS are merely acceptors for ANP,and not ANP receptors (Quirion et al., 1984). It is even possiblethat these binding sites may not be on neurons, but on glia orcapillaries, although the pattern of binding, for example, in theolfactory bulb would argue against this hypothesis. In addition ,one recent study did demonstrate that basal release of argininevasopressin from the neural lobe of the pituitary was decreasedwhen 2 nmol AP III were injected into the third ventricle ofconscious male rats (Samson, 1985). This would indicate thatANP receptors are indeed involved in brain function. Furtherelectrophysiological and microinjection studies are needed todemonstrate that these binding sites are biolog ically relevantreceptors that mediate the actions of ANP on the CNS.It has been established that, in some bioassay systems, theCOOH-terminal tyrosine of Pro-ANP-derived peptides is im-portant to the bioactivity of these peptides (Garcia et al., 1985).In the present study, (3-12SI-iodotyrosy128) rat ANP-28 was usedas the radiolabeled ligand. The presence of the large iodine atomattached to the COOH-terminal tyrosine might have resultedin a different affin ity for the receptor, as opposed to the nativepeptide, as well as a different pharmacological profile in com-peti tion experiments. Since *I-ANP was shown to have equa lpotency to noniodinated ANP in the rat and rabbit aorta bioas-say (Napier et al., 1984) and the IC,, values obtained in thepresent study using unlabeled ligand are similar to the lZSI-ANP-28 K, values, the modification of this tyrosine apparently doesnot radically change the properties of the peptide as a ligand.However, the Kd values obtained in the present study do seemto differ from those in some other studies. Napier et al. (1984)obtained K,, values of 50 PM for ANP-28 binding to partiallypurified kidney membranes, while our higher Kd and IC,, valueswere obtained by bind ing to sections of olfactory bulb and ad-renal mash and tissue sections. Significant differences in Kd val-ues have been obtained previously in other systems when these2 different binding protocols have been compared. For example,when binding of quinucl idinyl benz ilate to muscat-uric receptorswas examined, K, values of 14, 270, and 500 PM were obtainedin experiments using prepared membranes and 10 and 32 pmsections, respectively (Luthin and Wolfe, 1984; Rainbow et al.,1982; Wamsley et al., 198 1). Thus, it appears that the inabil ityof the ligand to penetrate into tissue sections may be impl icatedin the higher Kd values obta ined as tissue thickness increases.In the present study, specific binding to white matter struc-tures was observed, and this binding appeared to be discreteand heterogeneous as well. For example, while the corpus cal-losum and olfactory apparatus white matter contained moderateto moderately high levels of ANP receptors, the cranial nervesexamined and the cerebellar and cerebral peduncles containedmuch lower levels. In addi tion, within the corpus callosum itself,the media l corpus callosum contained higher levels than did themore lateral regions. Thus, it seems that these binding siteswithin white matter are not simply nonspecific binding, butrepresent actual specific binding sites. While the function ofANP b inding sites within white matter is not known, they couldrepresent receptors in transit to distal regions, as reported forother peptides and classical neurotransmitters (Laduron, 1980;Young et al., 1980; Zarbin et al., 1981, 1983b). For example,the receptors present in the lateral olfactory tracts could be intransit from or to the olfactory bulb, which contains high levelsof ANP receptors. It is possible that these binding sites mightnot be actual receptors, and may be of lower affin ity. Indeed,

layer of olfactory bulb; CR, granular layer of olfactory bulb; ON, olfactory nerve. B, Level 16: SFO, subfomical organ; CC, corpus callosum; VHC,ventral hippocampal commissure; CD, caudate. C, Level 21: HB, habenular nucleus of the thalamus; CC, corpus callosum; CA3, level CA3 of thehippocampus; FR, fasciculus retroflexus. D. Level 42: AP, area postrema; CB, cerebellum; MR, medullary reticular formation. E, Adrenal section:GM, zona glomerulosa; FS, zona fasc iculata; RT, zona reticulata; AM, adrenal medulla.


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2010 Gibson et al. Vol. 6, No. 7, Jul. 1986Quirion et al. (1984) found nondisplaceablebinding in whitematter, although a different peptide wasused o determine non-specificbinding than in the presentstudy.In conclusion, binding sites or ANP have been ocalized toboth peripheral and CNS regions. The localization of specificbinding sites o the circumventricular organssuggestshat ANP-related peptidesmay play a key role in integrating the abilitiesof the nervous and endocrinesystems o regulate luid and elec-trolyte balance. Further characterization of the factors respon-sible for the regulation of both peripheral and central ANP andANP receptor levels will facilitate an understandingof the roleof ANP in the heart and CNS asan important regulator of fluidbalance.ReferencesAtlas, S. A., H. D. Kleinert, M. J. Camargo, A. Januszewicz, J. E. Sealy,J. H. Laragh, . W. Schilling, . A. Lewicki,L. K. Johnson, nd T.Maack (1984) Purification, sequencing and synthesis of natriureticand vasoactive rat atria1 peptide. Nature 309: 7 17-7 19.Bogen, N., N. Brecha, C. Gall, and H. J. Karten (1982) Distributionof enkephalin-like immunoreactivity in the rat main olfactory bulb.

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autoradiographic localization and characterization of atrial natriuretic peptide binding sites in...

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