0013-7227/92/1301-0229$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine Society

Vol. 130,No. 1 Printed

Receptor Selectivity of Natriuretic Atria1 Natriuretic Peptide, Brain and C-Type Natriuretic Peptide* SHIN-ICHI YOSHIHIRO YOSHIKAZU

Peptide Natriuretic

in U.S.A.

Family, Peptide,

SUGA, KAZUWA NAKAO, KIMINORI HOSODA, MASASHI MUKOYAMA, OGAWA, GOTARO SHIRAKAMI, HIROSHI ARAI, YOSHIHIKO SAITO, KAMBAYASHI, KEN INOUYE, AND HIROO IMURA

Second Division, Department of Medicine, Kyoto University School of Medicine (S.-k%, Y.O., G.S., H.A., Y.S., Y.K., H. I.), Kyoto 606; and the Shiorwgi Research Laboratories, Shionogi Co. Ltd. (Y.K., K.I.), Osaka 553, Japan

K.N., K.H., M.M.,

marked species difference in the receptor selectivity of the natriuretic peptide family, especially among BNPs. Therefore, we investigated the receptor selectivity of the natriuretic peptide family using the homologous assay system with endogenous ligands and receptors of the same species. The rank order of binding affinity for the C-receptor was ANP > CNP > BNP in both humans and rats. The rank order of potency for cGMP production via the ANP-A receptor (GC-A) was ANP 2 BNP >> CNP. but that via the ANP-B recentor (GC-B) was CNP > ANP 2 BNP. These findings on the receptor selectivity of the natriuretic peptide family provide a new insight into the understanding of the physiological and clinical implications of the natriuretic peptide system. (Endocrinology 130: 229-239,1992)

ABSTRACT. To elucidate the ligand-receptor relationship of the natriuretic peptide system, which comprises at least three endogenous ligands, atria1 natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), and three receptors, the ANP-A receptor or guanylate cyclaseA (GC-A), the ANP-B receptor or guanylate cyclase-B (GC-B), and the clearance receptor (C-receptor), we characterized the receptor preparations from human, bovine, and rat tissues and cultured &is with the aid of the binding assay, Northern blot techniaue. and the cGMP nroduction method. Using these receptor preparations, we examined the binding affinities of ANP, BNP, and CNP for the C-receptor and their potencies for cGMP production via the ANP-A receptor (GC-A) and the ANP-B receptor (GC-B). These analyses revealed the presence of a

A

ll-13), as shown in Fig. 1. Porcine BNP (pBNP) is a 26-amino acid peptide (3); however, we and others demonstrated that rat BNP (rBNP) (ll), human BNP (hBNP) (12), and bovine BNP (bBNP) (13) are 45-, 32-, and 35-amino acid peptides, respectively. In addition, we have demonstrated that the biological potency (14) and tissue distribution (15, 16) of BNP are extraordinarily divergent among species. Recently, the third member of the natriuretic peptide family, C-type natriuretic peptide (CNP), has been isolated from the porcine brain (17). Subsequently, isolation of cDNA encoding a precursor for rat CNP has shown that rat CNP (rCNP) is identical to porcine CNP (pCNP) (18), while analysis of the human CNP (hCNP) gene has disclosed that hCNP is also identical to pCNP (19, 20). Thus, the sequence of mammalian CNP is highly conserved among species. Two types of receptors for natriuretic peptides have been hypothesized and identified. One type is coupled to particulate guanylate cyclase with a mol wt of 120,000140,000 (21, 22) and is designated the biologically active receptor. Molecular cloning has shown that the particulate guanylate cyclase itself is the biologically active

FTER the discovery of atria1 natriuretic peptide (ANP) in the heart (1,2), brain natriuretic peptide (BNP) was isolated from the porcine brain (3) and subsequently from the heart (4, 5). Since then, ANP and BNP have been considered to comprise the natriuretic peptide family responsible for body fluid homeostasis and blood pressure control as both cardiac hormones and neuropeptides (l-10). In contrast to the conserved amino acid sequence and similar biological action of mammalian ANP (1, 2), the molecular form of BNP is divergent among species (3, Received July 12, 1991. Address all correspondence and requests for reprints to: Kazuwa Nakao, M.D., Ph.D., Second Division, Department of Medicine, Kyoto Universitv School of Medicine. 54 Shogoin Kawahara-cho. Sakvo-ku. _ Kyoto 606, Japan. *This work was supported in part by research grants from the Japanese Ministry of Education, Science, and Culture; the Japanese Ministry of Health and Welfare “Disorders of Adrenal Hormone” Research Committee, Japan, 1990; Japan Tobacco, Inc.; the Yamanouchi Foundation for Research on Metabolic Disorders: the Cell Science Research Foundation, 1990; the Uehara Memorial Foundation; and research grants for cardiovascular diseases (63C-2 and 2A-3) from the Japanese Ministry of Health and Welfare. 229

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 December 2014. at 13:23 For personal use only. No other uses without permission. . All rights reserved.

RECEPTOR

230

SELECTIVITY

OF ANP, BNP, AND

CNP

Endo. Voll30.

1992 No 1

NATRIDIUYIC PEPYIDB FAMILY ANP a-rat a-human -BNP FIG. 1. Natriuretic peptides and receptors so far identified. The amino acid sequences of the natriuretic peptides ANP, BNP, and CNP are shown. Identical residues in ANP or BNP are boxed.

ANP ANP

1 rat BNP human BNP

porcine bovine -CNP

BNP BNP 1 22 GLSKGCFGLKLDRIGSMSGLGC

CNP

NAlltIlJREYIC PEPYIDE RJXXPYORS Biologically Clearance

Active

ANP-A receptor ANP-B receptor

or GC-A or GC-B

Receptor

receptor, which possesses an extracellular binding domain and an intracellular guanylate cyclase domain (23). Thereafter, the presence of two subtypes of the biologically active receptors, named the ANP-A receptor or guanylate cyclase-A (GC-A) and the ANP-B receptor or guanylate cyclase-B (G&B), have been demonstrated (24, 25). The other type of receptor forms a disulfidelinked homodimer composed of subunits with mol wt of 60,000-70,000 (26-28) and is not coupled to guanylate cyclase. Maack et al. (29) have proposed that this receptor is biologically silent and serves as a specific clearance binding site for natriuretic peptides. This receptor has been termed the clearance receptor (C-receptor) to denote its role in the clearance of natriuretic peptides. Thus, as shown in Fig. 1, the natriuretic peptide system is a complex system consisting of at least three endogenous ligands, ANP, BNP, and CNP, and three receptors, the ANP-A receptor (GC-A), the ANP-B receptor (GCB), and the C-receptor. However, little is known about the receptor selectivity of the natriuretic peptide family. In the present study, in order to investigate the ligandreceptor relationship of the natriuretic peptide system, selectivities of ANP, BNP, and CNP for the C-receptor and the biologically active receptor have been examined with the aid of a binding assay using C-receptor preparations and an assay for cGMP production via the biologically active receptor in cultured cells. Materials

Receptor

and Methods

Peptides a-rANP and cr-hANP werepurchasedfrom Peptide Institute, Inc. (Minoh, Japan). rBNP, hBNP, pBNP, and CNP were synthesizedby the solid phasemethod. All peptidessynthesized

were characterized by amino acid analysis, and the purity of eachpeptide wasconfirmed by reversephaseliquid chromatography. The ANP analog, Des-[Gln’s,Ser1g,Gly20,Leu2’,Gly22] rANP-(4-23)-NH2 [C-ANF-(4-23)] was donated by Prof. T. Maack, Cornell University Medical College (New York, NY). C-ANF-(4-23) is a synthetic ring-deleted analogof ANP which binds with a high affinity to the C-receptor, but with a quite low affinity (at least 3 orders of magnitude lower than ANP) to the biologically active receptor (28-30). Cell culture Rat aortic smooth musclecells (SMC) were prepared from explants of thoracic aorta of 20-week-oldmale Wistar rats and cultured in the mannerpreviously described(31). Bovine aortic endothelial cells were dispersedwith collagenaseand cultured as previously reported (32). Human mesangialcells were prepared from the glomeruli isolated from cortical renal tissueby differential sieving and cultured as previously reported (33). PC12 rat pheochromocytomacells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplementedwith 10% horseserum, 5% fetal bovine serum, 100 U/ml penicillin, and 100pg/ml streptomycin at 37 C in a humidified atmosphere containing 7.5% CO,. Preparation of receptors Confluent rat aortic SMC, bovine endothelial cells,and PC12 cellswere washedtwice with Dulbecco’sPBS and mechanically scraped.The cells were then centrifuged at 200 X g for 5 min. Fresh bovine lung tissueswere obtained from a local slaughterhouse.Human lung tissueswereobtained at operation from the patient with lung cancer and were free of tumor invasion. Tissueswere immediately frozen in liquid nitrogen and stored at -70 C. Membranes were prepared from the human lung, rat aortic SMC, PC12 cells, bovine endothelial cells, and bovine lung and solubilizedby the procedure of Shimonakaet al. (27). The purified bovine C-receptor from bovine lung (27) was

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 December 2014. at 13:23 For personal use only. No other uses without permission. . All rights reserved.

RECEPTOR

SELECTIVITY

generously provided by Prof. S. Hirose, Tokyo Institute Technology (Tokyo, Japan). Receptor binding

of

assays

a-rANP was radioiodinated by the chloramine-T method (34). The specific activity of [?]a-rANP ranged from 7001400mCi/rmol. Nonspecific binding wasdetermined using 0.1 pM unlabeleda-rANP and was lo-20% of the total binding. assays with membrane preparations. The binding assayswith solubilized receptorswere performed aswe previously reported (35). Five to 50 pg of the solubilized membranesor 200pg of the purified bovine C-receptor were incubated at 4 C for 48 h with [““I]a-rANP (10,000cpm) in 0.5 ml of a reaction mixture consisting of PBS (20 mM phosphate and 120 mM NaCl, pH 7.4), 10 mM EDTA, 5 pg/ml pepstatin (Peptide Institute), 5 rg/ml leupeptin (Peptide Institute), 0.5 mM phenylmethylsulfonylfluoride (Boehringer Mannheim, Mannheim, Germany), 0.2%Triton X-100,0.1% lysozyme (SigmaChemical Co., St. Louis, MO), and various amounts of the competing ligand when used. Bound and free ligands were separatedby adding 1.0 ml of a suspensionof dextran-coated charcoal consisting of 250 mg Norit SX Plus (N.V. Norit-Vereening, The Netherlands) and 25 mg Dextran T-70 (Pharmacia Fine Chemicals,Uppsala,Sweden)in 100ml 50 mM phosphatebuffer (pH 7.4). Binding reachedequilibrium at 24 h and was stable for at least 48 h (data not shown). The stability of all ligands used during incubation wasconfirmed by gelpermeation chromatography and reverse phase liquid chromatography, as we previously described(36). Binding

assays with intact cells.Binding assayswith PC12 cells were carried out according to the method of Rathinavelu and Isom (37) with slight modifications. In brief, PC12 cells (5 x lo”) were incubated at 4 C for 2 h with [“‘I]a-rANP (200,000 cpm) and various concentrations of ligands in 250 ~1 Hanks’ BalancedSalt Solution (HBSS) containing 0.1% BSA, 5 kg/ml pep&&in, 5 fig/ml leupeptin, and 0.5 mM phenylmethylsulfonylfluoride (binding buffer). At the end of the incubation, bound and free ligands were separated by rapid filtration through GF/C filters (Whatman, Clifton, NJ) treated with 0.05% Tween-20 in PBS. Binding assayswith rat aortic SMC and bovine endothelial cells were performed in 24-well plates. Confluent cells were washedtwice with 1 ml HBSS containing 0.1% BSA. Cells were incubated at 4 C for 2 h with [lLT’I]arANP (200,000cpm) in the presenceof various concentrations of ligands in 250 ~1 binding buffer. After the incubation, the cells were washedfour times with 1 ml HBSS containing 0.1% BSA and solubilized with 1 ml 0.5 N NaOH. Binding

RNA extraction

and Northern

blot analysis

RNA was extracted from PC12 cells and rat aortic SMC using the guanidinium thiocyanate-CsCl method. Cellular levels of ANP-A receptor (GC-A) mRNA and ANP-B receptor (GC-B) mRNA were measuredby Northern blot analysis, as we previously reported (38). The rat ANP-A receptor (GC-A) cDNA probe wasprepared by cDNA synthesisand the polymerasechain reaction method. Two oligonucleotides (5’-AGAGAACATCACTCAGCG-3’ and 5’-ATCAGAGAGAGGCTG-

OF ANP, BNP, AND CNP

231

CCC-3’), which are complementary to (-)- and (+)-strands of the rat ANP-A receptor (GC-A) cDNA sequence(23), respectively, were synthesized as primers, using an Applied Biosysterns 381A DNA synthesizer (Applied Biosystems, Inc., Foster, CA). cDNA synthesiswasperformed with 10 pg total RNA of PC12 cells by oligo(dT) priming and avian myeloblastosis virus reversetranscriptase (Boehringer Mannheim) in a volume of 20 ~1. The polymerasechain reaction wascarried out for 30 cycles, using 2 ~1of the cDNA mixture together with Taq DNA polymerase (Cetus Corp., Emeryville, CA) and 100 pmol each of the 2 primers (39). The amplified product of the expected size (321 basepairs)was isolated from 5% polyacrylamide gel and further subcloned into Bluescript (Stratagene, La Jolla, CA) for sequencing.The rat ANP-B receptor (GC-B) cDNA probe wasprepared in the sameway, using 2 synthetic oligonucleotides (5’-GAGAGTCTACGAGCAGGC-3’ and 5’AATTGGCCGGCCTGTCCA-3’) asprimers (24). These2 fragments were labeled by the random priming method with [a“‘P]dCTP (Amersham International, Aylesbury, Buckinghamshire, United Kingdom) and usedasprobes.The specificactivity of probeswas approximately 1 x 10” cpm/pg. cGMP production

in cultured

celkr

Rat aortic SMC, bovine endothelial cells, and human mesangial cells were grown to confluence in six-well plates. The cellswere washedtwice with 2 ml serum-freeDMEM and then preincubated at 37 C for 10 min with 900 ~1DMEM containing 0.1% BSA and 0.5 mM isobutylmethylxanthine (Sigma Chemicals). Peptideswere addedto the medium, and the cells were incubated for 30 min at 37 C. The medium was then rapidly removedand addedto tubescontaining an ice-cold 12%solution of trichloroacetic acid (TCA) to give a final concentration of 6% TCA. To each well containing the cells, 1 ml ice-cold 6% TCA was added. Both medium and cell sampleswere centrifuged to remove precipitated proteins, and the supernatant fractions wereextracted three times with water-saturatedether. PC12 cells were washed twice with serum-free DMEM and dislodgedfrom the dishesby forceful pipetting, as previously described(40). A suspensionof cells containing 1 million cells/ ml DMEM with 0.1% BSA and 0.5 mM isobutylmethylxanthine waspipetted as0.45-ml aliquots in 12 x 75-mmglasstubesand preincubated for 10 min at 37 C. Peptides were then addedto the medium, and the cells were incubated for 30 min at 37 C. The tubes were centrifuged at 200 X g for 5 min at 4 C. The supernatant fractions containing medium free of cells were removed and addedto tubes containing an equalvolume of icecold 12% TCA. The pellets containing the ceils were mixed with 1 ml ice-cold 6% TCA. Both media and cells were processedas describedabove. cGMP was determined by RIA after succinylation, as previously described(41). Data analysis

Resultsare presentedasthe meansof three separateexperiments, performed in triplicate. The SE was lessthan 10% of the mean.

Results Characterization of receptors in solubilized membranes To evaluate the relative proportions of the biologically active receptor and the C-receptor in receptor prepara-

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 December 2014. at 13:23 For personal use only. No other uses without permission. . All rights reserved.

232

RECEPTOR

SELECTIVITY

OF ANP,

tions, displacement curves of [‘251](r-rANP by a-rANP and C-ANF-(4-23) were examined in the binding assay with solubilized membrane preparations from the rat aortic SMC, human lung, PC12 cells, bovine endothelial cells, and bovine lung and compared to those in the binding assay with the C-receptor purified from bovine lung (Fig. 2 and Table 1). As shown in Fig. 2A, C-ANF(4-23) completely displaced [‘251]a-rANP from the purified bovine C-receptor with a high affinity. In the binding assay with solubilized membranes from the rat aortic SMC (Fig. 2B), C-ANF-(4-23) effectively competed for more than 99% of the specific binding sites of [‘251]a-rANP with a high affinity. C-ANF-(4-23) also occupied 98% of the binding sites of [‘251]a-rANP in solubilized membranes from the human lung with a high affinity (Fig. 2C). The binding affinity of C-ANF-(4-23) for the C-receptor varied among species, as clearly shown in Fig. 2 and summarized in Table 1. By contrast, in PC12 cells (Fig. 2D), C-ANF-(4-23) failed to compete for [‘251]a-rANP binding, except at extremely high concentrations (>200 nM). In solubilized membranes from bovine endothelial cells and bovine lung, as shown in Fig. 2, E and F, C-ANF-(4-23) competed for 12% and 72% of the binding sites of [‘251]a-rANP with similar affinities to a-rANP. However, concentrations of CANF-(4-23) approximately 4 orders of magnitude higher were necessary for displacement of [‘251]rANP from the remaining binding sites by C-ANF-(4-23). To iurther confirm the proportions of the biologically active receptor and C-receptor, binding assays using intact cells were also performed in PC12 cells, rat aortic SMC, and bovine endothelial cells. C-ANF-(4-23) competed for more than 95% of the ANP-binding sites in rat aortic SMC and 20% of the ANP-binding sites in bovine endothelial cells with high affinities. However, C-ANF(4-23) failed to displace [‘251]cu-rANP from the ANPbinding sites in PC12 cells. Thus, the proportions of biologically active receptor and C-receptor vary from tissue (cell) to tissue (cell). The overwhelming majority of the receptor in solubilized

FIG. 2. Displacement curves of [‘““I]curANP by cu-rANP (O), and C-ANF-(423) (0) in the binding assay with the purified bovine C-receptor (A) and solubilized membranes from rat aortic SMC (B), human lung (C), PC12 cells (D), bovine endothelial cells (E), and bovine lung (F).

BNP,

AND

Endo. 1992 Vol 130. No 1

CNP

membrane preparations from the human lung and rat aortic SMC is classified as C-receptor, and the proportions of C-receptor in the total binding sites in bovine lung and bovine endothelial cells are 72% and 12%, respectively. The C-receptor is minimal, if present, in PC12 cells. Binding affinities of the natriuretic peptide family for the C-receptor

To assess C-receptor selectivity of the natriuretic peptide family, displacement curves of [‘251]cu-rANP by ANP, BNP, and CNP were determined in the binding assay with bovine, rat, and human C-receptor preparations (Fig. 3). Table 1 summarizes the binding affinities of ANP, BNP, and CNP in the binding assay with Creceptor preparations. In all binding assays with C-receptors from three species, a-hANP and a-rANP showed the highest binding affinities among natriuretic peptides. In the binding assay with the purified bovine C-receptor (Fig. 3A), pBNP had essentially the same binding affinity as a-hANP. The binding affinity of hBNP was 1.5-fold lower than that of a-hANP. By contrast, rBNP was 6fold less potent than a-hANP. CNP was 3-fold weaker than a-hANP. As shown in Fig. 3B, pBNP bound the rat C-receptor from the rat aortic SMC with almost the same affinity as cr-rANP. The binding affinity of hBNP was 2-fold lower than that of a-rANP. rBNP bound the rat C-receptor with a 6-fold lower affinity than a-rANP. The binding affinity of CNP was 3-fold lower than that of a-rANP, as was the case for the bovine C-receptor. In the binding assay with the C-receptor prepared from human lung (Fig. 3C), pBNP exhibited almost the same binding affinity as hANP. In contrast, hBNP was 14fold less potent than a-hANP. rBNP was as weak as hBNP in binding to the human C-receptor. The binding affinity of CNP was 5-fold lower than that of a-hANP. Thus, as shown in Fig. 3 and Table 1, the binding affinity for the C-receptors can be summarized as follows. 1) ANP has the highest affinity, and no difference is present between ANPs. 2) The binding affinity of CNP

l:;p.i:i -13

-12

-11 -10 -9 log[PEPTIDE](M)

-8

-7

-6

-13

-12

-11 -10 -9 -8 IO~[PEPTIDE I(M)

-7

-6

-13

-12

-11 -10 -9 log[PEPTIDE](M)

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 December 2014. at 13:23 For personal use only. No other uses without permission. . All rights reserved.

-9

-7

-6

RECEPTOR

SELECTIVITY

TABLE 1. Binding affinities of ANP, BNP, CNP, and C-ANF-(4-23)

OF ANP, BNP, AND CNP for bovine, rat, and human C-receptors Natriuretic

Bovine C-receptor K, (PM) Relative potency Rat C-receptor K, (PM) Relative potency Human C-receptor K, (PM) Relative uotencv

233

peptides

cu-hANP

a-rANP

hBNP

rBNP

6.9 0.87

6.0 1.00

10 0.60

46 0.13

6.9 0.87

24 0.25

14 0.43

7.7 0.86

6.6 1.00

13 0.51

41 0.16

7.4 0.89

22 0.30

100 0.07

10 0.88

8.8 1.00

140 0.06

96 0.09

11 0.80

48 0.18

61 0.14

Receptor binding affinities (Ki) were determined as described in Materials a-rANP relative to the Ki for natriuretic peptide.

DBNP

CNP

C-ANF-(4-23)

and Methods. Relative potency is expressed as the ratio of the Ki for

lar rank orders of binding not shown).

affinity

were obtained

(data

Determination of subtype of biologically active receptor in cultured cells Northern blot analysis. To clarify which subtype of the

biologically active receptor was expressed in cultured cells, ANP-A receptor (GC-A) mRNA and ANP-B receptor (GC-B) mRNA levels were measured in PC12 cells and rat aortic SMC by Northern blot analysis with specific rat cDNA probes. Figure 4 shows the results of Northern blot analysis of ANP-A receptor (GC-A)

ANP-A Receptor (GC-A)

-13

-12

-11 -10 -9 log [PEPTIDE]

-8 CM)

-7

-6

FIG. 3. Displacement curves of [““I]cz-rANP by natriuretic peptides in the binding assay with the bovine (A), rat (B), and human (C) Creceptors. 0, a-hANP; Cl, a-rANP, 0, hBNP, W, rBNP; A, pBNP, A, CNP.

is lower than that of ANP. 3) A marked difference is observed among BNPs. 4) In the binding assay with natriuretic peptides and C-receptor preparations of the same species, the binding affinity of BNP is approximately 1 order of magnitude lower than that of ANP in both humans and rats, and the rank order of binding affinity is ANP > CNP > BNP. When [1251]pBNP or [‘251]Tyro-rBNP was used instead of [‘251](y-rANP, simi-

ANP-B Receptor (GC-B)

9.5 kb7.5 kb+ 4.4 kb2.4 kb-

FIG. 4. Northern blot analysis of ANP-A receptor (GC-A) mRNA and ANP-B receptor (G&B) mRNA in cultured rat cells. Five micrograms of total RNA from PC12 cells and rat aortic SMC were used for analysis. kb, Kilobases.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 December 2014. at 13:23 For personal use only. No other uses without permission. . All rights reserved.

RECEPTOR

234

SELECTIVITY

mRNA and ANP-B receptor (GC-B) mRNA from PC12 cells and rat aortic SMC. RNA extracted from PC12 cells contained an intense hybridizing signal of ANP-A receptor (GC-A) mRNA of approximately 4.0 kilobases, but it contained only a slight hybridizing signal of ANP-B receptor (GC-B) mRNA. By contrast, in rat aortic SMC, ANP-A receptor (GC-A) mRNA was not detectable, and an intense hybridizing band of ANP-B receptor (GC-B) mRNA was detected. It has been reported that COS-7 cells transfected with the ANP-A receptor (GCA) expression vector respond differently to ANP and pBNP than those transfected with the ANP-B receptor (GC-B) expression vector (24, 25). Therefore, to further verify the subtype of the biologically active receptor, the effects of ANP and pBNP on cGMP production were examined and compared with the results reported in transfected COS-7 cells (24,25). Figure 5 shows the doseresponse curves of ANP and pBNP in PC12 cells, rat aortic SMC, human mesangial cells, and bovine endothelial cells. In PC12 cells (Fig. 5A), a-rANP was 13-fold more potent than pBNP. The concentration of cu-rANP necessary to stimulate half-maximal production of cGMP (EC& was 2.88 nM. These observations were identical to the results reported for COS-7 cells transfected with the rat ANP-A receptor (GC-A) expression vector (24). In rat aortic SMC (Fig. 5B), pBNP was 6fold more potent than a-rANP. However, the EC,,, of pBNP in rat aortic SMC was 50-fold higher than that of a-rANP in PC12 cells (EC&,,, 147 us. 2.88 nM). These results were also identical to the previous findings in COS-7 cells transfected with the rat ANP-B receptor (GC-B) expression vector (24). Combined with the results of Northern blot analysis, these results clearly indicate that the majority of the biologically active reAnalysis by cGMPproduction.

OF ANP,

BNP,

AND

CNP

Endo. 1992 Vol 130. No 1

ceptors expressed in PC12 cells is classified as ANP-A receptor (GC-A), whereas almost all of the biologically active receptors expressed in rat aortic SMC are classified as ANP-B receptor (GC-B). We also examined the effects of a-hANP and pBNP on cGMP production in human mesangial cells and bovine endothelial cells. In human mesangial cells (Fig. 5C), pBNP was 30-fold more potent than a-hANP, and the patterns in cGMP production by a-hANP and pBNP were identical to previous observations in COS-7 cells transfected with the human ANP-B receptor (GC-B) expression vector (25). Figure 5D shows that pBNP is equipotent to cr-hANP in cGMP production in bovine endothelial cells, which accorded well with the previous report in COS-7 cells transfected with the human ANPA receptor (GC-A) expression vector (25). These observations for human mesangial cells and bovine endothelial cells suggest that the major subtypes of biologically active receptor expressed in human mesangial cells and bovine endothelial cells could be the ANP-B receptor (GC-B) and the ANP-A receptor (GC-A), respectively. Effects of BNP on cGMP production in cultured cells

Since the biological action and molecular form of BNP are known to be markedly divergent among species (3, ll-14), we examined the effects of pBNP, hBNP, and rBNP on cGMP production in cultured cells and compared them with that of ANP. Figure 6 illustrates the dose-response curves of BNPs and ANPs of different species in PC12 cells, rat aortic SMC, human mesangial cells, and bovine endothelial cells. The potencies of ANP and BNP for cGMP production varied from cell to cell (Fig. 6 and Table 2). In PC12 cells with the abundant ANP-A receptor (GC-A; Fig. 6A), a-rANP was the most potent peptide in cGMP production and was 5-fold

0 -11

0 -11

-10 -9 -8 IxIPEPTIDEI(M)

-7

-6

-11

-10 -9 -8 Iog[PEPTIDE](M)

-7

-6

FIG. 5. Effects of ANP and pBNP on cGMP production in cultured cells. PC12 cells (A), rat aortic SMC (B), human mesangial cells (C), and bovine endothelial cells (D) were exposed to various concentrations of a-hANP (O), a-rANP (Cl), and pBNP (A) for 30 min.

-10 -9 -8 IX[PEPTIDEJ(M)

-7

-6 IxfPEPTIDE

j(M)

FIG. 6. Effects of ANPs and BNPs on cGMP production in cultured cells. PC12 cells (A), rat aortic SMC (B), human mesangial cells (C), and bovine endothelial cells (D) were exposed to various concentrations of a-hANP (O), cu-rANP (Cl), hBNP (O), rBNP (W), and pBNP (A) for 30 min.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 December 2014. at 13:23 For personal use only. No other uses without permission. . All rights reserved.

RECEPTOR TABLE

SELECTIVITY

OF ANP,

BNP,

AND

CNP

235

2. Potencies of natriuretic peptides for cGMP production via biologically active receptor in cultured cells Natriuretic Cells

a-hANP

PC12 cells ECW bM) Relative potency Rat aortic SMC EC,,, (nM) Relative potency Bovine endothelial cells EC,,, bf) Relative potency Human mesangial cells EG, (nM) Relative potency

e-rANP

13.6 0.21

2.88 1.00

P-1000 co.92

926 1.00

1.68 0.93 >lOOO co.98

hBNP >lOOO lOOO co.003

>lOOO lOOO co.92

147 6.30

97 9.54

1.56 1.00

1.48 1.05

31.6 0.05

1.38 1.13

>lOOO co.002

977 1.00

>lOOO CO.98

>lOOO CO.98

85.7 11.4

34.1 28.7

Concentrations of peptides required for half-maximal stimulation of cGMP production (EC,& were determined as described in Materi& Methods. Relative potency is expressed as the ratio of the ECso of ol-rANP to that of natriuretic peptide.

stronger than cu-hANP. rBNP was the strongest among BNPs, but was 6-fold weaker than a-rANP. pBNP was 13-fold weaker than a-rANP. hBNP had no significant effect on cGMP production at concentrations below 100 nM and was at least 300-fold weaker than a-rANP. Thus, the rank order of potency for cGMP production of BNP was rBNP > pBNP > hBNP via the rat ANP-A receptor (GC-A) in PC12 cells. Figure 6B shows that in rat aortic SMC with the ANP-B receptor (GC-B), pBNP was the most potent peptide in cGMP production and was 6-fold more potent than a-rANP. a-rANP was 4-fold more potent than cr-hANP, as was the case in PC12 cells. rBNP was 2-fold weaker than a-rANP and 13-fold weaker than pBNP. hBNP was 6-fold less potent than rBNP. The rank order of potency of BNP was pBNP > rBNP > hBNP via the rat ANP-B receptor (GC-B) in aortic SMC. As shown in Fig. 6C, in human mesangial cells, which presumably contain a large proportion of the ANP-B receptor (GC-B), pBNP was the most potent peptide and was 30-fold stronger than a-hANP, similar to the results in rat aortic SMC. hBNP, which was much weaker than rBNP in rat cultured cells, was 2-fold more potent than rBNP and was equipotent to a-hANP. The rank order of potency was pBNP > hBNP 2 rBNP via the human ANP-B receptor (GC-B) in mesangial cells. In bovine endothelial cells, which presumably express the abundant ANP-A receptor (GC-A) (Fig. 6D), hBNP and pBNP were equipotent to a-hANP and a-rANP in cGMP production. However, rBNP was 20-fold weaker than a-hANP. The rank order of potency was pBNP = hBNP > rBNP via the bovine ANP-A receptor (GC-A) in endothelial cells. These results indicate that a marked species difference exists in the potency for cGMP production among BNPs and that the potency of BNP for cGMP production via the biologically active receptor depends not only on

and

subtypes of the biologically active receptor, but also on species difference in the molecular structure of receptors. cGMP production in the homologous assay system with ligands and receptors of the same species

As mentioned above, the potency of the natriuretic peptide family for cGMP production is divergent, especially that of BNP, among species. To investigate the physiological and clinical implications of the natriuretic peptide family, the potencies of ANP, BNP, and CNP for cGMP production were examined in the homologous assay system with endogenous ligands and receptors of the same species. a-rANP, rBNP, and CNP were used in the experiment with rat cells. a-hANP, hBNP, and CNP were used in the study with human mesangial cells. In the experiment with bovine endothelial cells, a-hANP, pBNP, and CNP were chosen because a-hANP is identical to bovine ANP, and pBNP is identical to the Cterminal 26-amino acid sequence of bBNP, which is known to be the portion of BNP essential to its biological action. The results are summarized in Fig. 7 and Table 2.

In PC12 cells with the ANP-A receptor (GC-A; Fig. 7A), a-rANP was the most potent among the three ligands, and rBNP was g-fold weaker than a-rANP. CNP exerted little effect on cGMP production at a concentration of 1 PM and was at least 300-fold weaker than CYrANP. The rank order of potency for cGMP production was cy-rANP > rBNP >> CNP. As shown in Fig. 7B, in bovine endothelial cells with the ANP-A receptor (GCA), a-hANP and pBNP were equipotent, and CNP was at least 700-fold weaker than a-hANP. The rank order of potency was cr-hANP > pBNP >> CNP. By contrast, in rat aortic SMC with the ANP-B receptor (GC-B; Fig. 7C), CNP was lo-fold more potent than cu-rANP. rBNP was 2-fold less potent than a-rANP. The rank order of potency for cGMP production was CNP > a-rANP 2

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 December 2014. at 13:23 For personal use only. No other uses without permission. . All rights reserved.

RECEPTOR

236

Ii 50

40

B

SELECTIVITY 0 6

30 20 10

z 60 c B u

40 20

z

J

600 Llslld -11

-10

-9

logi

PEPTIDE

-8

-7

-6

j(M)

FIG. 7. Effects of ANP, BNP, and CNP on cGMP production in the homologous assay system with endogenous ligands and receptors of the same species. PC12 cells (A), bovine endothelial cells (B), rat aortic SMC (C), and human mesangial cells (D) were incubated with various concentrations of a-hANP (O), ol-rANP (O), hBNP (O), rBNP (m), pBNP (A), and CNP (A) for 30 min.

rBNP in rat aortic smooth muscle cells. In cGMP production via the ANP-B receptor (GC-B) of human mesangial cells (Fig. 7D), CNP was go-fold stronger than cyhANP and hBNP. The rank order of potency was CNP > a-hANP = hBNP in human mesangial cells. Discussion To elucidate the ligand-receptor relationship of the natriuretic peptide system comprising at least three endogenous ligands (ANP, BNP, and CNP) and three receptors (ANP-A receptor or GC-A, ANP-B receptor or GC-B, and C-receptor), in the first half of the present study we characterized the C-receptor in solubilized membrane preparations from tissues and cultured cells with the aid of a binding assay and revealed the subtype of the biologically active receptor expressed in cultured cells by the Northern blot technique and the cGMP production method. Using these receptor preparations, we obtained the following findings. 1) The receptor proportion varies from tissue (cell) to tissue (cell) in the same species and is variable even in the same tissue among different species. 2) Among ANP, BNP, and CNP, the receptor selectivity of BNP is particularly divergent, presumably due to its divergent molecular form. Therefore, to assess the physiological and clinical implications of the natriuretic peptide system, the receptor selectivity of the natriuretic peptide family should be analyzed using the homologous assay system with endogenous ligands and receptors of the same species. Using the homologous assay system, the present study demonstrates that ANP is a selective ligand for the ANPA receptor (GC-A), that CNP possesses the highest selectivity for the ANP-B receptor (GC-B), but the lowest

OF ANP,

BNP,

AND

Endo. 1992 Vol 130. No 1

CNP

selectivity for the ANP-A receptor (GC-A), and that the selectivity of BNP for the biologically active receptor shows a marked species difference. Thus, in the homologous assay system, the rank order of potency for cGMP production via the ANP-A receptor (GC-A) is ANP L BNP >> CNP, but that via the ANP-B receptor (GCB) is CNP > ANP r BNP. The present study also demonstrates that ANP has the highest affinity for the C-receptor, that the rank order of binding affinity is ANP > CNP > BNP in both humans and rats, and that affinities of BNPs are variable among species. Conclusions based on ligand-receptor specificity in the same species elucidated in the present study are summarized in Table 3. Characterization of type of receptor is critical in the present study. Using the competitive inhibition of [1251] ANP binding by C-ANF-(4-23), the present study clearly indicates that the overwhelming majority of ANP-binding sites in crude receptor preparations from the human lung and rat aortic SMC are classified as C-receptors, and that these preparations are available to investigate the C-receptor selectivity of natriuretic peptides. The observation that the C-receptor is predominantly expressed in the rat aortic SMC is consistent with previous reports (26). As for the biologically active receptor which itself is particulate guanylate cyclase, studies on cGMP production in COS-7 cells transfected with receptor cDNA expression vector revealed that the ANP-A receptor (GCA) is more sensitive to ANP than to pBNP in rats (24) and is equally activated by ANP and pBNP in humans (29, whereas the ANP-B receptor is preferentially activated by pBNP than ANP in both humans (25) and rats TABLE

3. Receptor selectivity of natriuretic

peptide family

Biologically active receptor Species Rat Bovine Rat Human

Major receptor subtype __ ANP-A receptor (GC-A) ANP-A receptor (GC-A) ANP-B receptor (GC-B) ANP-B receptor (GC-B)

Clearance receptor Snecies

Cells

Rank order

PC12 cells

ANP >BNP

Endothelial cells Aortic SMC

ANP” = BNP* >> CNP

Mesangial cells

Tissue

>>C!NP

CNP > ANP

2 BNP

CNP >ANP

=BNP

Rank order

Rat

Aortic SMC ANP >CNP >BNP Bovine Lung ANP” > BNPb > CNP Human Lung ANP > CNP > BNP ’ hANP is identical to bANP. bpBNP is identical to the C-terminal 26-amino acid residues of bBNP.

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 December 2014. at 13:23 For personal use only. No other uses without permission. . All rights reserved.

RECEPTOR

SELECTIVITY

(24). These observations raised the possibility that the receptor (GC-B) is selective for BNP (25). In this context, we have determined the subtype of the biologically active receptor expressed in cultured cells. The present study elucidates, with the aid of Northern blot analysis and the cGMP production method, that the majority of the biologically active receptor expressed in PC12 cells belongs to the ANP-A receptor (CC-A) and that in cultured rat aortic SMC, the ANP-B receptor (GC-B) is predominantly expressed. In human mesangial cells, the patterns of cGMP production by ANP and pBNP suggest that the predominant biologically active receptor expressed is the ANP-B receptor (G&B), as in cultured rat aortic SMC. In contrast, the cGMP production patterns by ANP and pBNP in bovine endothelial cells indicate that the major biologically active receptor expressed is the ANP-A receptor (GC-A). Our preliminary results from Northern blot hybridization of RNA from cultured human mesangial cells and bovine endothelial cells are consistent with our interpretation of the subtype of the biologically active receptor. The next critical point for interpretation of the pattern in cGMP production by natriuretic peptides is the possibility that the C-receptor, which is expressed together with the biologically active receptor in cultured cells, affects the guanylate cyclase activity of the biologically active receptor. The expression of C-receptor in heterologous cells has been reported to have no effect on basal or ANP-stimulated cGMP production in cultured cells (42). Therefore, the C-receptor expressed in cultured cells is unlikely to affect cGMP production via the biologically active receptor. The weaker potency of a-hANP than cu-rANP for cGMP production in both PC12 cells and rat aortic SMC is consistent with a previous report (40) and can be explained by the substitution of a single amino acid at the 12th amino acid of ANP. Combined with the marked species difference in the potency for cGMP production among BNP, investigations using the homologous assay system with endogenous ligands and receptors of the same species are essential to assess the physiological and clinical implications of the natriuretic peptide system. It has been proposed that BNP could be a selective ligand for the ANP-B receptor (GC-B), using pBNP (25). However, as shown in Table 3, rBNP is less potent than (YrANP or CNP in cGMP production via the rat ANP-B receptor (GC-B) in aortic SMC. Similar results showing a much weaker action of hBNP than that of CNP were obtained for cGMP production via the human ANP-B receptor (GC-B) in mesangial cells. Thus, the previous proposal that the ANP-B receptor (GC-B) is one subtype of the biologically active receptor specific for BNP (25) is incorrect. Schulz et al. (24) reported that relatively high amounts of pBNP are required to stimulate the ANP-B

OF ANP,

BNP,

AND

CNP

237

ANP-B receptor (GC-B), which raised the possibility that an unknown ligand for ANP-B receptor (GC-B) is present other than ANP and BNP. The present study demonstrates that CNP is the most selective endogenous ligand so far identified for the ANP-B receptor (GC-B). We and others have revealed that CNP is distributed mainly in the brain (18, 43), and the ANP-B receptor (GC-B) is also present in the brain (24). It is likely, therefore, that CNP could act in the brain as a neuropeptide or a local regulator. Studies in the fine localization of ANP-B receptor (GC-B) will help us to confirm this hypothesis. During the preparation of this manuscript, Koller et al. (44) reported the more selective activation of the human ANP-B receptor (GC-B) by CNP than a-hANP and hBNP, similar to our results. Another important finding in the present study is that the binding affinity of hBNP for the human C-receptor and that of rBNP for the rat C-receptor were about 1 order of magnitude lower than those of ANP. Since the C-receptor serves as a specific clearance binding site for natriuretic peptides (29, 45), it is conceivable that the clearance of BNP from the circulation is slower than that of ANP. We recently reported that the half-times of plasma disappearance of hBNP are about 2-fold longer than those of cu-hANP in humans (35). Our unpublished observation reveals that the half-times of plasma disappearance of rBNP are also longer than those of a-rANP in rats and that preadministration of C-ANF-(4-23) prolongs the half-times of plasma disappearance of (YrANP more effectively than those of rBNP. These observations on the different clearances between ANP and BNP are consistent with the different binding affinities for the C-receptor between ANP and BNP elucidated in the present study. As we previously reported (14), rBNP was approximately 2-fold more potent than a-rANP in natriuretic-diuretic action in rats in ho. However, its potency for cGMP production in vitro was several-fold weaker than that of a-rANP in the present study. This discrepancy could be accounted for in part by the slower clearance of rBNP than Lu-rANP, which results in the higher plasma level of rBNP than a-rANP when the same doses of rBNP and a-rANP are administrated. This finding also raises the possibility of the presence of another subtype of the biologically active receptor selective for BNP. Accumulating evidence indicates tissue-specific distribution of the natriuretic peptide family (5,15, 16,18,35) and receptors (24, 25, 28, 30) and demonstrates the marked increases in plasma levels of ANP and BNP in cardiovascular disorders, such as congestive heart failure, renal failure, and hypertension (6,35,46-48). The receptor selectivity of the natriuretic peptide family elucidated in the present study provides new insight into under-

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 December 2014. at 13:23 For personal use only. No other uses without permission. . All rights reserved.

238

RECEPTOR

standing of the physiological and clinical the natriuretic peptide system.

SELECTIVITY

implications

of

Acknowledgments We thank Prof. T. Maack, Cornell University (New York, NY), for kindly providing synthetic peptide, Prof. S. Hirose, Tokyo Institute of Technology (Tokyo, Japan), for donating purified bovine C-receptor, and Dr. T. Yamamoto, Niigata University (Niigata, Japan), for advice on cell culture. We also acknowledge Ms. H. Kato, Ms. A. Kibune, and Ms. S. Takahashi for their secretarial assistance.

References 1. Kangawa K, Matsuo H 1984 Purification and complete amino acid sequence of a-human atria1 natriuretic polypeptide (a-hANP). Biochem Biophys Res Commun 118:131-139 2. Flynn TG, de Bold ML, de Bold AJ 1983 The amino acid sequence of an atria1 peptide with potent diuretic and natriuretic properties. Biochem Biophys Res Commun 117:859-865 3. Sudoh T, Kangawa K, Minamino N, Matsuo H 1988 A new natriuretic peptide in porcine brain. Nature 332:78-81 4. Minamino N, Kangawa K, Matsuo H 1988 Isolation and identification of a high molecular weight brain natriuretic peptide in porcine cardiac atrium. Biochem Biophys Res Commun 157:402409 5. Saito Y, Nakao K, Itoh H, Yamada T, Mukoyama M, Arai H, Hosoda K, Shirakami G, Suga S, Minamino N, Kangawa K, Matsuo H, Imura H 1989 Brain natriuretic peptide is a novel cardiac hormone. Biochem Biophys Res Commun 158:360-368 6. de Bold AJ 1985 Atria1 natriuretic factor: a hormone produced by the heart. Science 230:767-770 7. Nakao K, Morii N, Itoh H, Yamada T, Shiono S, Sugawara A, Saito Y, Mukoyama M, Arai H, Sakamoto M, Imura H 1986 Atria1 natriuretic polypeptide in the brain-implication of central cardiovascular control. J Hypertension [Suppl6] 4:S492-S496 8. Itoh H. Nakao K. Yamada T. Shirakami G. Kaneawa K. Minamino N, Matsuo H, Imura H 1988 Antidipsogenicaction’of a novel peptide, “brain natriuretic peptide,” in rats. Eur J Pharmacol 150:193-196 _ 9. Shirakami G, Nakao K, Yamada T, Itoh H, Mori K, Kangawa K, Minamino N, Matsuo H, Imura H 1998 Inhibitory effect of brain natriuretic peptide on central angiotensin II-stimulated pressor response in conscious rats. Neurosci Lett 91:77-83 10. Yamada T, Nakao K, Itoh H, Shirakami G, Kangawa K, Minamino N, Matsuo H, Imura H 1998 Intracerebroventricular injection of brain natriuretic peptide inhibits vasopressin secretion in conscious rats. Neurosci Lett 95:223-228 11. Kambayashi Y, Nakao K, Itoh H, Hosoda K, Saito Y, Yamada T, Mukoyama M, Arai H, Shirakami G, Suga S, Ogawa Y, Jougasaki M, Minamino N, Kangawa K, Matsuo H, Inouye K, Imura H 1989 Isolation and sequence determination of rat cardiac natriuretic peptide. Biochem Biophys Res Commun 163:233-240 12. Kambayashi Y, Nakao K, Mukoyama M, Saito Y, Ogawa Y, Shiono S, Inouye K, Yoshida N, Imura H 1990 Isolation and sequence determination of human brain natriuretic peptide in human atrium. FEBS Lett 259:341-345 13. Nguyen TT, Lazure C, Babinski K, Chretien M, Ong H, de Lean A 1989 Aldosterone secretion inhibitory factor: a novel neuropeptide in bovine chromaffin cells. Endocrinology 1241591-1593 14. Kambayashi Y, Nakao K, Kimura H, Kawabata T, Nakamura M, Inouye K, Yoshida N, Imura H 1991 Biological characterization of human BNP and rat BNP-species-specific actions of BNP. Biochem Biophys Res Commun 173:599-605 15. ltoh H, Nakao K, Saito Y, Yamada T, Shirakami G, Mukoyama M, Arai H, Hosoda K, Suga S, Minamino N, Kangawa K, Matsuo H, Imura H 1989 Radioimmunoassay for brain natriuretic peptide (BNP)-detection of BNP in canine brain. Biochem Biophys Res

OF ANP. BNP. AND

CNP

Endo. Vol130.

1992 No 1

Commun 158120-128 16. Ogawa Y, Nakao K, Mukoyama M, Shirakami G, Itoh H, Hosoda K, Saito Y, Arai H, Suga S, Jougasaki M, Yamada T. Kambavashi Y, Inouye K, Imura H-1996 Rat brain natriuretic peptide-&sue distribution and molecular form. Endocrinology 126:2225-2227 17. Sudoh T, Minamino N, Kangawa K, Matsuo H 1990 C-Type natriuretic peptide (CNP): a new member of natriuretic peptide family identified in porcine brain. Biochem Biophys Res Commun 168863-870 18. Kojima M, Minamino N, Kangawa K, Matsuo H 1990 Cloning and sequence analysis of a cDNAencoding a precursor for rat C&e natriuretic peptide (CNP). FEBS Lett 276:209-213 19. Tawaragi Y-, Fuchimura K, Tanaka S, Minamino N, Kangawa K, Matsuo H 1991 Gene and precursor structure of human C-type natriuretic peptide. Biochem Bionhvs Res Commun 175:645-651 20. Ogawa Y, Nakao K, Nakagawa- 0; Komatsu Y, Mukoyama M, Hosoda K, Suga S, Arai H, Shirakami G, Kishimoto I, Hama N, Imura H 1991 Human C-tvne natriuretic DeDtide (CNP)-structure of the gene and identification of the peptide in‘the brain. 45th Annual Fall Conference and Scientific Sessions of Council for High Blood Pressure Research. Chicaeo IL. 1991. ~18 (Abstract) 21. Waldman SA, Rapoport’ RM, Mu&d F I984 Atria1 natriuretic factor selectively activates particulate guanylate cyclase and elevates cyclic GMP in rat tissues. J Biol Chem 259:14332-14334 22. Kuno T, Andresen JW, Kamisaki Y, Waldman SA, Chang LY, Saheki S, Leitman DC, Nakane M, Murad F 1986 Co-purification of an atria1 natriuretic factor receptor and particulate guanylate cyclase from rat lung. J Biol Chem 261:5817-5823 23. Chinkers M, Garbers DL, Chang MS, Lowe DG, Chin H, Goeddel DV. Schulz S 1989 A membrane form of euanvlate cvclase is an atria1 natriuretic peptide receptor. Nature 33878-83 24. Schulz S, Singh S, Bellet RA, Singh G, Tubb DJ, Chin H, Garbers DL 1989 The primary structure of a plasma membrane guanylate cyclase demonstrates diversity within this new receptor family. Cell 581155-1162 25. Chang MS, Lowe DG, Lewis M, Hellmiss R, Chen E, Goeddel DV 1989 Differential activation by atria1 and brain natriuretic DeDtides of two different receptor guanylate cyclases. Nature 341:68-72 26. 1Schenk DB, Phelps MN, Porter JG, Scarborough RM, McEnroe GA, Lewicki JA 1985 Identification of the receptor for atria1 natriuretic factor on cultured vascular cells. J Biol Chem 260:14887-14890 27. Shimonaka M, Saheki T, Hagiwara H, Ishido M, Nogi A, Fujita T, Wakita K, Inada Y, Kondo J, Hirose S 1987 Purification of atria1 natriuretic peptide receptor from bovine lung. J Biol Chem 262:5510-5514 28. Fuller F, Porter JG, Arfsten AE, Miller J, Schilling JW, Scarborough RM, Lewicki JA, Schenk DB 1988 Atria1 natriuretic peptide clearance receptor. J Biol Chem 263:9395-9401 29. Maack T, Suzuki M, Almeida FA, Nussenzveig D, Scarborough RM. McEnroe GA. Lewicki JA 1987 Phvsioloeical role of silent receptors of atria1 natriuretic factor. Science 238:675-678 30. Martin ER, Lewicki JA, Scarborough RM, Ballermann BJ 1989 Expression and regulation of ANP receptor subtypes in rat renal glomeruli and papillae. Am J Physiol257:F649-F657 31. Kambayashi Y, Nakajima S, Ueda M, Inouye K 1989 A dicarba analog of B-atria1 natriuretic peptide (/3-ANP) inhibits guanosine 3’,5’-cyclic monophosphate production induced by a-ANP in cultured rat vascular smooth muscle cells. FEBS Lett 24828-34 32. Hagiwara H, Shimonaka M, Morisaki M, Ikekawa N, Inada Y 1984 Sitosterol-stimulative plasminogen activator production in cultured endothelial cells from bovine carotid artery. Thromb Res 33:363-370 33. Yaoita E, Kazama T, Kawasaki K, Miyazaki S, Yamamoto T, Kihara I 1985 In uitro characteristics of rat mesangial cells in comparison with aortic smooth muscle cells and dermal fibroblasts. Virchows Arch [Cell Pathol] 49:285-294 34. Nakao K, Sugawara A, Morii N, Sakamoto M, Suda M, Soneda J, Ban T, Kihara M, Yamori Y, Shimokura M, Kiso Y, Imura H 1984 Radioimmunoassay for a-human and rat atria1 natriuretic polypeptide. Biochem Biophys Res Commun 124~815-821 35. Mukoyama M, Nakao K, Hosoda K, Suga S, Saito Y, Ogawa Y,

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 December 2014. at 13:23 For personal use only. No other uses without permission. . All rights reserved.

I

~~

~-

-~-

RECEPTOR

36.

37. 38.

39.

40.

41.

SELECTIVITY

Shirakami G, Jougasaki M, Obata K, Yasue H, Kambayashi Y, Inouye K, Imura H 1991 Brain natriuretic peptide (BNP) as a novel cardiac hormone in humans-evidence for an exquisite dual natriuretic peptide system, atria1 natriuretic peptide and brain natriuretic peptide. J Clin Invest 87:1402-1412 Itoh H, Nakao K, Shiono S, Mukoyama M, Morii N, Sugawara A, Yamada T, Saito Y, Arai H, Kambayashi Y, Inouye K, Imura H 1987 Conversion of &human atria1 natriuretic polypeptide into (Yhuman atria1 natriuretic polypeptide in human plasma in vitro. Biochem Biophys Res Commun 143:560-569 Rathinavelu A, Isom GE, 1991 Differential internalization and processing of atrial-natriuretic factor B and C receptors in PC 12 cells. Biochem J 276493-497 Arai H, Nakao K, Saito Y, Morii N, Sugawara A, Yamada T, Itoh H, Shiono S, Mukoyama M, Ohkubo H, Nakanishi S, Imura H 1988 Augmented expression of atria1 natriuretic polypeptide gene in ventricles of spontaneously hypertensive rats (SHR) and SHRstroke prone. Circ Res 62:926-930 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA 1988 Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239487-491 Fiscus RR, Robles BT, Waldman SA, Murad F 1987 Atrial natriuretic factors stimulate accumulation and efflux of cyclic GMP in C6-2B rat glioma and PC12 rat pheochromocytoma cell cultures. J Neurochem 48522-528 Homma M, Satoh T, Takexawa J, Ui M 1977 An ultrasensitive method for the simultaneous determination of cyclic AMP and cyclic GMP in small volume samples from blood and tissue.

OF ANP, BNP, AND CNP

239

Biochem Med l&257-273 42. Porter JG, Wang Y, Schwartz K, Arfsten A, Loffredo A, Spratt K, Schenk DB, Fuller F, Scarborough RM, Lewicki JA 1988 Characterization of the atrial natriuretic peptide clearance receptor using a vaccinia virus expression vector. J Biol Chem 26318827-18833 43. Komatsu Y, Nakao K, Suga S, Ogawa Y, Mukoyama M, Arai H, Shirakami G, Hosoda K, Nakagawa 0, Hama N, Kishimoto I, Imura H 1991 C-Type natriuretic peptide in rats and humans. Endocrinology 129:1104-1106 44. Koller KJ, Lowe DG, Bennett GL, Minamino N, Kangawa K, Matsuo H, Goeddel DV 1991 Selective activation of the B natriuretic peptide receptor by C-type natriuretic peptide (CNP). Science 252:120-123 45. Almeida FA, Suzuki M, Scarborough RM, Lewicki JA, Maack T 1989 Clearance function of type C receptors of atria1 natriuretic factor in rate. Am J PhysioI256:R469-R475 46. Burnett Jr JC, Kao PC, Hu DC, Heser DW, Heublein D, Granger JP, Opgenorth TJ, Reeder GS 1986 Atria1 natriuretic peptide elevation in congestive heart failure in the human. Science 231:1145-1147 47. Sugawara A, Nakao K, Morii N, Yamada T, Itoh H, Shiono S, &to Y, Mukoyama M, Arai H, Nishimura K, Obata K, Yasue H, Ban T. Imura H 1988 Svnthesis of atria1 natriuretic nolvoeutide in human failing heart&evidence for altered processing of atrial natriuretic polypeptide precursor and augumented synthesis of @human ANP. J Clin Invest 81:1962-1970 48. Mukoyama M, Nakao K, Saito Y, Ogawa Y, Hosoda K, Suga S, Shirakami G, Jougasaki M, Imura H 1990 Human brain natriuretic peptide, a novel cardiac hormone. Lancet 335801802

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 December 2014. at 13:23 For personal use only. No other uses without permission. . All rights reserved.