Localization of Specific Binding Sites for AtrialNatriuretic Factor in Peripheral Tissues of the

Guinea Pig, Rat, and HumanCHRISTOPHER R. MANTYH, LAWRENCE KRUGER, NICHOLAS C. BRECHA,

AND PATRICK W. MANTYH

SUMMARY Specific, high affinity atrial natriuretic factor (ANF) binding sites were identified andlocalized by autoradiographic techniques in peripheral tissues of the guinea pig, rat, and human. Inthe guinea pig kidney, high concentrations of ANF binding sites were located in the glomerularapparatus, outer medulla, and small renal arteries. Other peripheral tissues containing ANF bindingsites included the zona glomerulosa of the adrenal cortex, the smooth muscle layer of the aorta andgallbladder, the lung parenchyma, the posterior lobe of the pituitary, the ciliary body of the eye,and the leptomeninges and choroid plexus of the brain. The distribution of ANF binding sites in therat and human kidney was nearly identical to those seen in the guinea pig kidney; high concentrationswere present in the glomerular apparatus, outer medulla, and small renal arteries. These results areconsistent with earlier physiological and pharmacological studies that suggested that ANF plays afunctional role in the regulation of extracellular fluid volume and blood pressure. There appears to belittle species variation in the location and concentration of renal ANF binding sites, suggesting that, atleast in the kidney, the results in experimental animals are relevant to the actions of ANF in humans.The finding that ANF binding sites were stable and present in high concentrations in human postmor-tem kidneys further suggests that these tissues may be amenable to testing for the involvement of ANFreceptor dysfunction in diseases such as hypertension and congestive heart failure.(Hypertension 8: 712-721, 1986)

KEY WORDSciliary body

atriopeptins •choroid plexus

receptors • kidney* hypertension

adrenal smooth muscle

A wide range of physiological experiments have/ \ indicated the existence of humoral factors

A. \ . that stimulate natriuresis and diuresis. Ex-tracts of heart atria, but not ventricular tissue, havebeen shown to exhibit natriuretic and diuretic activity,1

and studies of these crude extracts have indicated thatthe kidney is a major target tissue.2"5 The effect of atrialnatriuretic factor (ANF) on renal salt excretion is of

From the Center for Ulcer Research and Education (C. R. Man-tyh, N. C. Brecha, P. W. Mantyh), VA Medical Center — Wads-worth, and the Department of Medicine (N. C. Brecha, P. W.Mantyh) and the Brain Research Institute (L. Kruger, N. C. Brecha,P. W. Mantyh), UCLA School of Medicine, Los Angeles, Califor-nia.

P. W. Mantyh is a Smith, Kline and Beckman Fellow and anAlfred P. Sloan Fellow. N. C. Brecha is an Alfred P. Sloan Fellow(supported by National Institutes of Health Grant N5-5685).

Preliminary reports of these findings have been published {Na-ture 1984;312:756-757 and N Engl J Med 1985;312:1710).

Address for reprints: Dr. Patrick W. Mantyh, CURE/UCLA, VAWadsworth, Bldg 115, Room 219, Los Angeles, CA 90073.

Received September 3, 1985; accepted February 11, 1986.

particular interest since extracellular fluid volume isregulated by the balance between sodium intake andexcretion, and since a defect in renal sodium excretionhas been postulated to be an underlying abnormality insome patients with hypertension.6'7 Recently, a familyof peptides known as ANFs has been isolated,8'9 se-quenced,10"15 and cloned.16"19 These peptides appear tohave a common precursor16"20 and are released in re-sponse to changes in intra-atrial pressure and stretch ofthe atrial wall.21 Previous homogenate binding assayshave demonstrated specific, high affinity receptors inrabbit and rat kidney and aorta22'23 and bovine adrenalglomerulosa cells.24 These observations suggest thatANFs released from the atria play a role in the regula-tion of fluid volume, electrolyte balance, and bloodpressure.

In the present study, we examined several peripheraltissues in the guinea pig, rat, and human to determineif they contain specific ANF binding sites that maycorrespond to functional ANF receptors.22"24 The ob-jectives were threefold: 1) to assess which peripheral

712

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organs express specific ANF binding sites and thus arepossible target tissues for circulating ANF; 2) to definewhether the distribution and concentration of ANFbinding sites in experimental animals are similar tothose in humans; and 3) to determine the stability of theANF binding sites in human postmortem material.

Materials and MethodsThe quantitative autoradiographic receptor binding

technique25"27 was used to identify the specific ANFbinding sites. The incubation conditions used are amodification of a previous homogenate receptor bind-ing study.22 Male guinea pigs (weight, 500 g; Simon-sen Labs, Gilroy, CA, USA) and Sprague-Dawley rats(weight, 200 g; Simonsen Labs) were decapitated, andthe peripheral tissues were removed, embedded incryoform, serially sectioned at 30 /xm on a cryostat at— 20 °C, thaw-mounted on gelatin-coated microscopeslides, and stored at — 20°C until use. For analysis ofthe human material, postmortem kidneys were ob-tained from a 57-year-old man who died of myocardialinfarct, a 61-year-old man who died of cerebral aneu-rysm, and a 27-year-old man who died of head injury.The human tissue was obtained 12, 60, and 65 hoursafter death, respectively, and processed as described.

In initial experiments, we determined the optimalincubation conditions for ANF receptor binding in theguinea pig kidney in order to maximize the specific/nonspecific binding ratios. To measure the specific/nonspecific binding ratios, guinea pig kidney sectionswere scraped from the slide with a razor blade andplaced in a counting tube and the radioactivity wasdetermined using a gamma counter. Nonspecific ANFbinding was defined as that l23I-labeled ANF that wasnot displaced by an excess (1 /xM) of cold ANF. Afterextensive trial and error, it was determined that thefollowing incubation conditions consistently resultedin the highest specific/nonspecific binding ratios whileretaining optimal optical resolution of the bindingsites.

The slide-mounted tissue sections were first placedin a preincubation bath at 25°C containing 0.005%(vol/vol) polyethylenimine (Sigma Chemical, St.Louis, MO, USA) and 50 raMN-2-hydroxyethylpiper-azine-W-2-ethanesulfonic acid (HEPES) buffer for 10minutes. They were then incubated in 50 mM HEPES,5 mM MgCl2,0.3% bovine serum albumin, 0.1% baci-tracin, and 150 pM I23I-ANF at 25°C for 45 minutes.For rat and guinea pig tissue, l23I-rat ANF (Amer-sham, Chicago, IL, USA) was the radioligand, and forthe human tissue we used l25I-human ANF (Amer-sham). The slide-mounted tissue sections were thenwashed three times (5 minutes each) at 4°C in 50 mMHEPES, 5 mM MgCl2, 0.3% bovine serum albumin,and 0.1% bacitracin. Finally, the slides were rinsedtwice for 5 seconds at 4°C in distilled water. Controlslides were incubated similarly except for the secondstep, where either 1 /xM of cold rat ANF (1-28) (Ba-chem, Torrance, CA, USA), cold human ANF (1-28)(Bachem), or an unrelated peptide (substance P, so-

matostatin 1-14, sulfated cholecystokinin-8, or thyro-tropin releasing hormone; all from Bachem) was add-ed. The slides were placed in apposition to LKBUltrofilm (Bromma, Sweden) for 1 week and thendeveloped in Kodak D-19.

The autoradiograms in the figures are enlargementsof the LKB Ultrofilm negatives. To identify the cellu-lar location of the binding sites, the sections werestained with hematoxylin and eosin and coverslippedwith Permount. After development and fixation as justdescribed, the optical densities of the autoradiogramswere determined by projecting the autoradiograms at20 x on a white, horizontal surface and quantifying thedensity of the projected image with a photocell con-nected to a digital voltmeter, as described by Rainbowet al.27 This densitometer consisted of a Sharp BS-500A silicon blue photodiode (Paramus, NJ, USA)connected to a Radio Shack voltage amplifier (LosAngeles, CA, USA). At 20 x , the resolution of thedevice corresponded to a region 50 /xm in diameter onthe projected sections. Previous experiments had es-tablished that the LKB film does not respond linearlyto a linear increase in radioactivity.27 We thereforeconstructed a series of standards, exposed these to theLKB film, developed and fixed the firm, measured thisfilm densitometrically, and used these values with theTexas Instruments (Lubbock, TX, USA) automaticcurve-fitting program (Hewlett-Packard, Palo Alto,CA, USA) to obtain an equation that described thefirm's characteristics. Mean raw density values for theconcentration of ANF binding sites were then obtainedfrom a minimum of five separate readings and placedin this equation to correct for the nonlinearity of theLKB film. Thus, all values listed in Table 1 are cor-rected optical densities such that a doubling of densityvalues in this table corresponds to a doubling in theconcentration of ANF binding sites in the peripheraltissues.

ResultsSpecific, high affinity ANF binding sites were dem-

onstrated in rat, guinea pig, and human peripheral tis-sues using the autoradiographic technique and 125I-ratANF (1-28) for rat and guinea pig tissue, and l23I-hu-man ANF (1-28) for the human tissue (Table 1). Initialscrape-off experiments in the guinea pig kidney re-vealed that greater than 75% of the total binding wasspecific. No inhibition of specific binding was ob-served in adjacent sections where 1 /iM of unrelatedpeptide including substance P, somatostatin 1-14,cholecystokinin-8, or thyrotropin releasing hormonewas added to the incubation medium. These observa-tions are consistent with previous membrane homog-enate studies2224 and indicate that the two techniquesprobably are identifying the same specific, high affin-ity ANF binding sites that appear to correspond to theANF receptor.22-24

In the guinea pig urogenital system, a high concen-tration of specific ANF binding sites was present in theglomerular apparatus (GA) of the kidney (Figure 1).

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TABLE 1. Concentration* of Atrial Natriuretic Factor BindingSites in Peripheral Tissues of Humans, Rats, and Guinea Pigs

Optical Density

Tissue

Urogenital system

Kidney

Glomerularapparatus

Renal arteries(smooth muscle)

Outer medulla

Endocrine system

Pituitary gland(posterior lobe)

Adrenal gland (zonaglomerulosa)

Respiratory system

Lung (parenchyma)

Cardiovascular system

Aorta (smooth muscle)

Eye

Ciliary body(epithelial layer)

Choriocapillaris

Gastrointestinalsystem

Colon (smoothmuscle)

Other systems

Gallbladder (smoothmuscle)

Choroid plexus

Leptomeninges

Guinea pig

81.9±5.7

52.0±4.0

53.1±2.3

66.7±3.1

54.2±1.8

51.7±1.3

42.1 ±0.5

100.0+1.645.8±1.1

13.2±1.7

43.1 ±2.530.8±0.7

38.2±1.2

Rat

80.2±1.7

40.5 ±0.8

45.2 + 0.7

—

64.6 ±0.6

—

—

—

—

—

—33.4±0.9

41.6±1.5

Humant

74.1 ±1.3

62.7±2.3

39.2±2.0

—

—

—

—

—

—

—

——

—

Values are corrected optical densities ± SEM of at least fivedeterminations throughout the area under examination. A dash indi-cates no data; negligible levels were found for all regions of thefollowing guinea pig organs: heart, tongue, salivary glands, esoph-agus, stomach, small intestine, pancreas, liver, spleen, larynx,trachea, urinary bladder, testes, vas deferens, fallopian tubes,uterus, nose, and skin.

*The concentration is expressed as the corrected optical densityof LKB Ultrofilm exposed to guinea pig, rat, and human tissuesections after incubation with either '"I-rat ANF (guinea pig andrat) or l23I-human ANF. Background labeling on the film has anoptical density of 0.0, while the epithelial layer ciliary body of theguinea pig eye is defined as the highest optical value of 100.0.

tThe values for me human are those of a 27-year-old man with apostmortem autopsy time of 65 hours.

Comparison of the location and density of ANF bind-ing sites indicates that of the several thousand GAspecimens we have examined, all have a similar highconcentration of ANF binding sites. The smooth mus-

cle layer of the small renal arteries also had a highconcentration of ANF binding sites (see Figure 1). Theother region of the kidney with a high concentration ofANF binding sites was the outer medulla, which corre-sponds to the location of the cortical collecting tubules(see Figure 1). Other areas of the urogenital systemexamined displayed a negligible level of ANF bindingsites, including all layers of the bladder, testes, vasdeferens, seminal vesicles, uterus, and fallopian tubes.

In rat (Figure 2) and human (Figure 3) kidney, thedistribution of specific ANF binding sites was almostidentical to that seen in guinea pig kidney. High con-centrations of ANF binding sites were present in theGA, outer medulla, and renal arteries. As with theguinea pig, of the several thousand GA specimensexamined in either the rat or human, all displayed ahigh concentration of ANF binding sites.

In the guinea pig endocrine system, both the adrenalgland and the posterior pituitary revealed a substantialconcentration of ANF binding sites. In the adrenals,ANF binding sites were confined to the zona glomeru-losa of the adrenal cortex (see Figure 1). All otherlayers of the cortex and medulla showed a negligibleconcentration of ANF binding sites. In the pituitary,only the ventral aspect of the pars nervosa (posteriorpituitary) contained a significant concentration ofbinding sites in contrast to the negligible concentrationin the dorsal aspect of the pars nervosa, pars distalis,and pars intermedia (Figure 4). The rat adrenals dis-played a distribution of binding sites confined to thezona glomerulosa (see Figure 2) that was nearly iden-tical to that found in the guinea pig adrenals (see Fig-ure 1).

In the guinea pig cardiovascular system, a moderateconcentration of binding sites was seen predominantlyin the smooth muscle layer of the aorta (Figure 5). TheANF binding sites were also present in the lung paren-chyma (Figure 6), with no apparent proximal-distalconcentration gradient. Negligible concentrations ofbinding sites were present in all other layers of theaorta, all areas of the right and left atrium and ventri-cles, pulmonary arteries and veins, trachea, bronchi,bronchioles, and larynx.

In the guinea pig gastrointestinal system, only thelongitudinal muscle layer of the distal colon had ameasurable level of ANF binding sites. All other areasof the gastrointestinal tract examined, including alllayers of the esophagus, stomach, duodenum, ileum,jejunum, cecum, proximal colon, and rectum, hadnegligible concentrations of binding sites. The onlyother component associated with the digestive appara-tus that contained a high concentration of ANF bindingsites was the smooth muscle layer of the gallbladder(Figure 7). Negligible levels of binding sites werepresent in all other layers of the gallbladder, spleen,liver, pancreas, tongue, and salivary glands.

The ciliary body in the eye of the guinea pig dis-played the highest concentration of ANF binding sitesof all the tissues examined in guinea pig, rat, or human(Figure 8). These binding sites were clearly concen-trated in the epithelium of the ciliary body; the otheraspects of the ciliary body displayed negligible levels.

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The choriocapillaris also contained a moderate con-centration of binding sites. Of the intrinsic muscles ofthe eye, only the longitudinal muscle contained a sig-nificant concentration of binding sites (see Figure 8).All other layers of the eye, including the retina, sclera,lens, iris, and cornea, possessed a negligible concen-tration of ANF binding sites.

Other tissues in the guinea pig containing a distinc-tive concentration of specific ANF binding sites in-cluded the choroid plexus epithelium and the lepto-meninges (i.e., pia and arachnoid) of the brain, asreported in a previous study.28 The concentration ofANF binding sites appeared to vary within the differentregions of the choroid plexus; consistently high levelswere present in the fourth ventricle, whereas markedlydiminished or patchy concentrations were present inthe lateral cerebral ventricles (Figure 9). Additionaltissues examined that contained negligible concentra-tions of binding sites included all layers of the thyroidand the skin of the nose, hindpaw, and forepaw.

Examination of the concentration of ANF bindingsites in the three human postmortem kidneys revealedno significant differences in either concentration orlocation based on the subject's age (27, 57, and 61years) or time of autopsy (12, 60, and 65 hours afterdeath).

DiscussionThe distribution of ANF binding sites in the periph-

eral tissues examined in the present study appears con-sistent with the functional role generally ascribed toANF in regulating fluid volume, electrolyte balance,and blood pressure. Previous studies have suggestedthat ANF acts directly on the kidney, because injectionof ANF produces a marked increase in urine volumeand an accompanying increase in sodium, potassium,and chloride excretion.1"5 This effect is consistent withthe reports that specific ANF binding sites are found intissue homogenates of rabbit and rat kidney22'M andaorta, as well as in bovine adrenal glomerulosa cells.24

FIGURE 1. Dark-field photomicrographs ofLKBfilm autoradiograms of a guinea pig kidney and adrenal section(a), incubated in the presence of 150 pM l25I-atrial natriuretic factor (ANF), and its serial pair (b), incubated in150 pM I251-ANF plus 1 fiM ANF. In these and the following dark-field photomicrographs, ml-ANF bindingappears as white silver grains. Since nonspecific binding is defined as that I251-ANF binding that remains in thepresence of 1 (JLM ANF, Panel b is an image of the nonspecific binding. Thus, the specific binding in Panel a isobtained by subtracting the nonspecific binding in Panel b. A high concentration of receptors is present in theglomerular apparatus (GA), renal arteries (RA), and outer medulla (OM) of the kidney. The zona glomerulosa(ZG) is clearly outlined in the adrenal cortex. (Line bar = 7.5 mm.)

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716 HYPERTENSION VOL 8, No 8, AUGUST 1986

ANF 1 ANF + 1uM ANF

' - • ' ' • ; h

FIGURE 2. Dark-field photomicrograph showing the localization ofatrial natriuretic factor (ANF) binding sitesin a coronal section of the rat kidney and adrenal. Note the high concentration of ANF binding sites in theglomerular apparatus (GA), renal arteries (RA), outer medulla (OM), and zona glomerulosa (ZG). See Figure 1for explanation. (Line bar = 1.5 mm.)

In the present study, guinea pig, rat, and humankidney revealed a very high concentration of specificbinding sites localized to the GA, renal arteries, andouter medulla. The high concentration of ANF bindingsites in the GA and outer medulla (corresponding to thelocation of the cortical collecting tubules) suggests thatthese regions are primary sites of ANF's natriureticand diuretic actions on the kidney. The localization ofhigh concentrations of specific ANF binding sites onrenal arteries and moderate levels in the muscularislayer of the aorta suggests a direct action on vascularsmooth muscle, accounting for ANF's effects on vas-cular resistance.2'N'30 Recent studies suggest that ANFacts directly on the adrenals since ANFs inhibit basalaldosterone release as well as block the release of al-dosterone from isolated adrenal glomerulosa cellsstimulated by adrenocorticotropic hormone and angio-tensin II.31-33 The present report directly supports thisview by demonstrating high concentrations of specificANF binding sites in the zona glomerulosa of the ad-renal cortex, the region where aldosterone is principal-ly synthesized and secreted. Moderate concentrationsof ANF binding sites in the posterior pituitary mayaccount for the reported ANF stimulation of vasopres-sin release from the isolated posterior pituitary gland,36

but the significance of its presence in only a sector of

this endocrine organ remains obscure and deservesfurther study.

The kidney, the ciliary body of the eye, and thechoroid plexus of the brain are all involved in plasmafiltration and in specialized active transport. The cili-ary body is involved in the production of the vitreousfluid, and the choroid plexus is involved in the secre-tion of cerebrospinal fluid. The present finding of adistinctly localized, high concentration of ANF bind-ing sites in these structures suggests that ANF also maybe involved in regulating the environment and pressureof interocular and intracranial fluids.

In other peripheral tissues, however, it is not clearwhether the ANF binding sites are generally involvedin the regulation of fluid and electrolyte balance orwhether they are involved in specific active transportfunctions. Thus, binding sites in the colon and gall-bladder might be implicated in regulating the degree ofwater reabsorption by influencing the transit time ofthe meal and bile, respectively. In the lung, the highconcentration of ANF binding sites might be relatedmost logically to regulation of water reabsorption. In-terestingly, the BIO 14.6 hamster, which is known toexperience congestive heart failure, has a marked defi-ciency in the atrial content of its ANF.37 Coupled withthe present finding, this observation may indicate that

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FIGURE 3. Dark-field photomicrograph showing the autoradiographic localization of atrial natriuretic factor(ANF) binding sites in a coronal section of human kidney obtained from a 2 7-year-old man (autopsy time, 65 hoursafter death). Note the high concentration of binding sites in the glomerular apparatus (GA), renal arteries (RA),and outer medulla (OM). See Figure 1 for explanation. (Line bar = 1.7 mm.)

H & E

FIGURE 4. Light-field photomicrograph of a guinea pig pituitary stained with hematoxylin and eosin (H & E; a),and dark-field photomicrographs ofLKB autoradiograms of the same section (b) and its serial pair (c). See FigureIfor explanation. A moderate concentration of'25l-atrial natriuretic factor (ANF) binding sites can be seen in thepars nervosa (PN), while a negligible concentration of binding sites can be seen in the pars distalis (PD) and parsintermedia (PI). (Line bar = 0.6 mm.)

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HYPERTENSION VOL 8, No 8, AUGUST 1986

H&E H&E

TM

ANF

1251 ANF + 1>uM ANF

FIGURE 5. Light-field photomicrograph of a guinea pig aortastained with hematoxylin and eosin (H & E; a), and dark-fieldphotomicrographs of LKB film autoradiograms of the samesection (b) and its serial pair (c). See Figure 1 for explanation.A moderate concentration of atrial natriwetic factor (ANF)binding sites can be seen in the tunica media (TM; smoothmuscle layer), while a negligible concentration can be seen inthe tunica intermedia (Tl). (Line bar = 0.7 mm.)

FIGURE 6. Light-field photomicrograph of a guinea pig lungstained with hematoxylin and eosin (H & E; a), and dark-fieldphotomicrographs of LKB film autoradiograms of the samesection (b) and its serial pair (c). See Figure 1 for explanation.Note the heavy concentration of binding sites in the parenchyma(P), while no specific binding can be seen in the bronchus (Br)or the blood vessels (BV). (Line bar = 1.5 mm.)

the dysfunction in the BIO 14.6 hamster is due not onlyto the lack of ANF stimulation of natriuretic and di-uretic response in the kidney but also to the lack ofANF-induced water reabsorption by the alveoli.

The demonstration of comparable ANF bindingsites in human, guinea pig, and rat kidney suggests

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H& H & E

CjEp

FIGURE 7. Light-field photomicrograph of a guinea pig gall-bladder and liver stained with hematoxylin and eosin (H & E;a), and dark-field photomicrographs of LKB film autoradio-grams of the same section (b) and its serial pair (c). See Figure1 for explanation. A high concentration of specific binding sitescan be seen on the muscularis layer (Mus) but not on the mucosa(Muc) or the liver (L). (Line bar = 0.8 mm.)

that, at least in the kidney, the physiological actions ofANF in experimental animals are similar to those seenin humans.38'" The unexpected stability of ANF bind-ing sites in the postmortem human kidney also sug-gests that human kidneys could be assessed for ANFreceptor dysfunction in disease states such as hyperten-sion and congestive heart failure. The high concentra-tion and postmortem stability of ANF binding sitesfound in the human kidney indicate that this tissueshould prove an excellent source for the extraction,

FIGURE 8. Light-field photomicrograph of a guinea pig eyestained with hematoxylin and eosin (H & E; a), and dark-fieldphotomicrographs of LKB film autoradiograms of the samesection (b) and its serial pair (c). See Figure 1 for explanation.Note the high concentration of specific atrial natriuretic factor(ANF) binding sites in the ciliary body epithelium (Ep) and amoderate concentration in the choriocapillaris (Ch) and thelongitudinal muscle (LM). No specific binding can be observedin the circular muscle (CM), conjunctival epithelium (CjEp),sclera (S), or retina (R). (Line bar = 0.9 mm.)

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FIGURE 9. Dark-field photomicrograph of a horizontal sec-tion of the rat brain showing specific atrial natriuretic factor(ANF) binding sites in the olfactory bulb (OB), fasciculus retro-flexus (fr), pia-arachnoid (p/a), and choroid plexus (cp). Spe-cific binding sites were not observed in the cerebellum (Cb),caudate-putamen (CPu), or the hippocampus (H). (Line bar =1.6 mm.)

purification, and sequence analysis of the human ANFreceptor.40

While the present study provides information con-cerning the location and possible target tissues forANF, it cannot indicate whether these tissues are occu-pied by circulating ANF or by locally synthesizedANF. Immunohistochemical data have revealed that inaddition to the atrium, the salivary glands41 and severalareas of the brain42'43 display cells containing ANF-like immunoreactivity. However, recent RNA blot hy-bridization experiments44 have suggested that the atri-um is the only tissue examined that displays high levels(> 0.5%) of the total messenger RNA (mRNA) codingfor ANF. These histochemical and biochemical ex-periments are not necessarily in disagreement, sincethe RNA blot technique only detects very high levelsof ANF mRNA. Therefore, tissues that synthesize rel-atively small amounts of ANF mRNA could very wellbe below the limits of detection with this technique butdetectable with immunohistochemical techniques.

It is not clear whether all the binding sites identifiedin the present study correspond to functional ANF re-ceptors. Previous experiments strongly suggest thatseveral of these binding sites have the proper physio-logical location (i.e., kidney, adrenals, and aorta) andpharmacological characteristics to be considered ANFreceptors. Before many of the tissues we have shownto express ANF binding sites (i.e., lung, gallbladder,ciliary body, choroid plexus) can be properly consid-ered to express a functional ANF receptor, however,activation of these binding sites must be shown toproduce a relevant physiological response.

AcknowledgmentsWe thank Martin Harris, Tony Hill, Kevin McFarthing, and

Linda Zimnicki of the Amersham Corporation for the gift of rat andhuman '"I-ANF.

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C R Mantyh, L Kruger, N C Brecha and P W Mantyhguinea pig, rat, and human.

Localization of specific binding sites for atrial natriuretic factor in peripheral tissues of the

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