THE AMERICAN JOURNAL OF ANATOMY 190:182-191 (1991)

lmmunohistochemical Localization of Atrial Natriuretic Peptide in the Developing and Adult Mammalian Kidney JAMES C. McKENZIE, JANE N. SCOTT, AND TADASHI INAGAMI Department of Anatomy and Cancer Research Center, College of Medicine, Howard University, Washingotn, DC 20059 (J.C.M.);Department of Anatomy, Wright State University, Dayton, Ohio 45435 (J.N.S.);Department of Biochemistry and S.C.O.R.Hypertension Center, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232 (T.I.)

ABSTRACT The discovery, within the last decade, of atrial natriuretic peptide (ANP), a family of peptides with natriuretic/diuretic and vasorelaxant properties, has prompted much research into the mechanisms and sites of action of ANP within the kidney. In the present study, ANP was localized in the kidneys of several mammalian species by immunohistochemical techniques 1)to identify possible sites of synthesis; 2) to compare the localization of ANP to known physiological effects; 3) to determine species differences, if any, in ANP localization; and 4) to study the development of A N P immunoreactivity in the fetal and neonatal rat kidney. Using an antibody against rat ANP IV, ANP was localized exclusively on the proximal convoluted tubule (PCT) brush border and within intercalated cells of the outer medullary and cortical collecting tubules and ducts of adult mouse, rat, pig, monkey, and human kidneys. The development of ANP immunoreactivity paralleled the differentiation and maturation of collecting duct epithelium in rat fetal kidney. Atrial natriuretic peptide found within intercalated cells of the cortical and outer medullary collecting ducts may be the result of endogenous synthesis and, following secretion, may be available to receptors in the inner medullary collecting ducts.

1984a). Rat and human plasma atrial natriuretic peptides (ANPI-,,) differ only in the substitution of isoleucine in rat ANP for methionine in human ANP at position 12 (Flynn et al., 1983; Kangawa and Matsuo, 1984). Subsequent to the initial observation that the number of secretory granules in atrial myocytes changed following sodium or water deprivation (De Bold, 1979), De Bold and colleagues (1981)demonstrated that atrial extracts caused profound natriuresis and diuresis when injected into rats. Other investigators have confirmed these results using either atrial extracts, purified ANP, or synthetic ANP (Borenstein et al., 1983; Burnett et al., 1984; Seidah et al., 1984). From these studies, two distinct renal targets emerged as primary sites of action for ANP: 1)the vasculature and 2) the tubular epithelium. The importance of the pre- and intra-glomerular vasculature as sites of ANP action was suggested by the increase in glomerular filtration rate (GFRj observed in many studies following ANP injection (Burnett et al., 1984; Maack et al., 1984; Huang et al., 1985).It was hypothesised that ANP caused vasorelaxation in pre- and intra-glomerular vessels resulting in increased glomerular blood flow and increased GFR. Constriction of post-glomerular vessels was also assumed to play a role in this response (Marin-Grez et al., 1986; Loutzenhiser et al., 1988). It has also been hypothesized that natriuretic and diuresis may result from inner medullary washout following changes in intrarenal blood flow affecting the vasa recta (reviewed in Maack, 1987). However, oiher studies have demonstrated ANP-induced increases in natriuresis and diuresis without increased GFR or changes in inINTRODUCTION trarenal blood flow (De Bold et al., 1981; Pollock and Atrial natriuretic factor or peptide (ANF, ANP) is Banks, 1983; Seymour et al., 1985; Hansell and Ulfenthe generic term for a group of related peptides pos- dahl, 1986, 1987; Pollock and Arendshorst, 1986; sessing potent natriuretictdiuretic and vasorelaxant Hansell et al., 1987). The results of these studies sugproperties (De Bold et al., 1981; Borenstein et al., 1983; gested that ANP may inhibit sodium reabsorption at Grammer et al., 1984; Kleinert et al., 1984). ANP is the tubular level. Furthermore, the results of studies synthesized in the cardiac atria as a high-molecular- using micropuncture and similar techniques (Briggs et weight preprohormone (Maki et al., 1984; Seidman et al., 1982; Sonnenberg et al., 1982,1986)suggested that al., 1984) which is cleaved during subsequent process- ANP acted distally in the tubular system, probably in ing ((Vuolteenaho et al., 1985). The primary stimulus the collecting tubule or duct. for release of ANP from the atria appears to be atrial stretch or distention (Dietz, 1984; Ledsome et al., 1985; Goetz et al., 1986; McKenzie et al., 1986; Edwards et al., 1988).The predominant form of ANP circulating in the plasma is a peptide consisting of 28 amino acids derived from the carboxy-terminus of the preprohorReceived March 9,1990. Accepted September 17, 1990. mone (Kangawa and Matsuo, 19841, and containing a Address reprint requests to James C. McKenzie, Ph.D., Dept. Anatdisulfide bridge between residues 7 and 23 which is omy, Coll. Med., Howard University, 520 W St., NW, Washington, essential for the activity of the peptide (Misono et al., DC, USA, 20059. Q

1991 WILEY-LISS, INC.

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Biochemical studies have demonstrated the presence of small amounts of ANP in the kidney (Sakamoto et al., 1985).This amount was considered to be consistent with the amount of ANP which may be bound to receptors (Wiegand et al., 1987). Preliminary results from our laboratory have indicated the presence of immunoreactive ANP within rat renal collecting tubule cells (McKenzie et al., 1985). The purposes of the present study were to determine the precise distribution of ANP in the mammalian kidney by immunohistochemical localization and to attempt to correlate the results with the physiological actions of ANP. MATERIALS AND METHODS Tissue Preparation

Male Sprague-Dawley rats, 150-200 gm, were anesthetized with sodium pentobarbital (Nembutal), 50 mg/ kg IP, and were perfused via the left ventrieleiaorta with Tyrode's buffer (pH 7.4, 37"C, 200 ml) followed by Perfix (Fisher, Pittsburg, PA; 4"C, 200 ml). Kidneys were removed and further fixed by immersion in icecold Perfix for 1 hr with continuous agitation. Fixed kidneys were routinely dehydrated in ethanol, cleared in xylene, and embedded in Paraplast Plus. Sections were cut on a rotary microtome a t 5 Fm and mounted on acid-cleaned, gelatinfchrome alum-coated glass slides. Mouse kidneys were obtained in the same manner but required smaller quantities (50 ml) of buffer and fixative for perfusion. Human kidney was obtained at autopsy from the non-involved portion of a kidney with a renin-secreting pericytoma. The tissue was immediately placed on ice, minced into small cubes (2-3 mm2) and fixed by overnight immersion in ice-cold Perfix. Pig and monkey kidney sections were obtained from Bouin's fixed tissue. Fetal and neonatal rat kidneys were fixed by immersion in Bouin's solution for 24 h r and stored in 70% ethanol until embedded.

Fig. 1. Immunolocalization of ANP in rat heart. A Adult heart. Nearly all atrial myocytes demonstrate intense ANP immunoreactivity. No staining was observed in the ventricle (v).B: Sixteen-day fetal rat. Immunoreactivity for ANP is widely distributed in the heart at this stage of development. Bars = 250 pm.

Antibody Preparation

Rat ANP-IV (ANF-IV) with the amino-acid sequence H2N-Arg-Ser-Ser-Cys-Phe- Gly -Gly - Arg- Ile- Asp- Arg-

goat serum (NGS)/O.O2% Tween for 20 for 30 min at room Ile-Gly-Ala-Glyn-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe- temperature, sections were incubated with priArg-Tyr-COOH (Misono et al., 1984a,b) was synthe- mary antibody 11:1,000) €or 24-48 h r at 4°C. The secsized by a solid phase method and purified by tions were then washed twice in PBS (5 min each) and chromatography (Sugiyama et al., 1984). ANP-IV is incubated with biotinylated goat anti-rabbit IgG contained within the rat ANPI-,, sequence. ANP-IV (GARG: 1:200 in PBS containing 1.5% NGS) for 30 min was coupled with thyroglobulin and injected intrader- at room temperature. Following two washes in PBS, mally in Dutch belted rabbits (Tanaka et al., 1984). the sections were next incubated in avidin-biotin perAntisera against synthetic ANP-IV cross-reacted 100% oxidase complex (150 in PBS) for 30 min at room temwith natural ANP-IV, 100% with rat ANP-11, 42.5% perature and washed three times in PBS before incuwith human ANP (hANP4p28),and 28% with atriopep- bation for 5 min in 0.05 Tris-HC1 (pH 7.2) containing tin I, but did not cross-react with arginine-vasopressin diaminobenzidine (DAB: 0.05%)and H2 0 2(0.01%).The (AVP), angiotensin I (AI), or angiotensin I1 (AII) (Mc- sections were then washed exhaustively in running tap water, dehydrated in alcohol, cleared in xylene, and Kenzie et al., 1985). mounted in Permount. Controls for the immunohistochemical staining included 1) substitution of normal lmmunohistochemistry rabbit serum (NRS) for primary antibody and 2 ) preinFollowing routine deparaffinization in xylene and rehydration in a graded ethanol series, kidney sections cubation of primary antibody with excess ANP-IV (10 were stained for ANP using the avidin-biotin-perox- pglml, 48 hr, 4°C). idase complex (ABC) technique (Hsu et al., 1981) with RESULTS a kit purchased from Vector Laboratories (Burlingame, CA). Prior to immunohistochemical staining, the secUsing the antibody against ANP-IV as described tions were incubated in absolute methanol containing above, positive staining was observed in adult rat 1.5%hydrogen peroxide (H202) to inhibit endogenous atrial but not ventricular myocytes (Fig. 1A) and in the peroxidases. Following incubation in PBS/B.O% normal developing hearts of 16-day rat fetuses (Fig. 1B). Anti-

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Fig. 3. Adult rat renal cortex. Staining for ANP is typically observed in the brush border of proximal convoluted tubule cells (large arrowheads) and in a subpopulation of collecting tubule cells identified as intercalated cells by morphological characteristics (small arrowheads). Intense staining is usually either distributed throughout the cytoplasm or restricted to apical regions. Occasionally, a n intense bar of immunoreactivity is observed across the basal portion of these cells (arrow). Bar 50 pm.

Fig. 2. Immunostaining of ANP in adult rat renal cortex. A: Positive staining for ANP is observed in a subpopulation (intercalated or “dark” cells) of collecting tubuleiduct epithelium (large arrowheads). Light staining for ANP is seen in the brush border of the proximal convoluted tubules (small arrowheads). No staining is observed within cells of the glomerulus, juxtaglomerular apparatus, or proximal/distal convoluted tubules. B: Absorption control. Staining is completely abolished by pre-incubation of the antibody with ANF IV (10 Fg/ml). Bars = 100 pm.

densa, distal convoluted tubule, loop of Henle, vasa recta, or interstitial cells. The subpopulation of collecting duct cells which stained positively for ANP were identified as “dark” or intercalated cells based on their distribution and morphology (Hancox and Komender, 1963; Kriz and Bankir, 1988). In the cortical and medullary regions of rat and mouse kidneys, these cells were cuboidal to low columnar in shape and extended farther into the lumen of the duct than neighboring principal cells. In collecting tubules of the juxtamedullary region of the porcine (Fig. 4A) and human kidney cortex (Fig. 4B),positively stained cells were of medium to high columnar stature, as were stained cells in the collecting ducts of the inner stripe of the outer medulla of the monkey kidney (Fig.

5).

Sections containing all regions of the kidney (cortex, outer medulla, and inner medulla/papilla) were available for only rat and mouse and are described below. In both species, ANP-immunopositive cells were found at all levels of the collecting tubulelduct system extending from the region immediately subjacent to the renal rat ANP-IV antibody provided slightly more intense capsule (Fig. 2A) to the base of the papilla (Fig. 6). It staining of ANP in rat atrial myocytes than commer- was not possible to determine reliably whether ANPcially available antibodies in serial dilution tests, to a positive cells were present in the connecting piece bedilution of 1:128,000. Positive staining for ANP was tween the distal convoluted tubule and the initial colobserved in the kidneys of all species tested in this lecting tubule segment. In the cortex, the cytoplasm of study (rat, mouse, pig, monkey, and human). In all intercalated cells tended to be completely filled with cases, staining was abolished by substitution of NRS reaction product, In the majority of these cells, staining for primary antibody or by the use of antibody pre-ab- was either homogeneous or more intense in the apical sorbed with excess antigen (Fig. 2). Staining of rat region. A few cells, however, demonstrated particukidney was concentration dependent and disappeared larly intense staining in a bar-like pattern across the basal portion of the cell (Figs. 3, 9B). As the duct sysat dilutions greater than 1:10,000. ANP immunoreactivity was confined to two specific tem progressed through cortex and outer medulla, regions: 1)the brush border of proximal convoluted tu- staining appeared to be restricted more to the apical bule cells and 2) a subpopulation of collecting tubule and lateral portions of the intercalated cells. In the rat cells (Fig. 3). Positive staining for ANP was not ob- kidney, the number of positively stained cells diminserved in any other structures of the kidney including ished rapidly as the ducts entered the base of the pathe juxtaglomerular apparatus, glomerulus, macula pilla and were seen only seldom within the papilla

ANP IN THE MAMMALIAN KIDNEY

Fig. 4. A Porcine kidney. Intense immunoreactivity is observed in intercalated cells of a cortical collecting tubule. B: Human kidney. Staining is seen in intercalated cells of a juxtamedullary collecting duct and in the brush border of proximal convoluted tubules (arrowheads). Immunopositive duct cells appear less densely distributed than in rat, mouse, or pig. Bars = 50 pm.

Fig. 5. Monkey medullary collecting duct. Immunoreactivity for ANP appears most dense in the apical region of the intercalated cells. Adjacent light cells (1) are devoid of staining. Thick limbs of the loop of Henle (h) are adjacent. Bar = 15 Fm.

proper. This pattern is in excellent agreement with the reported distribution of intercalated cells in the rat renal collecting duct system (Hancox and Komender,

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Fig. 6. Mouse kidney. A: Numerous cells immunoreactive for ANP are seen distributed in collecting tubules and ducts throughout the cortex (c), outer medulla (om), and inner medulla (im). Bar = 250 pm. B: In a cross-section through the inner medulla, collecting ducts are observed interspersed among the vasa recta and intersititium. Most duct cross-sections demonstrate 1 or 2 ANP-positive cells. Hematoxylin counterstain. Bar = 30 p m ~

1963). In contrast, positively stained cells were seen throughout the collecting ducts of the mouse renal papilla (Fig. 6). Developing kidneys from 16-, 18-, and 20-day rat fetuses and 1-day-old neonatal rats were also examined for ANP immunoreactivity. No specific staining for ANP was evident in kidneys from 16-day fetuses (Fig. 7). In 18-day fetuses, weak ANP immunoreactivity was observed only occasionally in tubular epithelial cells near the corticomedullary border. Labelling was occasionally distributed throughout the cytoplasm but was more often observed in apical regions (Fig. 8). Distinct staining for ANP on the luminal aspect (brush border) of proximal convoluted tubule cells was also observed at this time. Bv the 20th dav ANP immu" of gestation. noreactivity wis present in many cells of the medullary collecting ducts and in cortical collecting tubules than a t 18 days. ImmunoreactivitY associated with the lumen of proximal convoluted tubules was also more intense and more widely distributed than at 18 days (Fig. 9). In the neonatal kidney, intense staining for ANP was observed in a subpopulation of cells in the medullary collecting ducts and also occasionally in cortical collecting tubules (Fig. 10). In addition, many

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Fig. 7. Sixteen-day fetal rat kidney. A Low-power view showing both developing kidneys. Nephrogenic tissue occupies the perimeter while the developing collecting tubuleslducts are observed in the center of the organ. Bar = 250 Fm. B: Higher power view. No positive staining for ANP was observed in the presumptive collecting ducts (asterisks) or associated with the PCT cells (arrowhead) a t this stage. g, Glomerulus, Bar = 50 prn.

cells of the epithelium lining the external surface of the papilla were also strongly positive as were many collecting duct cells within the papilla itself (Fig. 10B). Intense ANP immunoreactivity was also observed at the luminal membrane of maturing proximal tubule cells and often appeared to fill the narrowed lumen of this portion of the nephron (Fig 10D). DISCUSSION

Results of the present study have immunohistochemically demonstrated the presence of ANP in the intercalated cells of renal collecting tubules and ducts of several mammalian species. A small amount of immunoreactivity was also observed in the brush border of proximal convoluted tubule (PCT) cells. The antibody used in these studies was raised against a synthetic ANP-IV with composition and natriureticlvasorelaxant properties identical to those of natural ANP-IV. This antibody has been used previously to localize ANP in rat cardiac atria, adrenal medullary chromaffin cells, and cells of the anterior pituitary (McKenzie et al., 19851, as well as neurons and glia of the canine brain (McKenzie and Cowie, 1989; McKenzie et al., 1989; 1990) and has localized ANP in the secretory

Fig. 8. Eighteen-day fetal rat kidney. A Low power view of kidney showing renal sinus (s) and adrenal gland (a). Bar = 250 pm. B: Higher power. Immunoreactivity for ANP begins to appear in the apical regions of presumptive collecting tubules (small arrowheads) in the cortex. Staining was only very rarely seen in cortical collecting tubules a t this stage. Staining in the lumen of proximal convoluted tubules (large arrowhead) appears more intense than in the 16-day embryos. g, Glomerulus. Bar = 50 pm.

granules of atrial myocytes at the EM level (not shown). One other study has reported immunohistochemical localization of ANP in the rat kidney (Lindop et al., 1987). In t h a t case, a n antibody against human ANPIpSRstained only the cortical distal convoluted tubules of one Bouin's-fixed rat kidney. Granular staining was apparently localized in the infranuclear region of these cells. The difference between this pattern of staining and the localization of ANP in collecting duct cells reported herein is difficult to explain. Urodilatin, a 32 amino-acid peptide derived from the ANP precursor, has also been immunohistochemically localized in collecting tubules from porcine kidney, although the staining pattern appeared ubiquitous and not restricted to a particular cell type (Feller et al., 1989). Although renal glomeruli possess a high density of ANP receptors, ANP immunoreactivity was not detected in the glomeruli in this study. It is possible that ANP bound to glomerular receptors may be rapidly internalized and degraded by lysosomes as described in cultured vascular smooth muscle cells (Hirata et al., 1985). It is assumed that the large majority of degradation products would not be immunoreactive. How-

ANP IN THE MAMMALIAN KIDNEY

Fig. 9. Twenty-day fetal rat kidney. A: Low power. Intense immunoreactivity is observed in the lumen of proximal convoluted tubules (small arrowheads). Numerous ANP-positive cells are seen in the medullary collecting tubules, particularly in the forming papilla (asterisk). ANP-positive cells are still scarce in cortical collecting tubules but more numerous than at 18 days. a, Adrenal gland. Bar = 250 pm. B: Higher power. Numerous collecting tubule cells near the corticomedullary border possess ANP immunoreactivity (small arrowheads). Although most immunoreactivity appears in the apical regions of these cells, one cell demonstrates a basal bar (arrow). Bar = 50 pm.

ever, in an elegant study, Suzuki and colleagues (1987) found that, while renal cortex (glomeruli) contained greater than 90% of ANP binding sites, only minimal proteolysis occurred at these sites. Therefore, it is suggested that the binding of ANP to glomerular receptors may render it incapable of binding antibody or may represent a concentration of ANP undetectable by the immunohistochemical technique. Immunoreactivity for ANP observed on the luminal surface of PCT cells may represent ANP bound to specific receptors or ANP in the process of being degraded by peptidases attached to the lasma membrane. Labelling of PCT brush border by ‘51-ANP in vitro and in vivo has been detected by autoradiography (Bianchi et al., 1985; Healy and Fanestil, 1986; Yamamoto et al., 1987).However, other studies have provided somewhat contradictory anatomical and physiological evidence regarding the presence of specific, guanylate cyclaselinked ANP receptors in the PCT brush border (Ham-

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mond et al., 1985; Baum and Toto, 1986; Butlen et al., 1987; Chabardes et al., 1987). Whole kidney homogenates (Tang et al., 1984) and brush border membrane preparations (Olins et al., 1987a,b) have been shown to degrade atriopeptin I11 (AP 111)and rat ANPI-,,. It was hypothesized by these investigators that ANP would be proteolytically degraded by brush border peptidases of the PCT cells and the peptide fragments reabsorbed by these cells. Luft and co-workers (1986) were unable to detect ANP in the urine of rats given a bolus injection of AP 111. Apparently, very little of the ANP escaping the glomerular receptors to enter the nephron is excreted intact (Suzuki et al., 1987). This does not preclude the possibility that intact ANP or immunoreactive ANP fragments may pass into more distal nephron segments where it may be taken up by other cells. The presence of ANP in the brush border of the PCT upstream from a possible site of synthesis in the collecting tubules, as well as its early developmental appearance at this location, suggest an exogenous source, i.e., the plasma/glomerular filtrate. The distribution of ANP immunoreactivity in the rat and mouse kidney matches precisely the distribution of the “dark” or intercalated cells along the collecting tubule/duct system (Young and Wissig, 1964). However, the number of ANP-positive collecting tubule/duct cells appears slightly greater than the number of intercalated cells at various levels of the system as reported by Hancox and Komender (1963) and may include transitional cells between “light” and “dark’ types. ANP in intercalated cells of the collecting tubules and ducts may be of plasma (glomerular filtrate) origin and result from either non-specific endocytosis or a receptor-mediated process. Intercalated cells possess a high level of endocytotic activity and have a well-developed system of apical vesicles and canaliculi (Dorup, 1985a,b; Brown et al., 1987). However, autoradiographic studies at both the light and EM levels have reported binding sites for 1251-ANPon inner medullary but not outer medullary or cortical collecting ducts (Chai et al., 1986; Healy and Fanestil, 1986; Koseki et al., 1986a,b; Bianchi et al., 1987; Suzuki et al., 1987). The data suggest that, while ANP may be internalized by endocytosis in intercalated cells at some levels of the collecting tubule/duct system, only duct cells in the inner medulla possess specific receptors for ANP. This hypothesis has been questioned by a recent study demonstrating the effects of ANP on NaCl and fluid absorption in rat cortical collecting ducts (Nonoguchi et al., 1989). Most physiological evidence supports a role for inner medullary, but not outer medullary or cortical, collecting ducts in the natriuretiddiuretic response t o ANP (reviewed by Zeidel, 1990). Physiological concentrations of ANP significantly inhibited 0, consumption in cells of inner medullary collecting ducts (IMCD) but not in cells of outer medullary collecting ducts or of the thick ascending limb of Henle’s loop (Zeidel et al., 1986, 1987a).The change in metabolism in these cells may be related to the significant inhibition of “Na uptake from the medium of cultured rabbit IMCD cells following exposure to ANP (Zeidel et al., 1987a,b). In addition, Zeidel(1987) has reported the presence of specific high-affinity receptors for ANP on isolated intact IMCD cells. The changes in metabolism and Na’

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Fig. 10. One-day neonatal rat kidney. A Low power. ANP immunoreactivity is located in collecting tubulesiducts throughout the kidney but is most obvious in the developing papilla (asterisk). Bar = 250 pm. B Higher power view of the developing papilla. Numerous ANP-positive cells are seen lining the collecting ducts as well as on the exterior surface of the papilla (arrowheads). Bar = 25 ym. C: Many ANP-positive cells are seen in the collecting tubules of the

outer medulla. A juxtamedullary proximal convoluted tubule is indicated by the large arrowhead. Bar = 50 pm. D: In the cortex, several collecting tubules demonstrate intensely ANP-positive cells (small arrowheads). Immunoreactivity for ANP is particularly intense on the lurninal aspect of proximal convoluted tubules (large arrowheads). g, Glomerulus. Bar = 50 pm.

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transport following ANP treatment of IMCD cells may be modulated by cGMP, the intracellular second messenger for ANP (Ishikawa et al., 1985; Tremblay et al., 1985;Zeidel et al., 1987b).In addition, Appel and Dunn (1987) have recently demonstrated that ANP stimulates accumulation of cGMP in cells cultured from the renal papillary collecting ducts of Sprague-Dawley rats as well as salt-sensitive (Sj and salt-resistent (R) Dahl rats. A physiological role for ANP in the development of hypertension in Dahl-S rats is suggested by the hyporesponsiveness in renal cGMP production in Dahl-S compared to Dahl-R rats. These data suggest that, in addition to the natriuresis and diuresis resulting from increased GFR, ANP may directly inhibit tubular sodium transport in IMCD cells via a receptor linked to guanylate cyclase. The failure of intercalated cells to incorporate even a small amount of label following in vivo infusion of lZ5IANP suggests that the immunoreactive intracellular ANP may result from synthesis rather than uptake. The intensity of staining in these cells is comparable to that observed in anterior pituitary and adrenal medulla (McKenzie et al., 1985) at identical antibody concentrations. The latter tissues have been shown to contain small amounts of ANP mRNA (Gardner et al., 1986) o r ANP precursor (Ong et al., 1987). However, one study (Sakamoto et al., 1985) has reported that only the 28 amino acid circulating form of ANP (plasma ANP) was found in rat kidney. Further indirect support for endogenous renal ANP synthesis comes from a very recent study reporting the synthesis of endothelin, a potent vasoconstrictor, and pro-endothelin in renal endothelial cell lines (Shichiri et al., 1989). The function of intercalated cells remains speculative, particularly with regard to possible roles for ANP, and may be dependent on location within the collecting duct system. Intercalated cells in the cortical, but not medullary, collecting ducts have been shown to hypertrophy in response to mineralocorticoid (DOCA) treatment (Kennedy and Parker, 1963). ANP has been shown to induce significant kaliuresis along with natriuresis (DeBold et al., 1981; Seymour et al., 1985). This effect may involve intercalated cells because several studies have demonstrated hypertrophy and increased numbers of intercalated cells in the outer medullary collecting ducts of hypokalemic or waterdeprived rats (Oliver et al., 1957; MacDonald et al., 1962; Hancox and Komender, 1963; Kennedy and Parker, 1963). The hypertrophic response included a transfer of membrane from apical vesicles to the luminal surface ofthe cell (Stetson et al., 1980; Brown et al., 1987) and was correlated with decreased urine-concentrating ability in K -deficient animals. Specific stimuli for the release of ANP from intercalated cells of the cortical and outer medullary collecting ducts remain to be discovered but could include ionic, hormonal, or even neural mediators. In addition, the polarity of secretion remains speculative. There is little direct autoradiographic evidence for the presence of specific ANP receptors on either the apical or basolateral membranes of the collecting-duct epithelium. However, physiological evidence suggests that binding of ANP to sites on the basolateral, but not apical, surfaces of collecting-duct cells results in decreased so+

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dium and water reabsorption (Dllingham and Andersen, 1986; Nonoguchi et al., 1989). The reaction apparently involves decreased entry of luminal Na through channels in the apical membrane and, possibly, increased net secretion of Na' by a basolateral furosemide-sensitive Na/K/2Cl-~otransporter(Sonnenberg et al., 1986; Rocha and Kudo, 1988; Zeidel et al., 1988). If ANP is released from the basolateral pole of intercalated cells, it would then have access to basolatera1 binding sites on adjacent ANP-sensitive principal cells or, perhaps, to vascular and/or interstitial receptors (Koseki et al., 1986a,b). Possible roles for apically released ANP are presently undetermined. In fetal rats, the first appreciable ANP immunoreactivity is observed in the PCT lumen and in isolated collecting ducts a t 18 days gestation. However, ANP immunoreactivity appears widespread through the maturing collecting tubule/duct system by birth and may reflect the progress of development and differentiation in collecting-duct cells. The onset of ANP-immunostaining in the rat kidney is much delayed compared t o that in the cardiac atria which appears as early as 10 days gestation (Thompson et al., 1986; Scott and Jennes, 1988). Furthermore, ANP-binding sites appear in the rat kidney as early as 16 days gestation (Scott and Jennes, 1989)when binding sites are observed over punctate structures in the nephrogenic region of the cortex and are evenly distributed in the medulla. At later times (18 and 20 days gestation and 1 day postpartum), ANP-binding is observed in a reticular pattern near the renal pelvis. The label appears to be concentrated over the mesenchymal interstitiurn and not over the collecting ducts (Scott and Jennes, 1989). It is apparent that the pattern of ontogeny of ANP binding sites in the developing kidney does not match the developing pattern of ANP immunoreactivity. However, the developmental pattern of ANP immunoreactivity in the rat kidney does appear to coincide with the differentiation and maturation of the collecting tubule epithelium. This is further support for the hypothesis that ANP immunoreactivity in developing and mature collecting tubule intercalating cells is not a result of receptor-mediated uptake. In the present study, the presence of immunoreactive ANP was demonstrated in intercalated cells of cortical and outer medullary collecting ducts of adult rat, mouse, pig, monkey, and human and in the developing rat kidney. The high concentration of ANP immunoreactivity in the apical regions of intercalated cells, which possess a high degree of endocytotic activity, suggests that the intracellular ANP may be a result of endocytosis. However, in vivo autoradiographic studies indicate that lZ5I-ANPis not taken up from either plasma or glomerular filtrate by collecting duct cells. Moreover, the ontogeny of ANP-binding sites in the developing rat kidney does not match the ontogency of ANP immunoreactivity either spatially or temporally. Furthermore, the majority of evidence indicates that, unlike its effects on IMCD, ANP apparently has little or no direct effect on the metabolism or cyclic nucleotide levels of intercalated cells of cortical and outer medullary collecting ducts. These results suggest that intercalated cells of cortical and outer medullary collecting ducts may synthesize ANP for subsequent release to IMCD cells as part of a short feedback loop +

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mechanism. Conversely, basolateral release of ANP from these cells would suggest more local effects. The relative importance of these mechanisms to the overall natriureticldiuretic response to ANP remains to be determined. ACKNOWLEDGMENTS

This work was supported by a Faculty Research Support Grant from Howard University. The authors thank Dr. Loren Hoffman for his advice and assistance and Drs. Robert J. Cowie and Ivonne Lastra for their careful reading of the manuscript and their cogent criticisms. LITERATURE CITED Appel, R.G., and M.J. Dunn 1987 Papillary collecting tubule responsiveness to atrial natriuretic factor in Dahl rats. Hypertension, 10:107-114. Baum, M., and R.D. Toto 1986 Lack of a direct effect of atrial natriuretic factor in the rabbit proximal tubule. Am. J. Physiol., 250: F66-F69. Bianchi, C., J. Gutkowska, G. Thibault, R. Garcia, J. Genest, and M. Cantin 1985 Radioautographic localization of '"I-atrial natriuretic factor (ANF) in rat tissues. Histochemistry, 82:441-452. Bianchi, C., J. Gutkowska, R. Garcia, G. Thibault, J. Genest, and M. Cantin 1987 Localization of Iz5I-atrial natriuretic factor (ANF)binding sites in rat renal medulla. A light and electron microscope autoradiographic study. J . Histochem. Cytochem., 35t149153. Borenstein, H.B., W.A. Cupples, H. Sonneberg, and A.T. Veress 1983 The effects of natriuretic atrial extracts on renal haemodynamics and urinary excretion in anaesthetized rats. J. Physiol. (Lond.), 334t133-140. Briggs, J.P., B. Steipe, G. Schubert, and J. Schnermann 1982 Micropuncture studies of the renal effects of atrial natriuretic substance. Pflugers Arch., 3951271-279. Brown, D., P. Weyer, and L. Orci 1987 Nonclathrin-coated vesicles are involved in endocytosis in kidney collecting duct intercalated cells. Anat. Rec., 218t237-242. Burnett, J.C., Jr., J.P. Granger, and T.J. Opgenorth 1984 Effects of synthetic atrial natriuretic factor on renal function and renin release. Am. J. Physiol., 247rF863-F866. Butlen, D., M. Mistaoui, and F. Morel 1987 Atrial natriuretic peptide receptor along the rat and rabbit nephrons: 1lZ5I]-atrialnatriuretic peptide binding in microdissected glomeruli and tubules. Pflugers Arch., 408t356-365. Chabardes, D., M. Montegut, M. Mistaoui, D. Butlen, and F. Morel 1987 Atrial natriuretic polypeptide effects on cGMP and CAMP contents in microdissected glomeruli and segments of the rat and rabbit nephrons. Pflugers Arch., 408r366-372. Chai, S.Y., P.M. Sexton, A.M. Allen, R. Figdor, and F.A.O. Mendelsohn 1986 In vitro autoradiographic localization of ANP receptors in rat kidney and adrenal gland. Am. J. Physiol., 250tF753-757. De Bold, A.J. 1979 Heart atrial granularity effects of changes in water-electrolyte balance. Proc. SOC. Exp. Biol. Med., 161:508-511. De Bold, A.J., H.B. Borenstein, A.T. Veress, and H. Sonnenberg 1981 A rapid and potenti natriuretic response to intravenous injection of atrial myocardial extracts in rats. Life Sci., 28:89-94. Dietz, J.R. 1984 Release of natriuretic factor from rat heart-lung preparation by atrial distension. Am. J. Physiol., 247tR1093-Rl096. Dillingham, M.A., and R.J. Andersen 1986 Inhibition of vasopressin action by atrial natriuretic factor. Science, 23Lt1572-1573. Dorup, J. 1985a Ultrastructure of distal nephron cells in rat renal cortex. J. Ultrastruct. Res., 92:101-118. Dorup, J . 198513 Structural adaptation of intercalated cells in rat renal cortex to acute metabolic acidosis and alkalosis. J . Ultrastruct. Res., 92:119-131. Edwards. B.S., R.S. Zimmerman, T.R. Schwab, D.M. Heublein. and J.C. Burnett, J r . 1988 Atrial stretch, not pressure, is the principal determinant controlling the acute release of atrial natriuretic factor. Circ. Res., 62t191-195. Feller, S.M., M. Gagelmann, and W.G. Forssmann 1989 Urodilatin, a newly described member of the ANP family. Trends Pharm. Sci., 10:93-94. Flynn, T.G., M.L. De Bold, and A.J. De Bold 1983 The amino acid sequence of a n atrial peptide with potent diuretic and natriuretic properties. Biochem. Biophys. Res. Commun., I 1 7;859-865.

Gardner, D.G., C.F. Deschepper, W.G. Ganong, S. Hane, J. Fiddes, J.D. Baxter, and J. Lewicki 1986 Extra-atrial expression of the gene for atrial natriuretic factor. Proc. Natl. Acad. Sci. USA, 83t6697-6701. Goetz, K.L., B.C. Wang, P.G. Geer, R.J. Leadley, and H.W. Reinhardt 1986 Atrial stretch increases sodium excretion independently of release of atrial peptides. Am. J . Physiol., 25O:R946-R950. Grammer, R.T., H. Fukumi, T. Inagami, and K.S. Misono 1984 Rat atrial natriuretic factor. Purification and vasorelaxant activity. Biochem. Biophys. Res. Commun., 124;663-668. Hammond, T.G., A.N.K. Yusufi, F.G. Knox, and T.P. Dousa 1985 Administration of atrial natriuretic factor inhibits sodium-coupled transport in proximal tubules. J. Clin. Invest., 75t1983-1989. Hancox, N.M., and J . Komender 1963 Quantitative and qualitative changes in the dark cells of the renal collecting tubules of rats deprived of water. Q. J. Exp. Physiol., 48;346-354. Hansell, P., and H.R. Ulfendahl 1986 Atriopeptins and renal cortical papillary blood flow. Acta Physiol. Scand., 127r349-357. Hansell, P., and H.R. Ulfendahl 1987 Effects of atrial natriuretic peptide (ANP) during converting enzyme inhibition. Acta Physiol. Scand., 13Ot393-399. Hansell, P., A. Fasching, M, Sjoquist, N.-E. Anden, and H.R. Ulfendahl 1987 The dopamine receptor antagonist haloperidol blocks natriuretic but not hypotensive effects of atrial natriuretic factor. Acta Physiol. Scand., 130:401-407. Healy, D.P., and D.D. Fanestil 1986 Localization of atrial natriuretic peptide binding sites within the rat kidney. Am. J. Physiol., 250; F573-F578. Hirata, Y., S. Takata, M. Tomita, and S. Takaichi 1985 Binding, internalization and degradation of atrial natriuretic peptide in cultured vascular smooth muscle cells of rat. Biochem. Biophys. Res. Commun., 1291651-657. Hsu, S.M., L. Raine, and H. Fanger 1981 Use of avidin-hiotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody PAP procedures. J . Histochem. Cytochem., 29.577-580. Huang, C.-L., J. Lewicki, L.K. Johnson, and M.G. Cogan 1985 Renal mechanisms of action of rat atrial natriuretic factor. J. Clin. Invest., ?5:769 -773, Ishikawa, S., T. Saito, K. Okada, T. Kuzuya, K. Kangawa, and H. Matsuo 1985 Atrial natriuretic factor increases cyclic GMP and inhibits cyclic AMP in rat renal papillary collecting tubule cells in culture. Biochem. Biophys. Res. Commun., 130t1147-1153. Kangawa, K., and H. Matsuo 1984 Purification and complete amino acid sequence of -human atrial natriuretic polypeptide. Biochem. Biophys. Res. Commun., 118:131-139. Kennedy, G.C., and R.A. Parker 1963 The effect of hydrochlorothiazide on the kidney of the electrolyte deficient rat. Q.J. Exp. Physiol., 48r186-191. Kleinert, H.D., T. Maack, S.A. Atlas, A. Januszewicz, J.E. Sealey, and J.H. Laragh 1984 Atrial natriuretic factor inhibits angiotensin-, norepinephrine-, and potassium-induced vascular contractility. Hypertension, Ksuppl. I)tI143-1147. Koseki, C., Y. Kanai, Y. Hayashi, N. Ohnuma, and M. Imai 1986a Intrarenal localization of receptors for or-rat atrial natriuretic polypeptide: An autoradiographic study with LLZ51]-labeled ligand injected in vivo into the rat aorta. Jpn. J . Pharmacol., 42;27-33. Koseki, C., Y. Hayashi, S. Torikai, M. Furuya, N. Ohnuma, and M. Imai 198610 Localization of binding sites for a-rat atrial natriuretic polypeptide in rat kidney. Am. J . Physiol., 250:F210-F216. Kriz, W., and L.Bankir 1988 A standard nomenclature for structures of the kidnay. Kidney Intl., 33t1-7. Ledsome, J.R., N. Wilson, C.A. Courneya, and A.J. Rankin 1985 Release of atrial natriuretic peptide by atrial distension. Can J. Physiol. Pharmacol., 63;739-742. Lindop, G.B.M., E.A. Mallon, G.D. McIntyre, and A.M. McNicol 1987 Tissue atrial natriuretic peptide: Immunoreactivity in humans and in the rat. In: American Society of Hypertension Symposium Series, Vol. I, Biologically Active Atrial Peptides, B.M. Brenner and J.H. Laragh, eds. Raven Press, New York, pp. 187-191. Luft, F.C., R.E. Lang, G.R. Aronoff, H. Ruskoaho, M. Toth, D. Ganten, R.B. Sterzel, and T. Unger 1986 Atriopeptin 111kinetics and pharmacodynamics in normal and anephric rats. J. Pharmacol. Exp. Therapeut.,236:4 16-4 18. Loutzenhiser, R., K. Hayashi, and M. Epstein 1988 Atrial natriuretic peptide reverses afferent arteriolar vasoconstriction and potentiates efferent arteriolar vasoconstriction in the isolated perfused rat kidney. J. Pharmacol. Exptl. Therapeut., 246.522-528. Maack, T. 1987 ANF-induced increase in glomerular filtration rate and decrease in inner medullary hypertonicity are important determinants of ANFs natriuretic action. In: American Society of

ANP IN THE MAMMALIAN KIDNEY Hypertension Symposium Series, Val. I, Biologically Active Atrial Peptides, B.M. Brenner and J.H. Laragh, eds. Raven Press, New York, pp. 109-117. Maack, T., D.N. Marion, M.J.F. Camargo, H.D. Kleinert, J.H. Laragh, E.D. Vaughan, and S.A. Atlas 1984 Effects of auriculin (atrial natriuretic factor) on blood pressure, renal function, and the renin-aldosterone system in dogs. Am. J. Med., 77t1069-1075. MacDonald, M.K., M.S. Sabour, A.T. Larnbie, and J.S. Robson 1962 The nephrology of experimental potassium deficiency. An electron microscopic study. Q.J. Exp. Physiol., 47A.262-272. Maki, M., K.Takayanagi, K.S. Misono, K.N. Pandey, C. Tihbetts, and T. Inagami 1984 Structure of rat atrial natriuretic factor precursor deduced from cDNA sequence. Nature, 309:722-724. Marin-Grez, M., J.T. Fleming, and M. Steinhausen 1986 Atrial natriuretic peptide causes pre-glomerular vasodilatation and post-glomerular vasoconstriction in rat kidney. Nature, 324t473-476. McKenzie, J.C., and R.J. Cowie 1989 Atrial natriuretic peptide-like immunoreactivity (ANP-LIR) in glial cells of the parenchyma and glia limitans of the canine brain. Sac. Neuroscience Abstr., 15:228.14 (abstr.). McKenzie, J.C., I. Tanaka, K.S. Misono, and T. Inagami 1985 Irnmunocvtochemical localization of atrial natriuretic factor in the kidne, adrenal medulla, pituitary and atrium of rat. J. Histochem. Cytochem., 33328-832. McKenzie, J.C., I. Tanaka, T. Inagami, K.S. Misono, and R.M. Klein 1986 Alterations in atrial and plasma atrial natriuretic factor (ANF) content during development of hypoxia-induced pulmonary hypertension in the rat. Proc. SOC.Exp. Biol. Med., 181: 459-463. McKenzie, J.C., R.J. Cowie, and T. Inagami 1989 Mapping of atrial natriuretic peptide-like immunoreactivity (ANP-LIR) in the canine brain. Anat. Rec., 223.7'24 (abstr.). McKenzie, J.C., R.J. Cowie, and T. Inagami 1990 ANP-like immunoreactivity in neurons1 perikarya and processes associated with vessels of the PIA and cerebral parenchyma in dog. Neurosci. Lett., 117253-258. Misono, K.S., H. Fukumi, R.T. Grammer, and T. Inagami 1984a Rat alrial natriuretic factor: Complete amino acid sequence and disulfide linkage essential for biological activity. Biochem. Biophys. Res. Commun., 119,524-529. Misono, K.S., R.T. Grammer, H. Fukumi, and T. lnagami 1984b Rat atrial natriuretic factor: isolation, structure and biological activities of four major peptides. Biochem. Biophys. Res. Commun., 123:444-451. Nonoguchi, H., J.M. Sands, and M.A. Knepper 1989 ANF inhibits NaCl and fluid absorption in cortical collecting duct of rat kidney. Am. J . Physiol., 256:F179-F186. Olins, G.M., K.L. Spear, N.R. Siegel, E.J. Reinhard, and H.A. Zurcher-Neely 1987a Atrial peptide inactivation by rabbitkidney brush-border membranes. Eur. J. Biochem., 170t431434. Olins, G.M., K.L. Spear, N.R. Siegel, and W.A. Zurcher-Neely 1987b Inactivation of atrial natriuretic factor by the renal brush border. Biochirn. Biophys. Acta, 901:97-100. Oliver, J., M. MacDowell, L.G. Welt, M.A. Holliday, W. Hollander, Jr., R.W. Winters, T.F. Williams, and W.E. Segar 1957 The renal lesions of electrolyte imbalance. I. The structural alterations in potassium-depleted rats. J . Erp. Med., 106r563-575. Ong, H., C. Lazure, T.T. Nguyen, N. McNicoll, N. Seidah, M.Chretien, and A. De Lean 1987 Bovine adrenal chromaffin granules are a site of synthesis of atrial natriuretic factor. Biochim. Biophys. Res. Commun., 147:957-963. Pollock, D.M., and W.J. Arendshorst 1986 Effect of atrial natriuretic factor on renal hemodynamics in the rat. Am. J. Physiol., 251: F795 -F801. Pollock, D.M., and R.O. Banks 1983 Effect of atrial extract on renal function in the rat. Clin Sci., 65.47-55. Rocha, A S . , and L.H. Kudo 1988 Direct effect of atrial natriuretic factor on Na, C1 and water transport in the papillary collecting duct. Proc. Tenth Int. Congr. Nephrol., 1Or218. Sakamoto, M., K. Nakao, M. Kihara, N. Morri, A. Sugawara, M. Suda, M. Shimokura, Y.Kiso, Y. Yamori, and H. Imura 1985 Existence of atrial natriuretic polypeptide in kidney. Biochem. Biophys. Rcs. Commun., 128:1281-1287. Scott, J.N., and L.H. Jennes 1988 Development of immunoreactive atrial natriuretic peptide in fetal hearts of spontaneously hypertensive and Wistar-Kyoto rats. Anat. Embryol., 178:359-363. Scott, J.N., and L.H. Jennes 1989 Ontogeny of atrial natriuretic peptide receptors in fetal rat kidney and adrenal gland. Histochemistry, 91:395-400. Seidah, N.G., C. Lazure, M. Chretien, G. Thibault, R. Garcia, M. Cantin, J. Genest, R.F. Nutt, S.F. Brady, T.A. Lyle, W.J. Paleveda, C.D. Colton, T.M. Ciccarone, and D.F. Veber 1984 Amino acid sequence of homologous rat atrial peptides: Natriuretic activity of

191

native and synthetic forms. Proc. Natl. Acad. Sci. USA, 8126402644. Seidman, C.E., AD. Duby, E. Choi, R.M. Graham, E. Haber, C. Honey, J.A. Smith, and J.G. Seidman 1984 The structure of rat preproatrial natriuretic factor as defined by a complimentary DNA clone. Science, 225r324-326. Seymour, A.A., E.H. Blaine, E.K. Mazack, S.G. Smith, 1.1. Stabilito, A.B. Haley, M.A. Napier, M.A. Whinnery, and R.F. Nutt 1985 Renal and systemic effects of synthetic atrial natriuretic factor. Life Sci., 36r33-44. Shichiri, M., Y.Hirata, T. Emori, K. Ohta, T. Nakajima, K. Sato, A. Sato, and F. Marumo 1989 Secretion of endothelin and related peptides from renal endothelial cell lines. FEBS Lett., 253t203206. Sonnenberg, H., W.A. Cupples, A.J. De Bold, and A.T. Veress 1982 Intrarenal localization of the natriuretic effect of cardiac atrial extract. Can. J. Physiol. Pharmacol., 60:2249-2252. Sonnenberg, H., U. Honrath, C.K. Chong, and D.R. Wilson 1986 Atrial natriuretic factor inhibits sodium transport in medullary collecting duct. Am. J. Physiol., 250tF963-F966. Stetson, D.L., J.B. Wade, and G. Giebisch 1980 Morphologic alterations in the rat medullary collecting duct following potassium depletion. Kidnet Intl., 17r45-56. Sugiyama, M., H. Fukumi, R.T. Grammer, K.S. Misono, Y. Yabe, Y. Morisawa, and T. Inagami 1984 Synthesis of atrial natriuretic peptides and studies on structural factors in tissue specificity. Biochem. Biophys. Res. Commun., 123:338-343. Suzuki, M., F.A. Almeida, D.R. Nussenzveig, D. Sawyer, and T. Maack 1987 Binding and functional effects of atrial natriuretic factor in isolated rat kidney. Am. J. Physiol., 253:F917-F928. Tanaka, I., K.S. Misono, and T. Inagami 1984 Atrial natriuretic factor in rat hypothalamus, atria and plasma: Determination by specific radioimmunoassay. Biochem. Biophys. Res. Commun., 124t663668. Tang, J., R.J. Webber, D. Chang, J.K. Chang, J. Kiang, and E.T. Wei 1984 Depressor and natriuretic activities of several atrial peptides. Regulat. Peptides, 9:53-59. Thompson, R.P., J.A.V. Simpson, and M.G. Currie 1986 Atriopeptin distribution in the developing rat heart. Anat. Embryol., 175: 227-233. Tremblay, J., R. Gerzer, P. Vinay, S.C. Pang, R. Beliveau, and P. Hamet 1985 The increase of cGMP by atrial natriuretic factor correlates with the distribution of particulate y a n y l a t e cyclase. FEBS Lett., 181:17-22. Vuolteenaho, O., 0. Argamaa, and N. Ling 1985 Atrial natriuretic polypeptide (ANP): Rat atria store high molecular weight precursor but secrete processed peptides of 25-35 amino acids. Riochem. Biophys. Res. Commun.. 129:82-88. Wiegand, R.C., M.L. Day, C.P. Rodi, D. Schwartz, and P. Needleman 1987 Atriopeptin expression in the ventricle. In: Atrial Hormones and Other Natriuretic Factors, P.J. Mulrow and R. Schrier, eds. Waverly Press, Baltimore, pp. 33-38. Yamamoto, l., T. Ogura, and Z. Ota 1987 In vitro macro- and microautoradiographic localization of atrial natriuretic peptide in the rat kidney. Res. Commun. Chem. Path. Pharmacol., 56r185-198. Young, D., and S.L. Wissig 1964 A histological description of certain epithelial and vascular structures in the kidney of the normal rat. Am. J. Anat., 115r43-70. Zeidel, M.L. 1987 Direct transport effects of atrial natriuretic peptides (ANP) on inner medullary collecting duct (IMCD) cells. Am. Sac. Hypertens. Abst., 185:(abstr). Zeidel, M.L. 1990 Renal actions of atrial natriuretic peptide: Regulation of collecting duct sodium and water transport. Annu. Rev. Physiol., 551747-759. Zeidel, M.L., J.L. Seifter, S. Lear, B.M. Brenner, and P. Silva 1986 Atrial peptides inhibit oxygen consumption in kidney medullary collecting duct cells, Am. J. Physiol., 251:F379-F383. Zeidel, M.L., J.L. Seifter, B.M. Brenner, P. Silva, and S. SaribanSohraby 1987a Atrial natriuretic peptide and amiloride inhibit apical N a ' flux in cultured rabbit medullary collecting duct cells. In: American Society of Hypertension Symposium Series, Val. I, Biologically Active Atrial Peptides, B.M. Brenner and J.H. Laragh, eds. Raven Press, New York, pp. 420-422. Zeidel, M.L., P. Silva, B.M. Brenner, and J.L. Seifter 1987b Role of cGMP in atrial natriuretic peptide inhibition of Na+ transport by rabbit inner medullary collecting duct cells. In: American Society of Hypertension Symposium Series, Vol. I, Biologically Active Atrial Peptides, B.M. Brenner and J.H. Laragh, eds. Raven Press, New York, pp. 422-425. Zeidel, M.L., D. Kikeri, P. Silva, M. Burrowes, and B.M. Brenner 1988 Atrial natriuretic peptides inhibit conductive sodium uptake by rabbit inner medullary collecting duct cells. J . Clin. Invest., 82: 1067-1074.