PHYSIOLOGY AND CELL BIOLOGY UPDATE

The Natriuretic Peptides and Their Receptors Rex L. Jamison, MD, Sima Canaan-KOhl, MD, and Richard Pratt, PhD • Atrial natriuretic factor (ANF) is released from the cardiac atrium in response to stretch and acts through receptors to cause an increase in urinary flow and sodium excretion, vasodilatation, and a reduction in blood volume. Recently, two new natriuretic peptides, brain natriuretic peptide (BNP) and C-type natriuretic peptide (C-typeNP), have been isolated, and three different natriuretic peptide receptors have been identified. Two of the receptors, ANP-ROC(A) and ANP-RGc(B), mediate biologic actions. The natural ligand of ANP-Roc(A) is ANF, whereas that of ANP-RGC(B) is C-typeNP. In view of clear differences in ligand specificity and tissue distribution of these receptors, it has been proposed that ANF and its receptor, ANP-RGC(A), and C-typeNP and its receptor, ANP-RGC(B), represent two distinct natriuretic peptide regulatory systems. Whether a separate system exists that incorporates BNP awaits clarification of its natural receptor that mediates a biologic action. The third receptor, ANP-Rc , binds all three natriuretic peptides. Its messenger RNA lacks the guanylyl cyclase sequence present in the mRNA of the other natriuretic peptide receptors, suggesting that the principal function of ANP-Rc is to remove natriuretic peptides from the circulation, that is, to regulate plasma levels of the natriuretic peptides. However, ANP-Rc may also mediate a biologic effect. These findings raise several intriguing questions about the functional role of this family of natriuretic peptides. © 1992 by the National Kidney Foundation, Inc. INDEX WORDS: Atrial natriuretic factor; atrial natriuretic peptide; brain natriuretic peptide; C-type natriuretic peptide; atrial natriuretic peptide receptor; ANP-RGC(A); ANP-RGC(B); ANP-Rc .

T

HE FINDING of de Bold et all in 1981 that administration of extracts of cardiac atrium caused a natriuresis, diuresis, and decrease in blood pressure launched an exciting hunt. The quarry is a group of natriuretic peptides that are well conserved in the animal kingdom-molluscs (snail), echinoderms (sea urchin), and a variety of chordates from shark to man-and synthesized in several organs besides the heart. Three distinct receptors have been identified, two that mediate biological effects and one thought to serve primarily a clearance function. The purpose of this editorial is to review recent progress in the study of natriuretic peptides and their receptors. Several reviews2-7 and two books8.9 have recently been published. NATRIURETIC PEPTIDES

Atrial Natriuretic Factor

The first natriuretic peptide isolated circulates in human plasma primarily as a 28-amino acid peptide,1O called atrial natriuretic factor* (ANF) (Fig 1). Encoded by a 3-exon gene located on the short arm of chromosome 1,11,12 the messenger RNA (mRNA) directs the synthesis of a 152-amino acid pre-pro-peptide form, preproANF. 5,12-14 In cardiac myocytes, this primary translation product is processed to proANF (126 amino acids) and stored in secretory granules. 5,15

During or soon after secretion, proANF is cleaved to its mature circulating form, ANF. 5,1O,16 The amino terminal pro-fragment (98 amino acids), ANF I _98 , is cosecreted with ANF and reaches plasma levels 20-fold higher than that of ANF. 17,18 Besides the atria, the ANF gene is expressed in several other tissues during fetal development and in the mature organism (Table 1).12,19 In the course of development, the ANF gene is expressed in cardiac ventricles, as well as atria,20,21 but on

* Atrial natriuretic factor (ANF) is the official name of the predominant circulating form in rat and humans. 100 The lowercase letter preceding ANF designates species: h = human, p = porcine, r = rat. Atrial natriuretic peptide (ANP), a-ANP, and cardionatrin are synonyms. Several natriuretic peptides, auriculin A and Band atriopeptins I, II, and III, with similar structures of varying lengths, are probably artifacts of the extraction procedures. 3 From the Nephrology Unit, Department of Medicine, University ofRochester School ofMedicine and Dentistry, Rochester, NY; and the Divisions ofNephrology and Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA. Received April 22, 1992; accepted in revised form June 26, 1992. Supported by National Institutes of Health Research Grants No. 2R01 DK 29985 and HL 42663. Address reprint requests to Rex L. Jamison, MD, Division of Nephrology, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305. © 1992 by the National Kidney Foundation, Inc. 0272-6386/92/2005-0015$3.00;0

American Journal of Kidney Diseases, Vol XX, No 5 (November), 1992: pp 519-530

519

520

JAMISON, CANAAN-KOHL, AND PRAIT

15

32

C-typeNP 15

Fig 1. Structure of human natriuretic peptides. The shaded circles indicate identical amino acids in the ring of the three peptides. The black area indicates the cysteine-cysteine disulfide bond.

reaching maturation, expression decreases to 2% of that in the atria. However, in failing or pressure-overloaded hearts, ventricular expression of ANF is increased. 22.24 In other tissues that express ANF, the peptide may regulate a local function. For example, rat kidney cells in culture synthesize and secrete an ANF-like peptide. 25 Greenwald et ae 6 localized ANF and proANF to the distal cortical nephron in the rat kidney. The predominant form was ANF. However, in puromycin-induced nephrotic

syndrome, proANF predominated; since it was not detectable in plasma, proANF must have been synthesized by the renal tubule. The authors speculate that either renal ANF synthesis increases as an adaptation in the nephrotic syndrome to block sodium reabsorption in the distal nephron, or the increased cellular proANF is due to inhibition of ANF secretion,26 contributing to the intrinsic sodium retention characteristic of the syndrome. 27 ANF 1.30 , ANF31 -67, and ANF have been identified in the proximal tubule by immunocytochemical techniques,28 but it is not clear if this indicates ANF synthesis or merely binding to degradative enzymes present in this segment of the nephron. Infusion of ANF into cerebral ventricles suppresses arginine vasopressin (A VP), thirst, salt intake, and sympathetic outflow, and reduces blood pressure,3.29.32 reinforcing the notion that ANF has a fundamental role in salt and water regulation. ANF also inhibits growth of several cell lines in culture, including glomerular mesangium, adrenal zona glomerulosa, and vascular smooth muscle. 33.35 In broad terms, its role may be to oppose all actions of the vasoconstrictor peptides, angiotensin II (A II), A VP, and endothelin-ranging from vasoconstriction, antidiuresis, and antinatriuresis to cell proliferation. Classic experiments of Henry et al 36 set the stage for the finding that increasing right atrial pressure or stretch releases ANF. 18 ,37 Clinical studies have found a close relationship between atrial pressure or distention and ANF plasma levels. 3,5.18 Head-out water immersion, which expands vascular volume and distends the right atrium, increased plasma ANF levels and caused a natriuresis, diuresis, decrease in renal vascular resistance, and increase in cyclic guanylyl monophosphate (cGMP) excretion .38-40 Patients with tachyarrhythmias, congestive heart failure, diabetic glomerulopathy, and acute and chronic renal failure exhibit elevated plasma ANF levels. 3.22•24,40,41 End-stage renal disease (ESRD) patients were studied before and after ultrafiltration (without dialysis), which removed 2.3 kg of fluid. 42 Plasma ANF levels preultrafiltration (100 pg/mL) declined to 31 pg/mL; 48 hours later, when fluid had reaccumulated, ANF increased to 125 pg/mL. The changes correlated with changes in right atrial size.

521

NATRIURETIC PEPTIDES AND THEIR RECEPTORS Table 1. Location of Natriuretic Peptides and Their Receptors Natriuretic Peptide Receptors t

Natriuretic Peptides' Tissue

Heart Right atrium Right ventricle Left atrium Left ventricle Kidney Brain Cerebral hemispheres Basal ganglia Diencephalon Midbrain, pons, medulla Cerebellum Spinal cord Pituitary Anterior Intermediate Posterior Aorta Aortic arch vaso vasorum Abdominal Renal artery Saphenous vein Lung Adrenal cortex Glomerulosa Fasciculata Reticularis Adrenal medulla Pancreas Testis Thymus Spleen Liver Small intestine Large intestine

ANF

BNP

xxx::j: x xxx x x

x x x x

x x x x

x x x x

x

x

C-typeNP

x

x

ANP-RGC(A)

ANP-RGC(B)

ANP-R c

x~

XXii

x~

x~

x~

x~

xx

x

x

xl1 x x

X·

x x x

x

x

x

+/-

x xx x

x x

x

x

x

x

x xx

x

x

x§ x§ x§ x§

x x

• Peptides identified by immunoreactive techniques and/or presence of mRNA. Receptors identified by presence of mRNA in the Rhesus monkey/6 except for receptor-B mRNA in human kidney.65 ::j: xxx, highest-intensity hybridization; x, positive hybridization; +/-, positive signal in one experiment not reproduced; -, no hybridization signal. If blank, not tested. § Discrete clusters of cells. 11 Endocardial endothelial cell localization. II Both myocardium and endocardial endothelial cell localization. Data obtained from the following references: ANF: 5, 12, 15, 19, 26; BNP: 5, 49-51, 54, 55; C-typeNP: 5,14,63; ANPRGC(A): 82; ANP-RGC(B): 65, 82; ANP-R c : 82. Receptor section reprinted with permission. 82

t

Several forms of ANF exist in the circulation 41 ,43,44: ANF is the predominant form; other forms include the amino terminal fragment ofproANF (ANF I _98 ); is-ANF, an antiparallel dimer of ANF with 56 residues; 'Y-ANF, also called proANF (ANF I _126 ); and ANF I _30 and ANF31 _67 ,

two fragments of ANF I _98 . In the plasma of healthy subjects, ANF was present in all, is in none, and proANF in half.41 As already noted, the concentration of ANF I _98 is 20-fold higher than that of ANF. 17 ,18 In congestive failure, ANF levels increased as a function ofseverity22,41,45; in

522

the worst cases, {3 was the most common form. 41 ,45 These findings were reversed by correction of the failure. 41 In a study of chronic renal insufficiency, ANF was the major circulating form; in several patients, {3 and proANF were also present. 41 Plasma from patients with ESRD contains ANF; most also have proANF, and a few have {3. ANF may be removed by dialysis. 41 Wilson et al46 have identified a high-molecular weight form of ANF in rabbit plasma called "big ANF." It weighs 60 to 70 kDa, has a negative charge, and is not dialyzable. Immunoreactive ANF is released when big ANF is acidified. Analysis of the dissociated ANF using high-performance liquid chromatography disclosed ANF and ANF103-126 (also called atriopeptin III) in a ratio of 2: 1 and sometimes other ANF fragments, too. These findings are similar to those of Schwartz et al lO in rat plasma. The authors concluded that big ANF is a bound form of ANF found in normal plasma. It remains to be estabI lished if human plasma has big ANF. The physiological and pathophysiological significance of the variety of forms of atrial natriuretic peptides other than ANF is uncertain. It appears that specific peptide fragments of ANF I _98 may have effects not mediated by currently identified ANF receptors. For example, Gunning et al47 recently studied the mechanism whereby ANP 31 -67 , like ANF, stimulates natriuresis and diuresis. In suspensions of rabbit inner medullary collecting duct cells, ANF31 _67 inhibited basolateral Na-K-adenosine triphosphatase (ATPase), an effect mediated by the prostaglandin, PGE2. This contrasts with their previous findings that ANF inhibited passive sodium entry across the apical membrane of collecting ducts and that this inhibition was mediated by cGMP,3 the second messenger of the natriuretic peptide biological receptors. However, Needleman et ae have cautioned that, although ANF I _98 has been shown to induce a natriuresis,43,48 the lack of evidence that it has cardiovascular effects in vitro or in vivo raises doubts as to the physiological relevance of the several amino terminal peptide fragments-ANF I _30 , ANF31 _67 , and ANF79 _98 derived from it. Moreover, binding specificities by the three natriuretic peptide receptors are in general not known.

JAMISON, CANAAN-KOHL, AND PRATT

Brain Natriuretic Peptide In 1988, Sudoh et al49 identified a second pep-

tide in porcine brain, brain natriuretic peptide (BNP), containing 26 amino acids and a ring with 13 of 17 residues identical to those in ANF's ring. A second form ofBNP with six more amino acids at the N-terminus, BNP 32, was isoiated50 and shown to be the circulating form in humans51 (Fig 1). There is marked structural variability of BNP among species. However, species-specific BNP is equipotent to ANF in its renal effects.31 The actions ofBNP are similar to those of ANF52 ; maximally effective concentrations of ANF and BNP do not have an additive effect. 53 This finding suggests they bind the same biologically active receptors, although the affinities need not be the same (see below). BNP is synthesized in the brain and heart by a process strikingly similar to that for ANF5,14,54,55 (Table 1). Its highest concentration (in pmol/g wet tissue) is in the atrium, 570 (pig) and 250 (human), compared with concentrations of ANF, 8,500 and 9,600, respectively?4 The atrial myocyte can synthesize both ANF and BNP. 50 In the failing human heart, the ventricle is apparently more important than the atrium in the synthesis and release of BNP. 24,45 In normal subjects, plasma BNP averaged 0.9 pmol/L (2.7 pg/mL) and ANP 5.6 pmol/L (17 pg/mL).24 Removal ofBNP from the circulation is 10 times slower than that of ANF. 57 The affinity of the biologically active receptor for BNP is also less than that for ANF by an order of magnitude (see below). In short, plasma levels of BNP are lower than ANF; although BNP circulates longer, it is bound weakly by the biologically active receptor. C-Type Natriuretic Peptide Sudoh et al 58 isolated a 22-amino acid peptide,

C-type natriuretic peptide, or C-typeNP, similar to ANF, except that it has no carboxy-terminal extension beyond the 17-amino acid ring (Fig 1). A 53-residue peptide was identified as another form ofC-typeNP in the human brain. 59 Except for one or two amino acid substitutions, the structure of both forms is virtually invariant among species. Its synthesis is similar to that of other natriuretic peptides. 14,60,61 Like other natriuretic peptides, it activates guanylyl cyclase. 60

523

NATRIURETIC PEPTIDES AND THEIR RECEPTORS

In causing diuresis and natriuresis, C-typeNP was 1/100th as potent as rANF in the rat, but its rectum-relaxant activity was three to four times higher, comparable to that of BNP. 58 Recently, Stingo et al 62 studied C-typeNP in the dog, using concentrations equal to those of ANF that produced natriuresis, ie, doses 100 times lower than those used to cause natriuresis in the rat. 58 Mean arterial pressure and cardiac output decreased by approximately 25%, while renal blood flow (RBF), glomerular filtration rate (GFR), and sodium excretion decreased. When C-typeNP was administered as a bolus injection rather than continuously, to avoid hypotension, cardiac output declined as before, RBF and GFR did not change, but sodium excretion still decreased. In sum, C-typeNP had similar cardiovascular effects as ANF, but lacked the classic renal actions of the natriuretic peptides. Aword of caution may be in order. Stingo et al observed no change in the urinary excretion of cGMP, which could mean that the dog kidney lacks the natural receptor for C-typeNP (ANP-~(B» (see below), insufficient concentrations of C-typeNP reached ANP-RaC(B) to activate it (compare to Sudoh et aI's findings), or that the canine ANP-RoC(B) receptor mediates an effect other than inhibition of sodium reabsorption in the collecting duct. In contrast, plasma cGMP levels increased, which implies that the cardiovascular effects of CtypeNP were mediated by binding ANP-RaC(B) elsewhere. In the brain, C-typeNP is more abundant than ANF or BNp 14,63 (Table 1); it appears to be widely expressed in the rat and human central nervous system. The highest concentrations (in pmol/g wet tissue) were found in the cerebellum, 3.1, and anterior lobe of pituitary, 19.8, of the rat. 63 C-typeNP is also found in the rat kidney (0.66), ileum (0.46), and colon (0.31), but not in the heart or adrenal gland.63 Attempts to identify CtypeNP in circulating blood have failed. 63 Since it is not present in the circulation, CtypeNP may act as a neuropeptide/ 4.64 a view supported by evidence that its natural receptor, ANP-~(B), is abundant in the brain64 (see below). However, the presence ofC-typeNP in kidney and intestine, and the recent discovery of the mRNA for the C-typeNP natural receptor in the kidney65 (see below), suggest it may have other

roles. Moreover, C-typeNP is expressed in the heart of the spiny dogfish shark, Squalus acanthias,66 an elasmobranch that is hyperosmotic and hyponatremic to sea water, rendering the shark vulnerable to volume expansion and hypematremia. It regulates its NaCI composition and fluid volume by a rectal salt gland that secretes hypertonic NaCI fluid. ANF stimulates NaCl secretion and elevates cGMP in the rectal gland67 and the effect of C-typeNP is 50 to 100 times more potent than ANF in this regard (unpublished data, quoted in ref 66). In the shark, C-typeNP may be a circulating natriuretic peptide, while in other species, it may serve a paracrine function in such organs as the brain and kidney. Urodilatin

Urodilatin, a 32-amino acid peptide identical to the C-terminal sequence ofproANF, was originally found in urine, but not in plasma. 68 ,69 It is probably synthesized only by the kidney.6,69 Unlike other natriuretic peptides, it is resistant to proteolysis by endopeptidase (see below). Urodilatin binds ANF receptors with the same affinity as ANF, activates guanylate cyclase and induces natriuresis and diuresis.6,69 However, unlike ANF, urodilatin does not suppress renin or aldosterone secretion. 70 Thus, urodilatin appears to be a uniquely intrarenal natriuretic peptide with undetermined physiological importance. 6 NATRIURETIC PEPTIDE RECEPTORS

ANF stimulates guanylate cyclase (g.c.), which catalyzes the formation of cGMP from guanosine triphosphate (GTP).4,71 The enzyme exists in both soluble and particulate (or membrane-bound) forms.4 Since the soluble form, but not the membrane-bound form, is activated by nitrates like nitric oxide, the two forms are clearly distinct. 4 The membrane-bound g.C. constitutes the cytoplasmic tail of many peptide receptors.4 It was first cloned from sea urchin spermatozoa. Since ANF activates membrane g.C., it was suggested the ANF receptor may also contain g.c. activity.72 Using cDNA of the sea urchin particulate g.c., Chinkers et aC3 cloned the ANF receptor from rat brain; Lowe et aC4 simultaneously reported similar experiments using a human kidney cDNA library. Expression of these cDNAs in cultured

524

JAMISON, CANAAN-KOHL, AND PRATT

cells proves they encode particulate g.c and that g.c and natriuretic peptide receptor are parts of the same molecule. Direct formation of cGMP by a receptor is a new form of receptor signal transduction. 74 Based on the work of Light et al 75 (and references cited therein), Lowe et al suggested that binding of ligand to extracellular receptor allosterically regulates cytoplasmic catalytic domains. 74 ANF binding unfolds protein kinase, exposing its catalytic site to adenosine triphosphate (A TP). Hydrolysis of ATP further alters the structure to uncover the catalytic site of g.c. to GTP, leading to cGMP formation and the biological response. To date, three types of receptor have been identified. Two mediate biological effects: (1) atrial natriuretic peptide receptor A (ANPRooA», alias ANPR-A or guanylate cyclase-A (GC_A)73.74; and (2) atrial natriuretic peptide receptor B (ANP-RoC(B», alias ANPR-B or GCB. 76.77 The third receptor, atrial natriuretic pep-

PEPTIDES ANF BNP C-typeNP

tide receptor C (ANP-Rc), alias ANPR-C, has no g.c. sequence. 78 •79 The designation, ANP-RoqA), etc, indicates that it is a receptor that binds a member of the natriuretic peptide family, and the subscript indicates whether it contains g.c. and the specific receptor sequence, (A), (B), etc. Ligand specificies for the receptors are shown in Fig 2. 57 .64 Human ANP-Roc(A) contains 1,057 amino acids (74) (Fig 2). It has an extracellular ANF-binding domain of 441 residues, a transmembrane domain of 21 residues, and 568 intracellular residues consisting of a protein kinase and g.c. sequence domains. 74 It mediates the biological actions of ANF. 80 An excellent summary of the distribution of ANF receptors before it was known that two distinct biologically active receptors exist was written by Stewart et al. 81 Few studies have yet been done to identify the tissue distribution of the three types of receptors. In situ hybridization in the Rhesus

RECEPTORS

• C

X • /'• ## ##

J (

X

)

C

0

(

)

C

J (

A

X

)

Kinase Homology Guanylyl Cyclase Domain Domain

B

EXTRACELLULAR INTRACELLULAR Fig 2.

Human natriuretic peptide receptors. Receptor A

= ANP-RGC(A),

receptor B

= ANP-RGC(II),

and receptor C

= ANP-Rc . On the left, the peptide affinity is indicated by the arrows with the solid lines. The arrow with the dashed

line indicates that BNP also binds receptor A, but has a lower affinity than ANF. The vertical dashed line delineates the cell membrane. The extracellular domains of the three receptors have structural similarities (see text). In contrast, while the two biologically active receptors have intracellular domains for protein kinase and guanylyl cyclase, the clearance receptor has a very short cytoplasmic domain without kinase or guanylyl cyclase sequences. (Adapted with permission; Copyright 1991 by the AAAS....)

525

NATRIURETIC PEPTIDES AND THEIR RECEPTORS

monkey has shown the predominant sites of ANP-Roc(A) mRNA expression are the adrenal and kidney82 (Table 1). This is the first localization of sites of the three receptor mRNAs in the primate. However, it should be cautioned that the data were obtained postmortem from a single monkey and need to be confirmed by further studies. ANP-Roc(A) is expressed in the glomerulus and throughout the renal tubule. Using the highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay, Terada et al 83 demonstrated that the mRNA for ANP-Roc(A) was present in every tubule segment. It was most abundant in the inner medullary collecting duct and second most prominent in the thin loop of Henle. There was close correspondence between levels of ANF-stimulated cGMP and ANP-Roc(A) mRNA. These results are consistent with the thesis that ANF has several renal effects. 84,85 Mejia et al85 proposed that the short-term effects of ANF are to increase glomerular filtration and antagonize A-II-stimulated sodium reabsorption by the proximal tubule, while the long-term effects are to inhibit sodium transport in the collecting duct and thick ascending limb. (The effect of ANF on Henle's thin loops is uncertain.) ANP-RoC(A) mRNA is the predominant receptor mRNA in the zona glomerulosa of the adrenal cortex, where ANF inhibits aldosterone secretion,86 and in the adrenal medulla, Thus far, no role in the regulation of catecholamine release from the adrenal medulla has been attributed to ANF, although ANF has been shown to inhibit norepinephrine release from nerve terminals by means of ANP-Rc (see below). ANP-Roc(A) mRNA is also present in the endocardium ofleft and right atrium and right ventricle, perhaps to mediate the decrease in cardiac output induced by ANF. 3,87 ANP-RGC(B)

ANP-RoC(B) mRNA is structurally similar to ANP-RoC(A) mRNA, encoding 1,048 amino acids, including a 442-amino acid extracellular, a 21-residue transmembrane, and a 569-amino acid cytoplasmic domain 76,77,8o (Fig 2). Compared with ANP-RaC(A), there is high homology (74%) in the g.c. and protein kinase domains, but lower homology (44%) in the extracellular domain. The latter finding indicates receptor structural differ-

~

"

,.....

>< .c A B C a.

nnn n

... • ..-.

bp

•

•

310 "

271,281 234

Fig 3. Agarose gel showing the presence of three different natriuretic peptide receptors in human kidney tissue. RNA was extracted from human kidney and subjected to reverse transcription. The cDNA was amplified by the polymerase chain reaction, using specific primer sets designed to yield a product of a certain size. In the first lane (A), primers specific for receptor-A (ANP-RGC(A)) were used, disclosing the expected 600-base pair (bp) fragment of this receptor. In the second lane (B), specific receptor-B primers yielded the expected 228-bp fragment for receptor B (ANP-RGC(B))' A 378-bp specific fragment for receptor C (ANP-Rc) was obtained in lane C. The lane marked Phi X 174 is a standard calibration set of fragments of known basepair length. (Reprinted with permission of S. Canaan-KOhl, R.L. Jamison, B.D. Myers, and R.E. Pratt, "Identification of 'B' receptor for natriuretic peptide in human kidney," Endocrinology, vol 130, issue 1, pp 550-552,1992, © The Endocrine Society.65)

ences, consistent with the different ligand specificities of the two receptors 57 ,64 (see below). These findings are of great interest, since they imply there are two separate regulatory systems in which natriuretic peptides participate. The different tissue distribution of ANP-RaC(A) and ANP-RGC(B) reinforces this idea (Table 1). Wilcox et al found no ANP-RaC(B) mRNA in the Rhesus kidney.82 Recently, however, mRNA for ANP-Roc(B) was identified in human kidney tissue using the RT-PCR technique 65 (Fig 3). Moreover, C-typeNP, which has high affinity for ANP-RGC(B), binds selectively to human glomerular mesangial cells in culture and stimulates the

526

formation of cGMP. 57 The rat kidney contains mRNA for ANP-RoC(B)76 and, as noted above, infusion of C-typeNP in the rat causes diuresis and natriuresis. 58 Taken together, these findings suggest that C-typeNP acts on the kidney via ANP-RoC(B), possibly as a paracrine hormone. Other sites of ANP-RGC(B) mRNA are listed in Table 1.

ANP-Rc ANP-Re is half the size of ANP-Roc(A) and ANP-RoC(Bh with only 496 amino acid residues. 3,5,78,79,88.9o It has an extracellular domain that has 35% homology with ANP-Roc(A) and ANP-Roc(Bh a transmembrane domain, and a very short 37-amino acid cytoplasmic domain with no kinase or cyclase homologous sequences. 79,90 The proposition that ANP-Re functions as a clearance receptor to remove natriuretic peptides from the circulation stemmed from differences between in vitro and in vivo effects of ANF.88 Although there was concordance between ANF binding sites in the renal cortex and its functional effects in the perfused rat kidney, discrepancies were noted between ANF binding kinetics and cGMP formation. In cell cultures, ANF analogues with high affinity for binding sites were weak agonists and did not antagonize ANF-induced increases in cGMP. These sites were postulated to represent an ANF receptor that has only a clearance function. A ring-deleted analogue, C-ANF4-23, was shown to bind to most ANF receptors, but not to affect renal function or antagonize ANF actions in the in vitro perfused rat kidney, ie, it selectively blocked ANPRe. In contrast, C-ANF4•23 induced a decrease in blood pressure, increase in GFR, and natriuresis in the intact rat. This discrepancy between a lack of effect in vitro and an effect in vivo was resolved by the finding that C-ANF4. 23 increased endogenous ANF plasma levels in vivo, presumably by displacing ANF only from clearance receptors without blocking ANF binding to biologically active receptors. 88 Most evidence favors the view that ANP-Re functions to remove ANF from the circulation, and is mainly responsible for the large volume of distribution and rapid metabolic clearance rate of ANF (see below). ANP-Re, accompanied by

JAMISON, CANAAN-KOHL, AND PRATI

its ligand, ANF, was found to be rapidly internalized.91 It was recycled to the surface while the ligand was hydrolyzed in lysosomes. The total population of ANP-Re was recycled every hour in the presence or absence of ANF, that is, the process is constitutive, as it is in other clearance receptors, eg, the low-density lipoprotein receptor,91 rather than inducible. These findings imply that plasma ANF levels cannot regulate ANPR C •3,91 On the other hand, changes in total number of clearance receptors could regulate levels of plasma ANF. For example, Canaan-Kiihl et al92 observed a decrease in the number of ANP-Res per platelet in diabetic glomerulopathy and suggested the effect is to make more ANF available to the biological receptors. However, the view that ANP-Re is only a clearance receptor is not universal. Johnson et al 93 have shown that both ANF and C-ANF4.23 inhibit norepinephrine release from nerve terminals and thereby prevent contraction of the rabbit vas deferens. In pheochromocytoma cells in culture, ANF and C-ANF4-23 inhibited vasoactive intestinal peptide-induced cyclic adenosine monophosphate (cAMP) generation. 94 ANF and C-ANF4. 23 suppressed adenylate cyclase activity in platelets (which contain only ANP_Re95 ), and in rat aorta, brain, anterior pituitary, and adrenal cortical membranes. 96 The effect was blocked by pertussis toxin, which is traditionally a test that indicates coupling to adenylate cyclase by an inhibitory guanine nucleotide-binding protein. In brief, ANP-Re may have a biological as well as a clearance function. The distribution of ANP-Re mRNA in the Rhesus monkey is given in Table 1.82 It was the only natriuretic peptide receptor mRNA found in the monkey's cerebral cortex. ANP-Re mRNA was present throughout the myocardium and endocardium of the right atrium, the major site of ANF synthesis, but was found only in endocardial cells of the left atrium, and right and left ventricles. These intriguing findings in the heart imply that ANF, after secretion from the cardiac myocyte, immediately encounters ANP-Re. At this site, the receptor may serve to minimize fluctuations in circulating ANF.82 It may also directly mediate a biological effect of ANF on the heart. 87,89

NATRIURETIC PEPTIDES AND THEIR RECEPTORS

527

Endopeptidase and Pharmacokinetics ofANF

eated. Of the two receptors that mediate biological actions, the natural ligand of ANP-RcJC(A) is ANF, while that of ANP-Roc(B) is C-typeNP. In view of these differences in ligand specificity and tissue distribution, it has been proposed that ANF and its receptor, ANP-Roc(A). and C-typeNP and its receptor, ANP-RcJC(B), represent two separate natriuretic peptide regulatory systems. The third receptor, ANP-Rc, is a ubiquitous receptor that binds all three natriuretic peptides. Its lack of a cytoplasmic g.c. domain suggests that the principal function of ANP-Rc is to regulate plasma levels of ANF and BNP, but it may also mediate a biological effect. Rapid progress in this field has inevitably raised new questions. What is the biological significance of two separate natriuretic peptide-receptor systems? What actions are regulated by ANP-RcJC(B) and its ligand, C-typeNP? Where is C-typeNP bound in the kidney and what effect does its receptor mediate? What is the function of BNP? Why are ANF and other natriuretic peptides synthesized in the kidney and other non cardiac tissues? Why is the clearance receptor, ANP-Rc , located in the heart adjacent to the site of the principal synthesis and release of ANF? What is the role of natriuretic peptides and their receptors in the central nervous system? Answers to these questions should clarify the physiological and pathophysiological functions of the natriuretic peptides.

ANF is removed from the circulation by ANPRc, glomerular filtration, and endopeptidase, an enzyme that has an extracellular location. 97 The plasma half-time of ANF is approximately 1.5 minutes in the rat,98,99 and 2 to 4 minutes in humans. 3 The volume of distribution of ANF in the rat is very large, ranging from 500 to 970 mL/kg body weight. 97 ,98 Values for the metabolic clearance rate (MCR) varied from 237 to 500 mL/ min/kg body weight. 97 -99 These values indicate ANF is rapidly synthesized and released into the circulation and quickly bound by receptors. Most of the peptide is removed by clearance receptors, since administration of the ANP-Rc blocker, CANF4 _23 , decreased the MCR by 76%,99 while an endopeptidase inhibitor reduced the MCR by only 30% to 33%.98 Infusion of an endopeptidase inhibitor to nephrectomized rats increased the half-life of atriopeptin III (similar to ANF) from 2 to 7.8 minutes,99 indicating that endopeptidase has an extrarenal as well as a renal location. CONCLUSION

The basic hypothesis that has stimulated a great deal of research is that ANF is released from the atrium in response to stretch and acts through biological receptors in the kidney, adrenal gland, and blood vessels to promote diuresis, natriuresis, vasodilatation, and reduction in blood volume. That hypothesis must now be revised. Two new natriuretic peptides, BNP and C-typeNP, have been identified, and three natriuretic peptide receptors with different affinities have been delin-

ACKNOWLEDGMENT The authors wish to express their appreciation to Bryan Myers for his support and advice.

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