A New Member of the Natriuretic Peptide Family Is Present in the

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1 THE JOURNAL OF BIOLOGICAL CHEMISTRY 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Val. 267, No. 2, Issue of July 15, pp ,1992 Printed in U. S. A. A New Member of the Natriuretic Peptide Family Is Present in the Venom of the Green Mamba (Dendroaspis angusticeps)* (Received for publication, February 6, 1992) Hugues Schweitz, Paul Vigne, Danielle Moinier, Christian Frelin, and Michel LazdunskiS From the Institut de Pharmacologie Mokculaire et Cellulaire, 66 Route des Lucioles, Sophia Antipolis 656 Valbonne, France This paper describes the purification, sequence, and biological properties of a 38-amino acid residue peptide from the venom of Dendroaspis angusticeps which shared important sequence homologies with natriuretic peptides. Dendroaspis natriuretic peptide (DNP) relaxed aortic strips that had been contracted by 4 mm KC1 with a potency = 2 nm) similar to that of atrial natriuretic peptide (ANP) and larger than that of C type natriuretic peptide (CNP). The relaxing actions of ANP and DNP (both at 1 nm) were mutually exclusive. Bovine aortic endothelial cells responded to ANP (Ko.~ = 3 nm) and DNP = 3 nm) but not to CNP by a large activation of guanylate cyclase. Rat aortic myocytes showed larger cgmp responses to CNP = 1 nm) than to ANP or DNP = 1 nm). Finally, DNP completely prevented the specific lz6i-anp binding to clearance receptors in cultured aortic myocytes with a potency (& = 1 nm) that was less than that of ANP (& =.3 nm). It is concluded that DNP is a new member of the family of natriuretic peptides and that it recognizes ANPA receptors and clearance, ANPc receptors, but not CNP-specific ANPB receptors. The atrial natriuretic peptide (ANP) plays an important role in the regulation of blood pressure, electrolyte homeostasis and other physiological functions (1, 2). Its major action is mediated by an increased formation of cgmp through the activation of a particulate guanylate cyclase (3,4). Two other peptides with structures similar to ANP have been described recently. Brain natriuretic peptide (BNP) has been purified from brain and then found to be present at high concentrations in the heart (5, 6). The profile of actions of BNP is similar to that of ANP, and the two peptides are thought to recognize a common ANPA receptor that possess guanylate cyclase activity (7, 8). More recently, a third peptide called CNP has been characterized from porcine brain (9). CNP is the most abundant natriuretic peptide in the brain and is not present in peripheral tissues (1,ll). It has lower hypotensive and natriuretic properties than ANP and BNP (9, 11). CNP * This work was supported by the Centre National de la Recherche Scientifique and by Ministere de la DCfense Nationale Grant DRET 9/192. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be sent. Tel.: / 2; Fax: The abbreviations used are: ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; CNP, C type natriuretic peptide; DNP, Dendroaspis natriuretic peptide; HPLC, high performance liquid chromatography; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. recognizes a different receptor subtype called ANPe (12) that is structurally distinct from the ANPA receptor but also possesses a guanylate cyclase domain (13). This paper describes the presence in the venom of the Green Mamba (Dendroaspis angusticeps) of a 38-residue peptide called DNP (for Dendroaspis natriuretic peptide), which is structurally homologous to other natriuretic peptides. Biological properties of DNP were analyzed and compared with those of other natriuretic peptides. EXPERIMENTAL PROCEDURES Materials-The venom from D. angusticeps (Green Mamba) was obtained from Latoxan (Rosans, France). Rat a-anp (1-28) was from Neosystem (Strasbourg, France). Rat brain natriuretic peptide- 45 and porcine C type natriuretic peptide were from the Peptide Institute (Osaka, Japan). Isobutylmethylxanthine was from Sigma. Rat 3-[ 251]iodotyrosyl ANP(1-28) (2, Ci/mmol) was from Amersham. Sephadex G-5 fine and Sephadex G-25 fine were purchased from Pharmacia LKB Biotechnology Inc. DNP was purified by HPLC using a Waters system (pump 51, injector U6K, automated gradient controller and spectrophotometer 481) equipped with a Shimadzu CR3A integrator-recorder. Endolysin fragments of DNP were purified by HPLC using a pump 14A solvent delivery system (Applied Biosystems), a spectrophotometer monitor 5 (LDC Analytical), and a PU 6 integration system (Philips). Amino Acid Sequence Analysis-The peptide (1 pg) was reduced with 2-mercaptoethanol and pyridilated with 4-vinylpyridine (14). The reaction product was immediately desalted by chromatography onto a reverse phase support (Aquapore RP-3, 2.1 X 3 mm, Brownlee) and eluted as a single peak. Sequence information was obtained using an Applied Biosystems sequenator (model 477A) equipped with an on-line phenylthiohydantoin analyzer (model 12OA). Endoproteinuse Lys-C Treatment-About 1 pmol of peptide, dissolved into 5 pl of.5 M sodium phosphate buffer (ph 7.8) were treated for 48 h at room temperature with 1 pmol of endoproteinase Lys-C (Promega Corporation, Madison, WI). The reaction mixture was lyophilized, dissolved into 1 p1 of.1% trifluoroacetic acid, and loaded onto an Aquapore RP-3 column. Elution was performed using a trifluoroacetic acid/acetonitrile/water solvent system ( A.1% trifluoroacetic acid; B:.1% trifluoroacetic acid, 7% acetonitrile) and a gradient program (.5% of B/min at a flow rate of 1 pl/min). The two major peaks were collected, sequenced, and their mass determined by desorption mass spectrometry. Contraction Experiments-Thoracic aortae were isolated from 2- month-old female Wistar rats, cut into 5-mm strips, and the intima was denuded. Arterial rings were mounted in an organ chamber and equilibrated at 37 C for 1 h in a Krebs-Ringer solution (12 mm NaC1, 4.8 mmkc1, 1.2 mmcac12, 1.3 mm MgS4, 25 mm NaHC3, 1.2 mm K2HP4, 5.8 mm glucose) gassed with 95% 1, 5% CO,. Strips under 2 g of resting tension were conditioned by two contraction relaxation cycles with 4 mm KCI, contracted by a third application of 4 mm KC1, and then relaxed with different combinations of natriuretic peptides. Aortic strips were fixed to a UL5 microbalance screwed to a UC2 isometric force transducer which was connected to a model 5 amplifier (Gould). Changes in isometric tension were recorded on a Gould 26 polygraph. The cumulative dose-response curves for the relaxing actions of DNP and ANP were performed in the presence of 1 p~ captopril and 1 p~ phosphoramidon. Cell Cultures-Cultures of rat aortic myocytes were prepared as 13928

2 described previously (15). Cells were grown in M199 medium (GIBCO) supplemented with 2% fetal calf serum (Dutscher, Strasbourg, France), 1 units/ml penicillin, and 1 pg/ml streptomycin. Cultures were dissociated using.1% trypsin and passaged weekly. Cells at passages 2-1 were used. Bovine aortic endothelial cells were prepared as described previously (16) and grown in Dulbecco's modified Eagle's medium supplemented with 1% fetal bovine serum and antibiotics. Measurements of cgmp-confluent cell layers in 12-well Falcon plates were washed twice with an Earle's salt solution (14 mm NaC1, 5 mm KCl, 1.8 mm CaC12,.8 mm MgS4, 5 mm glucose buffered at ph 7.4 with 25 mm Hepes/Tris) and further incubated for 6 min in the same solution supplemented with.3 mm isobutylmethylxanthine at 37 "C. Cells were then stimulated with the peptides. After 6 min, the incubation solution was aspirated off, and cells were treated with an ethanol, 5 mm EDTA (2:l) solution for 3 min at 4 "C. Extracts Dendroaspis Natriuretic Peptide were lyophilized, and cgmp was assayed by radioimmunoassay using reagents provided by Amersham. Cell proteins were determined according to Bradford (17) using bovine serum albumin as standard. Data are expressed in pmol of cgmp/6 min/mg of protein. Binding Assays-Confluent layers of aortic myocytes in 12-well Falcon plates were washed twice with an Earle's salt solution and further incubated at 25 "C in the same solution supplemented with 2 mg/ml bovine serum albumin,.2% sodium azide, 1 p~ captopril, 1 p~ phosphoramidon, and the desired concentrations of peptide. After 3 min of equilibration, 9-ANP (9 PM final) was added to each well, and the incubation was allowed to proceed for an additional 8 min. Cells were then washed three times with 1 ml of Earle's salt solution, digested into.1 N NaOH, and the cell-associated radioactivity was determined. Means & S.E. are indicated. RESULTS Purification of DNP-The crude venom (536 mg) was dissolved into 7.5 ml of 1% acetic acid and loaded onto a Sephadex G-5 column (3 x 4 cm). The material was eluted using 1% acetic acid at a flow rate of 7 ml/h. The fraction corresponding to the second peak of absorbance (88% of the starting material) was recovered and further purified by HPLC using a cation exchange column eluted with a linear gradient of ammonium acetate. Fig. 1 shows the elution profile that had been obtained. The fraction that eluted at 3% ammonium acetate (peak E) relaxed KC1-contracted aortic strips. This fraction was lyophilized and dissolved into 3 ml of a solution consisting of.1% trifluoroacetic acid and.1% triethylamine in water. The material was loaded onto a C-18 reverse phase column and the column eluted with a discontinuous acetonitrile gradient. Fig. 2 shows the elution profile that had been obtained. Most of the vasorelaxing activity was associated with the overlined peak eluting at 3% acetonitrile. The fraction was lyophilized and desalted by chromatography on a Sephadex G-25 fine (18 x 2.6 cm) column using.1% acetic acid as eluent. The final yield was 1.2 mg, i.e..22% of total venom components. 5 1W FlelenUcm hme (mm) FIG. 1. Purification of DNP from the venom of D. angusticeps. HPLC profile of elution of a crude venom preparation. The column was a Beckman TSK SP 5PW column (21.5 X 15 mm), equilibrated with 1% acetic acid adjusted at ph 3. with ammonia (solution A). A linear gradient of solution A to solution B (1 M ammonium acetate) in 1 min was applied. The flow rate was 8 ml/ min. Peak E showed a vasorelaxing action and was used for further purification. 2 4 Retention time (min) s!e 28 ae FIG. 2. Reverse phase HPLC of the active fraction from Fig. 1. A Spherosorb ODSl5 pm Interchrom column (25 X 4.1 mm) was eluted at a flow rate of 1 ml/min with linear gradients of acetonitrile containing.1% trifluoroacetic acid and.1% triethylamine (22-33% for 25 min, 33-4% for 1 min, and 4% for 15 rnin). The vasorelaxing fraction is overlined. Structure of DNP-The amino acid sequence of the vasorelaxing peptide, as determined by automated sequence analysis, is shown in Fig. 3. The sequence was confirmed by sequencing and mass analysis of the peptides generated by endolysin treatment. Fig. 3 compares the structures of the peptide isolated from the venom of the Green Mamba with those of natriuretic peptides. Remarkable similarities are observed with ANP, BNP, and CNP. Eleven residues are shared with CNP, 12 with human a-anp, 13 with rat a-anp, 14 with human BNP, and 15 with rat BNP. In comparison human ANP and BNP have 14 residues in common, and rat ANP and BNP have 16 residues in common. For these reasons, the peptide isolated was named Dendroaspis natriuretic peptide. Most homologies between DNP and other natriuretic peptides were observed in the 17-residue sequence that separates the two cysteine residues. In the N-terminal parts of the sequence, only Val-2 was common to DNP and human BNP. DNP a 15-residue has C terminal extension beyond the second cysteine residue. A 6-residue extension is found in both ANP and BNP. No extension is found in CNP. Homologies concerning amino acids 25 and/or 26 and/or 27 were found in ANP, BNP, and DNP. Finally, the structure of DNP is characterized by the presence of 4 proline residues, 3 of them being clustered in the C-terminal part of the molecule. No proline is found in other natriuretic peptides. The Relaxing Action of DNP-The addition of 1 nm DNP to rat aortic strips that had been precontracted with 4 mm KC1 resulted in a rapid relaxation (Fig. 4). A similar action was observed with ANP (Fig. 4). The maximum relaxation produced by 1 nm DNP was 81 f 7% (n = 5). An identical value was observed for 1 nm ANP (79 k 7%, n = 5). Moreover, addition of 1 nm ANP to aortic strips that had been relaxed with 1 nm DNP did not promote a further relaxation (Fig. 4). Conversely, DNP (1 nm) had no action on aortic strips that had already been relaxed with 1 nm ANP (Fig. 4). The vasorelaxing action of CNP was much less pronounced than those of ANP and DNP (Fig. 4). Addition of either ANP or DNP to aortic strips that been had partially relaxed with 1 nm CNP induced a further relaxation (Fig. 4). The dose-response curves for the relaxing action of the different natriuretic peptides are presented in Fig. 5. values were observed at 1 and 2 nm for ANP and DNP, respectively. CNP, at a dose of 3 nm, only produced a 2% relaxation. DNP had no relaxing action on aortic strips that had not been precontracted with KC1. It had no action on the contractility of isolated rat atria (data not shown). The Action of DNP on Guanylate Cyclase-Natriuretic peptides produce a vasorelaxation by stimulating a particulate guanylate cyclase (3, 4). Incubation of cultured aortic myocytes for 6 min with 1 FM DNP in the presence of.3 mm

3 1393 Dendroaspis Natriuretic Peptide FIG. 3. Compared amino acid se DNP E V K Y D P C I G H K I D R I N H V S N L G C P S L R D P R P N A P S T S A quences of DNP, human ANP and H-MP S L R R S S C P G G R M D R I G A Q S G L G C N S F R Y BNP, rat ANP and BNP, and por- K-ANP S L R R S S C P G G R I D R I G A Q S G L G C N S F R Y cine CNP. ~~i~~ acid residues ofdnp H-BNP S P K M V Q G S G C P G R K M D R I S S S S G L G C K V L R R H r-bnp N S K M A H S S S C I G Q K I D R I G A V S R L G C D G L R L F that are common to other natriuretic CNP G L S K G C I G L K L D R I G S M S G L G C peptides are shown in bald. 2 minutes FIG. 4. Relaxing actions of ANP, DNP, and CNP on rat aortic strips. Aortic strips were precontracted with 4 mm KC1 and then treated with natriuretic peptides (1 nm) as indicated. greater efficacy (Fig. 6). In the absence of isobutylmethylxanthine, only CNP increased cgmp levels in a significant manner. An 8-fold stimulation by 1 p~ DNP of guanylate cyclase was observed in aortic myocytes of the A7r5 cell line. The KO., value for DNP action in A7r5 cells was 1 nm (data not shown). The action of DNP on the formation of cgmp was also analyzed in bovine aortic endothelial cells with different results (Fig. 7). In aortic endothelial cells, ANP was much more potent than CNP for stimulating guanylate cyclase, indicating the presence of a receptor site distinct from the one present in aortic myocytes. In these cells, low concentrations (.1-1 nm) of DNP induced large (up to 16-fold) increases in cgmp. The dose-response curve for DNP action was superimposable that of ANP (Fig. 7). The value for ANP and DNP actions was 3 nm, i.e. a value 3 times lower than the corresponding value found in aortic myocytes (1 nm, Fig. 6). The Action of DNP on lz51-anp Binding to Aortic Myocytes-Fig. 8 presents the results of binding experiments in which cultured aortic myocytes were exposed to * I-ANP in the presence of increasing concentrations of unlabelled ANP or DNP. DNP prevented the binding of I-ANP to cultured aortic myocytes with a KO, value of 1 nm. In comparison, LOG [PEPTIDE (M)] FIG. 5. Cumulative dose-response curves for the relaxing actions of ANP, CNP, and DNP. Rat aortic strips were precontracted with 4 mm KCl., DNP;, ANP; M, CNP. Means + S.E. (n = 4) are shown uxi [DNP(M)1 - uxi PEPTIDE (M)] FIG. 6. Dose-response curves for the actions of natriuretic peptides on the formation of cgmp by cultured aortic myocytes. Left panel: DNP; right panel: M, ANP;, BNP;, CNP. Experiments were performed as described under Experimental Procedures. Means of duplicates in a typical experiment are shown..e3 B e a +I r LOG [PEPTIDE (M)] FIG. 7. Dose-response curves for the actions of natriuretic peptides on the formation of cgmp by cultured bovine aortic endothelial cells. Experiments were performed as described under Experimental Procedures., ANP; W, CNP;, DNP. Means of duplicates in a typical experiment are shown. isobutylmethylxanthine resulted in an 11-fold increase in cgmp levels from.43 k.1 to 4.81 f.83 pmol/mg of protein (n = 3). In comparison, increases in cgmp formation were 29-, 17-, and 61-fold for ANP, BNP, and CNP, respectively (all at 1 p ~). The dose-response curves for the actions of the different natriuretic peptides on cgmp formation are shown in Fig. 6. ANP, BNP, and DNP acted in the same - LOG [PEPTIDE (M)] range of concentrations, between 1 nm and 1 pm with a FIG. 8. ANP and DNP prevent lz5i-anp binding to cultured value of 1 nm. CNP = 1 nm) was 1 times more aortic myocytes. Experiments were performed as described under potent than other natriuretic peptides. CNP also showed a Experimental Procedures., ANP; and, DNP.

4 Dendroaspis Natriuretic Peptide the value for ANP was.3 nm (Fig. 8). The concentration that they bind to a 7-kDa receptor in aortic smooth muscle of 12,51-ANP used in these experiments (9 PM) being much cells (ll), corresponding to the size of a receptor with no lower than the Kd value of ANP for its receptor (.3 nm), the KO, value determined for DNP is close to the true Kd value. guanylate cyclase domain. Therefore, binding experiments identify clearance ANPc receptors, the overwhelming majority of total receptor sites for natriuretic peptides. DISCUSSION As a vasorelaxant, DNP = 2 nm) was about as potent This paper describes the isolation and structural character- as ANP = 1 nm) and much more potent than CNP ization of a peptide from the venom of D. angwticeps which is homologous to natriuretic peptides. The peptide relaxed (Fig. 5), suggesting that the vasorelaxing action of DNP is likely to be mediated by ANPA receptors rather than by ANPB aortic strips that had been precontracted with KC1 (Fig. 4); it receptors. stimulated guanylate cyclase in cultured aortic myocytes (Fig. DNP stimulated guanylate cyclase in both cultured aortic 6) and in bovine aortic endothelial cells (Fig. 7), and it myocytes (Fig. 6) and in bovine aortic endothelial cells (Fig. prevented the binding of 'T-ANP to aortic myocytes (Fig. 8). 7). However, although the potency of DNP was similar to Thus, structural, physiological and biochemical data indicate that of ANP in the two cell types, the value for its action that the peptide isolated from the venom of D. angusticeps on cgmp formation was much lower in aortic endothelial has all characteristics of a natriuretic peptide. cells = 3 nm, Fig. 7) than in cultured aortic myocytes Three distinct types of receptor sites for natriuretic peptides = 1 nm, Fig. 6). Knowing that CNP is a selective have been identified, cloned, and their properties analyzed in agonist of ANPB receptors (12) and that CNP a is more potent transfected cells (12). ANPA receptors are specific for ANP agonist of guanylate cyclase in aortic myocytes (Fig. 6) than and BNP (7, 8). They are responsible for the natriuretic, in endothelial cells (Fig. 7), these resultsuggest that DNP is diuretic, and hypotensive effects of natriuretic peptides. a potent agonist of ANPA receptors and a poor agonist of ANPR receptors are more selective for CNP. They recognize CNP with a 5-5 higher affinity than ANP or BNP, respectively (12, 13). Another class of receptors, ANPc receptors, recognize all natriuretic peptides; but unlike otherecep- tors, they lack an intracellular domain with guanylate cyclase activity (18) and act as clearance receptors (19, 2). There is no obvious evidence from sequence data that DNP is more closely related to either ANP or CNP, except for the poorly conserved short segment situated in the C-terminal extension that is found in ANP, BNP, and DNP but not in CNP. Yet physiological and biochemical data presented in this paper provide some information about the specificity of interaction of DNP with the different receptor sites for natriuretic peptides. Contraction experiments merely assay the functions of ANPA receptors. CNP is well known to have much lower diuretic/natriuretic and hypotensive actions in rat than ANP or BNP (9). It is less potent than ANP for relaxing aortic strips that have been precontracted with KC1 (Fig. 5) or with norepinephrine (11). The sites identified from measurements of guanylate cyclase differ in different cell types. Activation by natriuretic peptides of guanylate cyclase was analyzed in two different cell types, rat aortic myocytes and bovine aortic endothelial cells, and has led to two different series of results. In aortic smooth muscle cells, the rank order of potency of natriuretic peptides for inducing the formation of cgmp was CNP > > ANP > BNP (Fig. 6) as also recently reported by Furuya et al. (21). In aortic endothelial cells, the profile of activation is different, ANP being much more potent than CNP (Fig. 7). A comparison of these data with results of transfection experiments using cloned ANPA or ANPR receptors (12) indi- cates that aortic endothelial cells express ANPA receptors, whereas cultured aortic myocytes mainly express CNP specific ANPB receptors. The apparent discrepancy between contraction experiments, which show a greater vasorelaxing action of ANP (Fig. 4), and cgmp measurements, which show that CNP was a more potent activator of guanylate cyclase in aortic myocytes (Fig. 6, Ref. 21), can be easily explained if after in vitro transplantation, aortic myocytes acquire ANPB receptors. In favor of this hypothesis Furuya et al. (21) ob- served that in intact aortic strips, CNP was much less potent than ANP for raising cgmp levels. What are the sites identified in binding experiments? Cross-linking experiments with Iz5I-ANP and T-CNP show ANPB receptors. Since the value for DNP action on cgmp formation via ANPA receptors (K.5 = 3 nm, Fig. 7) is close to the Koa value observed for its vasorelaxing action (2 nm, Fig. 5), the formation of cgmp in response to the occupancy of ANPA receptors is likely responsible for the vasorelaxing action of DNP. Finally, the interaction of DNP with clearance, ANPc receptors, defined using T-ANP binding experiments, shows that the affinity of DNP for clearance receptors (1 nm) was less than that of ANP (.3-1 nm, Fig. 8, Refs. 11 and 22), BNP (.5 nm, Ref. 23), and CNP (4 nm, Ref. 11). Sequence homologies among ANP, BNP, CNP, and DNP are mainly found in the 17-amino acid residue sequence that separates the 2 cysteine residues. This highly conserved sequence forms a loop structure that is thought to be important for the biological activity of the peptides. No sequence ho- mology is found in the N-terminal parts of the molecules. ANP and BNP have a 6-residue-long terminal extension that follows the 2nd cysteine residue. This extension is 15 residues long in DNP. CNP has no C-terminal extension. The observation that the biological properties of DNP are similar to those of ANP and BNP and unlike those of CNP might suggests that the C-terminal part of natriuretic peptides is important for the recognition of ANPA receptors. It should be noted, however, that only very limited structural homologies are observed in the C-terminal parts of ANP, BNP, and DNP. Envenomation is usually associated with a local vasodilatation and an increased capillary permeability that may facilitate a rapid diffusion of toxic substances of the venom. One mechanism by which vasodilatation is achieved is via proteolytic enzymes that release bradykinin in the bloodstream (24). Bradykinin acts on vascular endothelial cells and promotes the liberation of nitric oxide, a potent activator of soluble guanylate cyclase in vascular smooth muscle cells (25, 26). Natriuretic peptides provide another means of achieving the same action. DNP may diffuse to vascular smooth muscle cells, activate particulate guanylate cyclase, hence resulting in a vasodilatation. The vasorelaxing action of DNP may be further potentiated by the relatively low affinity of the peptide for clearance receptors. There is a 3-fold difference between the Kd value of ANP for clearance receptors (.3 nm, Fig. 8) and the value for ANP induced relaxations (1 nm, Fig. 4). There is only a 2-fold difference between the Kd value of DNP for clearance receptors (1 nm, Fig. 8) and KO.h the value for its vasorelaxing action (2 nm, Fig. 4). As a consequence,

5 13932 Dendroaspis Natriuretic Peptide clearance receptor sites are likely to be less limiting for DNP action than for ANP action. Natriuretic peptides have been identified in lower vertebrates such as amphibia and fish (27), but their functions are still unknown. It would be interesting to know whether DNP is only produced in the venom of D. angusticeps or also serves normal (presumably natriuretic and hypotensive) functions for the snake. It has been shown years ago that endogeneous equivalent of bee venom toxins exist in mammalian brain (28,29). It has been shown later that sarafotoxin (3), a potent vasoconstricting peptide from snake (Atructaspis engaddensis) venom, and endothelins (31) from endothelial cells are closely related peptides. It is now particularly interesting to observe that vasorelaxing mammalian natriuretic peptides also have structural and functional equivalents in snake venom. Acknowledgments-We are grateful to Dr. P. Paroutaud (Applied Biosystems) for advice in sequence analysis; to H. Drobecq and Dr. A. Tartar (Institut Pasteur, Lille) for mass analysis; and to N. Boyer, V. Friend, and C. Roulinat for expert technical assistance. REFERENCES 1. Genest, J., and Cantin, M. (1987) Circuhtion 76, (Suppl. l), De Bold, A. J. (1985) Science 23, Waldman, S. A., Rapoport, R. M., and Murad, F. (1984) J. Biol. Chem. 269., ~ ~." "" ~ 4. Wkquist, R. J., Faison, E. P., Waldman, S. A,, Schwartz, K., Murad, F., and Rapoport, R. M. (1984) Proc. Natl. Acad.Sci. U. S.A. 81, Sudoh, T., Kangawa, K., Minamino, N., and Matsuo, H. (1988) Nature 332, Kambayashi, Y., Nakao, K., Mokoyama, M., Saito, Y., Ogawa, Y., Shiono, S., Inouye, K., Yoshida, N., and Imura, H. (199) FEBS Lett. 269, Schultz. S.. Sineh. S.. Bellet. R. A.. Sineh. G.. Tuhb. D. J., Chin.. H.,. and Garbers,'D. Ly (1989) Cell 68,1155-lf62 8. Lowe, D. G., Chang, M. S., Hellmiss, R., Chen, E., Singh, S., Garbers, D. L., and Goeddel, D. V. (1989) EMBO J. 8, Sudoh, T., Minamino, N., Kangawa, K., and Matsuo, H. (199) Biochem. Biophys. Res. Commun. 168, Koiima. M.. Minamino. N.. Kangawa. K.. and Matsuo. H. (199) Biochem. hiophys. Res. Commun. 168, g ' 11. Furuya, M., Takehisa, M., Minamitake, Y., Kitajima, Y., Hayashi, Y., Ohnuma, N., Ishihara. T., Minamino. N.. Kangawa, K., and Matsuo, H. (199) Biochem. Biophys. Res. Commun. 17, Koller, K. J., Lowe, D. G., Bennett, G. L., Minamino, N., Kangawa, K., Matsuo, H., and Goeddel, D. V. (1991) Science 262, Chang, M. S., Lowe, D. G., Lewis, M., Hellmiss, R., Chen, E., and Goeddel, D. V. (1989) Nature 341, Andrews, P. C., and Dixon, J. E. (1981) J. Biol. Chem. 266, Marsault. R.. Viene. P.. Breittmaver. J. P.. and Frelin. C. (1991) Am. J. " I I,,, Physioi. 261, e986-c Vigne, P., Marsault, R., Breittmayer, J.-P., and Frelin, C. (1989) Biochem. J. 266, Bradford, M. M. (1976) Anal. Biochem. 72, Fuller, F., Porter, J. G., Arfsten, A. E., Miller, J., Schilling, J. W., Scarboroueh. R.M..Lewicki. J. A,. and Schenk. D. B. (1988) J. Biol. Chem. 26-3,' Maack, T., Suzuki, M., Almeida, F. A., Nussenzveig, D., Scarborough, R. M., McEnroe, G. A,, and Lewicki, J. A. (1987) Science 238, Nussenzveig, D. R., Lewicki, J. A,, and Maack, T. (199) J. Biol. Chem. 266, Furuya, M., Yoshida, M., Hayashi, Y., Ohnuma, N., Minamino, N., Kangawa, K., and Matsuo, H. (1991) Biochem. Biophys. Res. Commun. 177, Chahrier, P. E., Roubert, P., Lonchampt, M. O., Plas, P., and Braquet, P. (1988) J. Biol. Chem. 263, Song, D. L., Kohse, K. P., and Murad, F. (1988) FEBS Lett. 232, Diniz, C. R. (1968) in Venomous Animals and Their Venoms (Bucherl, W., Bucklev. E. E.. and Deulofeu., V.., eds). Vol. 1.. KID Academic Press, New York 25. Moncada, S., Palmer, R. M., and Higgs, E. A. (1991) Phnrmacol. Reu. 43, Vanhoutte, P. M., Rubanyi, G. M., Miller, V. M., and Houston, D. G. (1986) Annu. Reu. Physiol. 48, Takei, Y., Takahashi, A,, Watanabe, T. X., Nakajima, K., and Sakakibara, S. (1991) FEBS Lett. 282, Fosset, M., Schmid-Antomarchi, H., Hugues, M., Romey, G., and Lazdunski M. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, Chedbini, E., Ben Ari, Y., Gho, M., Bidard, J.-N., and Lazdunski, M. (1987) Nature 328, Wollberg, Z., Shabo-Shina, R., Intrator, N., Bdolah, A., Kochva, E., Shavit, G., Oron, Y., Vidne, B. A., and Gitter, S. (1988) Tonicon 26, Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., and Masaki, T. (1988) Nature 332,

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