Modulation of mannose receptor activity by proteolysis.

The binding, internalization and degradation of 200 pM monoiodinated human atrial natriuretic factor-(99-126) (125I-hANF) by cultured bovine aortic endothelial cells (BAECs) were studied at 37 degrees C. 125I-hANF was rapidly cleared from the extracellular medium (t1/2 approximately 10 min), whereas preincubation of the cells in the presence of 20 mM-NH4Cl or 0.2 mM-chloroquine resulted in a significant inhibition of this process. The BAECs rapidly produce three major degradation products of 125I-hANF, namely [125I]iodotyrosine 126 (125I-Y), Arg125-[125I]iodotyrosine126 (125I-RY) and Phe124-Arg125-[125I]iodotyrosine126(125I-FRY), which were detected in the extracellular medium. NH4Cl and chloroquine acted to inhibit the generation of 125I-Y and 125I-RY, but not that of 125I-FRY. Furthermore, excess unlabelled hANF (300 nM) completely blocked the rapid production of 125I-Y and 125I-RY in the first 5 min, but only partially (49%) inhibited the generation of 125I-FRY. Thus, in contrast with our previous findings with cultured smooth-muscle cells [Johnson, Arik & Foster (1989) J. Biol. Chem. 264, 11637-11642], BAECs bind, internalize and rapidly degrade 125I-hANF, resulting in the release of 125I-Y and 125I-RY into the extracellular medium. Similarly to smooth-muscle cells, the BAECs generate 125I-FRY from 125I-hANF via an extracellular proteolytic event. The rapidity of the receptor-mediated process and its sensitivity to NH4Cl and chloroquine suggest that the 125I-hANF is proteolytically processed in the endosomes of BAECs and that its receptors cycle between the cell surface and intracellular stores.     

Partial characterization of a metalloendopeptidase activity produced by cultured endothelial cells that removes the COOH-terminal tripeptide from 125I-atrial natriuretic factor

The presence of the COOH-terminal region of human atrial natriuretic factor-(99-126) (hANF) is necessary for the full expression of its biological activity. Here, we report on the partial characterization of a proteolytic activity in the conditioned medium from cultured bovine aortic endothelial cells that cleaves the Ser123-Phe124 bond of 125I-hANF generating the COOH-terminal tripeptide. The concentrated conditioned medium was fractionated by gel filtration high performance liquid chromatography and fractions were assayed for the ability to generate the COOH-terminal tripeptide from 125I-hANF. This analysis indicated that the protein responsible for this activity had an approximate molecular weight of 200,000 daltons. Of 16 protease inhibitors tested, only 1,10 phenanthroline, EDTA, EGTA and N-ethylmaleimide significantly inhibited the endopeptidase activity. Thus, we conclude that cultured bovine aortic endothelial cells produce a potentially novel phosphoramidon-insensitive metalloendopeptidase that removes the COOH-terminal tripeptide from 125I-hANF.     

Identification and characterization of atrial natriuretic factor receptors in the rat retina

The characteristics of atrial natriuretic factor (ANF) receptors where studied in rat retinal particulate preparations. Specific 125I-ANF binding to retinal particulate preparations was greater than 90% of total binding and saturable at a density (Bmax) of 40 +/- 8 fmol/mg protein with an apparent dissociation constant (Kd) of 6.0 +/- 2.0 pM (n = 3). Apparent equilibrium conditions were established within 30 min. The Kd value of 125I-ANF binding calculated by kinetic analysis was 4.0 pM. The Bmax of 60 +/- 10 fmol/mg protein and the Kd of 5 +/- 2 pM, calculated by competition analysis, were in close agreement with the values obtained from Scatchard plots or kinetic analysis. The 125I-ANF binding to retinal particulate preparations was not inhibited by 1 microM concentration of somatostatin, vasopressin, vasoactive intestinal peptide, adrenocorticotropin, thyrotropin releasing hormone, or leu-enkephalin. The rank order of potency of the unlabelled atrial natriuretic peptides for competing with specific 125I-ANF (101-126) binding sites was rANF (92-126) greater than rANF (101-126) greater than rANF (99-126) greater than rANF (103-126) greater than Tyro-Atriopeptin I greater than hANF (105-126) greater than rANF (1-126). Similar results have been obtained in peripheral tissues and mammalian brain, indicating that central and peripheral ANF-binding sites have somewhat similar structural requirements. Affinity cross-linking of 125I-ANF to retinal particulate preparations resulted in the labelling of two sites of molecular weight 140 and 66 kDa, respectively. This demonstration of specific high-affinity ANF receptors suggests that the peptide may act as a neurotransmitter or neuromodulator in the retina.     

Binding sites of atrial natriuretic peptide in tree shrew adrenal gland

Adrenal gland binding sites for atrial natriuretic peptide-(99-126) (ANP) were quantitated in tree shrew (Tupaia belangeri) by incubation of adrenal sections with (3-[125I]-iodotyrosyl28) atrial natriuretic peptide-(99-126), followed by autoradiography with computerized microdensitometry. In the adrenal glands, there are three types of ANP binding sites. One is located in the zona glomerulosa (BMax 84 +/- 6 fmol/mg protein; Kd 122 +/- 9 pM); the second in the zona fasciculata and reticularis (BMax 29 +/- 2 fmol/mg protein; Kd 153 +/- 6 pM) and the third in the adrenal medulla (BMax 179 +/- 1 fmol/mg protein; Kd 70 +/- 2 pM). Besides the influence of ANP on the regulation of adrenocortical mineralcorticoid and glucocorticoid secretion our findings raise the possibility for a local site of action of atrial natriuretic peptide in the regulation of adrenomedullary catecholamines in the tree shrew, primates and man.     

Atrial Natriuretic Peptide Modulates Amiloride-Sensitive Na+ Transport Across the Blood-Brain Barrier

We obtained evidence that amiloride specifically potentiates 125I-labeled alpha-rat atrial natriuretic peptide (1-28) [atrial natriuretic peptide (ANP)-(99-126); rANP] binding to cerebral capillaries isolated from the rat cerebral cortex. The binding parameters, KD of 173 pM and Bmax of 159 fmol/mg of protein, became 33 pM and 88 fmol/mg of protein, respectively, when 10(-4) M amiloride was added to the incubation medium. When the effect of rANP was investigated on in vitro 22Na+ uptake into isolated cerebral capillaries, 10(-7) M rANP significantly inhibited the uptake in the presence of 1.0 mM ouabain, 1.0 mM furosemide, and 2.0 mM LiCl in the uptake buffer, a finding suggesting a specific inhibitory effect of rANP on amiloride-sensitive Na+ transport. Thus, the possibility that ANPs control amiloride-sensitive Na+ transport at the blood-brain barrier by interacting with specific receptors has to be considered.     

Degradation of human atrial natriuretic peptide by human brain membranes

Human atrial natriuretic peptide (Ser 99-Tyr 126) was rapidly degraded by both choroid plexus and hypothalamic membranes with a complex pattern of cleavage. The use of protease inhibitors allowed a preliminary characterization of the enzymes involved in the hydrolysis of the Ser-Phe and Phe-Arg bonds of iodine-labelled atrial natriuretic peptide.The C-terminal tripeptide was generated by three different enzymatic activities acting on the Ser-Phe bond: endopeptidase 24.11, a phosphoramidon-insensitive metallopeptidase and a thiol protease. Peptides like substance P, neurotensin, bradykinin inhibited the cleavage of the Ser-Phe bond of atrial natriuretic peptide. The C-terminal tripeptide was further degraded by aminopeptidases. Cleavage of the C-terminal dipeptide was inhibited by aprotinin, suggesting the contribution of brain kallikrein in the formation of this metabolite.These results show that many different proteases were involved in the hydrolysis of the C-terminal sequence of atrial natriuretic peptide, at least in vitro and underline the complexity of neuropeptide catabolism by brain preparations.     

The post-translational processing of rat pro-atrial natriuretic factor by primary atrial myocyte cultures

Primary cultures of neonatal rat atrial myocytes were maintained in two different serum-free media for up to 25 days. Reversed-phase high performance liquid chromatography coupled with atrial natriuretic factor (ANF)-specific radioimmunoassay demonstrated that the cultures maintained in our previously described serum-free medium (Glembotski, C.C., and Gibson, T. R. (1985) Biochem. Biophys. Res. Commun. 132, 1008-1017) secreted primarily ANF-(1-126)-like material, whereas those cultures maintained in a different formulation of medium secreted mostly ANF-(99-126)-like material. Cultures that secreted ANF(99-126)-like material were biosynthetically labeled with [35S]cysteine followed by immunoprecipitation of secreted ANF and analysis by reversed-phase, size exclusion, and ion-exchange high performance liquid chromatography. The labeled ANF-(99-126)-like peptide was shown to be chromatographically indistinguishable from other synthetic peptides related to ANF-(99-126). Labeled ANF purified from extracts of the cultured cells was chromatographically indistinguishable from authentic ANF-(1-126), and could be cleaved specifically by thrombin into labeled ANF-(99-126)-like material. These results indicate that primary atrial myocytes maintained under certain serum-free conditions are capable of secreting ANF-related material that is chromatographically indistinguishable from ANF-(99-126), the known circulating form of the hormone. Additional preliminary studies suggest that the presence of glucocorticoids in the culture medium may confer ANF processing ability on cultured myocytes.     

Atrial natriuretic peptide, ANP(99-126), receptors in rat thymocytes and spleen cells

A single class of saturable, specific binding sites for the circulating form of atrial natriuretic peptides, ANP(99-126), was identified in rat thymus and spleen and in isolated thymocytes and spleen cells using quantitative autoradiographic techniques. In the thymus, the relative potency of ANP analogs to inhibit [125I] ANP(99-126) binding was ANP(99-126) = ANP(103-126) greater than ANP(111-126) greater than ANP(103-125). ANP(103-123) could not displace [125I]ANP(99-126) binding. Addition of ANP(99-126) stimulated the formation of cyclic GMP in isolated thymocytes and spleen cells in a dose-dependent manner. Our results indicate that immune cells have specific ANP receptors which could be coupled to guanylate cyclase activation and may play a role in the regulation of the immune response.     

Clearance receptor-binding atrial natriuretic peptides inhibit mitogenesis and proliferation of rat aortic smooth muscle cells.

The current studies were designed to explore the effects of C-receptor-binding atrial natriuretic peptide analogues on serum-induced mitogenesis in cultured rat aortic smooth muscle cells. To this end, rANF99-126 and a series of truncated (rANF103-126, rANF103-125), ring-deleted (des[Gln116, Ser117, Gly118, Leu119, Gly120]rANF102-121-NH2 (c-ANF) and linear des(Cys105, Cys121)rANF104-126 peptide analogues were used. The latter two peptides have been reported to be selective for the ANF-C receptor. In cells subcultured between passage 3 to 19, rANF99-126, rANF103-126, and rANF103-125 concentration-dependently (0.1-1000 nM) inhibited serum-induced (3H) thymidine incorporation with maximal inhibition observed at 1 microM for each peptide (approximately 40, 31 and 56%) respectively. Furthermore, des[Cys105, Cys121]rANF104-126 inhibited serum-induced (3H)thymidine incorporation concentration-dependently without altering basal or elevated cellular cAMP or cGMP levels. Moreover, the reduction in thymidine incorporation was associated with inhibition of serum-induced clonal cell proliferation. In contrast, c-ANF failed to inhibit serum-induced mitogenesis, yet at a concentration of 100 nM it antagonized the antimitogenic effects of des[Cys105, Cys121]rANF104-126 or rANF99-126 without having any effect on basal or elevated cellular cyclic nucleotide levels. We conclude that the antimitogenic effect of atrial peptides is mediated through interaction with the ANF-C receptor and may be independent of changes in cellular cyclic nucleotide levels.     

Atrial natriuretic factor-induced cGMP accumulation in rat anterior pituitary cells in culture is not coupled to hormonal secretion

While atrial natriuretic factor (ANF) does not influence ACTH secretion, it was reported to have a marked stimulatory effect on the intracellular accumulation of cGMP in rat anterior pituitary cells in culture. Since many biological actions of ANF appear coupled to its excitatory action on target cell guanylate cyclase, the current study was designed to characterize the ANF-induced cGMP response in anterior pituitary with a view to determining whether the nucleotide plays a regulatory role in the secretory function of this gland. A 3 min exposure of cells in primary culture to 300 nM ANF (99-126) or 100 microM sodium nitroprusside (SNP), a stimulator of guanylate cyclase, causes maximal 10- and 3-fold elevations of cGMP levels, respectively. Following a progressive decrease, 6- and 2-fold increases over basal cGMP levels are still observed after 180 min of incubation with ANF (99-126) and SNP, respectively. The half-maximal stimulation of cGMP accumulation induced by a 10 min exposure to ANF (99-126), or rat atriopeptin II (ANF 103-125) is observed at 9 +/- 2 and 125 +/- 22 nM, respectively. ANF fragments (99-109) and (111-126), as well as human cardiodilatin (hANF 1-16), do not alter cGMP levels. Basal and ANF-induced cGMP levels are at least 10-fold higher in cell populations enriched in gonadotrophs compared to gonadotroph-impoverished preparations. A 3 h incubation of cells with ANF (0.1-1000 nM), however, fails to modify spontaneous or LHRH-induced LH secretion. Similarly, ANF does not alter spontaneous release of GH, TSH or PRL. The data suggest indirectly that gonadotrophs represent a principal site at which ANF acts to stimulate cGMP synthesis, but that the nucleotide is not a specific regulator of the LH secretory process; nor is it generally involved as a second messenger in the secretory function of any cell type of the anterior pituitary gland.     

Atrial natriuretic factor recognizes two receptor subtypes in endothelial cells cultured from bovine pulmonary artery

In this study specific high affinity binding sites for atrial natriuretic factor (rANF(99-126] have been identified on cultured endothelial cells of bovine pulmonary artery origin (BPAEC). A time-dependent rise in cellular cGMP levels stimulated by rANF(99-126) was followed by release of the nucleotide into the incubation medium. The use of truncated, ring-deleted and linear atrial peptide analogs in competitive displacement analysis and measurement of cGMP accumulation indicated that only a minor proportion (5-11%) of the available receptor pool was of the ANF-B receptor subtype, linked to guanylate cyclase, with the remaining major proportion possibly of the ANF-C (clearance) receptor subtype. The existence of two ANF receptor subtypes in this cell culture model would suggest a significant role for the circulating peptide in modulation of pulmonary endothelial cell function, which would influence or complement its direct actions on the underlying vasculature of the pulmonary circulation.     

The effect of hypovolemia on the renal and cardiovascular responses to atrial natriuretic factor (ANF) infusion.

The renal and cardiovascular effects of ANF infusion have been examined in separate series of experiments; in conscious instrumented sheep following either hemorrhage (10 mL/kg body weight) or removal of 500 mL of plasma by ultrafiltration. Renal arterial infusion of hANF (99-126) at 50 micrograms/h increased sodium excretion from 99 +/- 30 to 334 +/- 102 (p less than 0.05) in normal animals, and from 77 +/- 31 to 354 +/- 118 mumol/min in hemorrhaged animals. Similarly in sheep following ultrafiltration, cardiac output and stroke volume were reduced by intravenous infusion of ANF (100 micrograms/h), although these effects were less marked than those observed in normal animals. The rapid modulation of natriuretic responses to ANF observed in volume expanded animals is not seen in this model of acute volume depletion suggesting that the mechanism through which the renal response to ANF is modulated in low sodium or volume states is not simply the reverse of that which produces rapid enhancement of response following blood volume expansion.     

Tissue-specific determinants of human atrial natriuretic factor gene expression in cardiac tissue

Elements controlling tissue-specific expression of the human atrial natriuretic factor gene have been examined in primary cultures of neonatal rat cardiocytes. When a 68-base pair fragment from human atrial natriuretic factor (hANF) 5'-flanking sequence (positions -400 to -333) was placed upstream from the herpes simplex thymidine kinase promoter linked to a bacterial reporter gene (chloramphenicol acetyltransferase), a tissue-specific positive regulatory effect was observed in atrial as well as ventricular cardiocytes but not in nonmyocardial cells. The cis-acting element in this fragment was orientation- and position-dependent. Examination of nuclear protein extracts for the presence of factors capable of interacting with the 5'-flanking sequence of the hANF gene revealed a cardiocyte-specific factor which bound to the 68-base pair fragment. This association was both tissue- and sequence-specific. These findings indicate that a cis-acting element present in the proximal 5'-flanking sequence confers tissue-specific expression upon the hANF gene, possibly through association with a cardiac-specific nuclear protein.     

Atrial granules contain an amino-terminal processing enzyme of atrial natriuretic factor

At least three enzymes have been identified in atrial tissue homogenates that are capable of processing pro-atrial natriuretic factor to active atrial peptides. The atrial peptides possess potent natriuretic, diuretic, vasorelaxant, and hemodynamic properties, and their existence has implicated the mammalian heart as an endocrine organ. We have purified and characterized a serine proteinase (Mr approximately equal to 70,000) associated with atrial granules that preferentially hydrolyzes the Arg-Ser bond in the synthetic substrates Gly-Pro-Arg-Ser-Leu-Arg, benzoyl-Gly-Pro-Arg-Ser-Leu-Arg, and benzoyl-Gly-Pro-Arg-Ser-Leu-Arg-Arg-2-naphthylamide, the Arg-2-naphthylamide bond in the substrate benzoyl-Gly-Pro-Arg-2-naphthylamide, and the Arg-Ser bond in a 31-residue substrate (Gly96-Tyr126 peptide) corresponding to residues Arg98-Ser99 in pro-atrial natriuretic factor. The Gly96-Tyr126 peptide contains the putative processing site in pro-atrial natriuretic factor and the sequence for the bioactive peptides. Our results indicate that the minimum processing site sequence is -Gly-Pro-Arg-Ser-Leu-Arg-Arg- and that the Ser99-Tyr126 natriuretic peptide is the predominant hydrolytic product. After prolonged incubation or at high enzyme concentrations, the Ser103-Tyr126 natriuretic peptide may also be formed. The Ser103-Arg125 natriuretic peptide was only a very minor product. The doublet of basic amino acids is not the primary processing site in pro-atrial natriuretic factor, but their presence may influence cleavage at the single Arg residue "upstream." Our findings are consistent with the idea that the pro-protein and the processing enzymes are packaged into the secretory granule and in response to the proper stimulus, the pro-protein is processed to the active peptides, probably during the process of secretion. The processing pathway of pro-atrial natriuretic factor is discussed.     

Contribution of blood and systemic circulation to the processing of pro-(atrial natriuretic factor).

Atrial natriuretic factor-(Asn1-Tyr126)-peptide, the 13.6 kDa propeptide of atrial natriuretic factor (ANF), is stored in the secretory granules of atrial cardiocytes. ANF-(Ser99-Tyr126)-peptide, the 28-amino-acid species, is the circulating form of this hormone in the rat. As the site of maturation of the prohormone is still unknown, the present study was undertaken to understand the contribution of the circulation to the maturation process of pro-ANF. 125I-ANF-(Asn1-Tyr126)-peptide was incubated with whole rat blood, plasma or serum for different time intervals, and the products were analysed. There was minimal activation of the propeptide in either whole blood or plasma. Incubation with serum, however, resulted in the formation of an 11 kDa and a 3 kDa peptide which corresponded respectively to the N-terminal and C-terminal parts of the propeptide. These results suggest that hydrolysis of the propeptide in serum is brought about by enzymes that may be stimulated during coagulation but which may not play a major role in the activation of pro-ANF in the circulation. Plasma analysis at different time intervals after prohormone injection indicated a non-specific hydrolysis of the pro-ANF molecule. The disappearance rate curves, obtained with radiolabelled pro-ANF, suggested the presence of two components with half-lives of 2.1 +/- 0.4 min and 52.5 +/- 8.4 min respectively. A metabolic clearance rate of 1.49 +/- 0.22 ml/min and an initial distribution volume of 47.4 +/- 8 ml were calculated. These results indicate that the maturation of pro-ANF to its active circulating form takes place before it is released into the circulation.     

Identification and characterization of three distinct atrial natriuretic factor receptors. Evidence for tissue-specific heterogeneity of receptor subtypes in vascular smooth muscle, kidney tubular epithelium, and Leydig tumor cells by ligand binding, photoaffinity labeling, and tryptic proteolysis

Three distinct atrial natriuretic factor (ANF) receptors have been identified and characterized from rat thoracic aortic cultured vascular smooth muscle (RTASM) cells, kidney tubular epithelium (MDCK), and Leydig tumor (MA-10) cells. These include 1) a disulfide-linked 140-kDa protein found in RTASM cells, which was reduced by dithiothreitol (DTT) to 70 kDa, 2) a 120-135-kDa single polypeptide protein, specific to MDCK and MA-10 cells whose Mr was not reduced by DTT, and 3) a 66-70-kDa protein prevalent in both RTASM and MDCK cells, which was not reduced by DTT. After incubation of RTASM cells with 4-azidobenzoyl 125I-ANF, labeling of the 140-kDa protein was blocked by both full-length ANF(99-126) and truncated ANF103-123. In contrast, the labeling of the 120-kDa receptor in MDCK cells was blocked only by full-length ANF(99-126). However, labeling of the 68-70-kDa receptor in both RTASM and MDCK cells was blocked by full-length ANF(99-126) and truncated ANF(103-123). Binding of 125I-ANF(99-126) to RTASM and MDCK cells was rapid, specific, and saturable with a Kd of 1.5 x 10(-10) M and binding capacity (Bmax) of 2.1 x 10(5) sites/RTASM cell and Kd 4.5 x 10(-10) M and Bmax 5 x 10(4) sites/MDCK cell, respectively. Binding of 125I-ANF(99-126) to RTASM cells was displaced with both full-length ANF(99-126) and truncated ANF(103-123), however, binding to MDCK cells was efficiently displaced only with full-length ANF. Both ANF(99-126) and ANF(103-123) stimulated cGMP in RTASM cells but only ANF(99-126) elicited cGMP in MDCK cells. Tryptic proteolysis of the high Mr single chain receptor produced only a 68-kDa fragment, whereas disulfide-linked 140-kDa receptor yielded 52-, 38-, 26-, and 14-kDa fragments. These data provide direct biochemical evidence for three distinct ANF receptors which might be linked to diverse physiological functions of ANF such as natriuresis in the kidney, vasorelaxation in vascular smooth muscle, and steroidogenic responsiveness in Leydig cells.     

The responses of atrial natriuretic factor concentrations to acute volume changes in conscious rats

A rapid and sensitive radioimmunoassay has been developed for measurements of atrial natriuretic factor (ANF) in rat plasma. The antiserum, raised to rat ANF (99-126), cross-reacts with rat ANF (103-123), ANF (103-125), ANF (103-126) but not with smaller fragments, human ANF (99-126), angiotensin II, bradykinin or vasopressin. The plasma ANF concentration is 181 +/- 24 pg/ml (N = 24) in the unstressed conscious rats (Charles River CD, male). The ANF immunoreactivity in the plasma extracts was verified by HPLC analysis, which displayed one major immunoreactive peak of ANF corresponding to rat ANF (99-126) and some smaller fragments. Intravenous injection of saline elevated circulating ANF, whereas acute volume depletion by hemorrhage, water deprivation and furosemide diuresis greatly lowered plasma ANF. The prompt response of plasma ANF levels to acute changes in volume status is consistent with the proposed role of ANF as a volume-regulatory hormone.     

Vasorelaxant effects of and receptors for atrial natriuretic peptides in the mesenteric artery and aorta of the rat

Vasorelaxant effects of different atrial natriuretic peptides (ANP) were measured on rat aortic strips and mesenteric artery rings. These results were compared with the potency of the same peptides to displace 125I-labelled ANP (101-126) on membrane preparations of aorta and of mesenteric vascular bed. In aortic strips and mesenteric artery rings precontracted with phenylephrine (3 X 10(-8) and 10(-6) M, respectively), the order of potency of ANP was as follows: ANP (99-126) greater than ANP (101-126) greater than ANP (103-126) = ANP (103-125) much greater than ANP (103-123). In the displacement binding assays, the order of potency of ANP peptides was similar to that of the relaxation experiments: ANP (99-126) = ANP (101-126) greater than ANP (103-126) = ANP (103-125) much greater than ANP (103-123). When the vessels were precontracted by a smaller concentration of phenylephrine (10(-7) M in mesenteric artery and 10(-8) M in aorta), the IC50 of ANP (101-126) was significantly lower than when the higher concentration of phenylephrine was used. These results show that ANP receptors in the mesenteric artery and in the aorta have similar structural requirements, according to the order of potency of different length ANP, both for binding and myotropic responses.     

Expression and secretion of human atrial natriuretic α-factor in Bacillus subtilis using the subtilisin signal peptide

Using the signal peptide of the Bacillus subtilis subtilisin gene (aprE) and a synthetic cDNA corresponding to the mature region of the human atrial natriuretic α-factor (hANF), we have constructed a secretion vector. B. subtilis cells, when transformed with this vector, secrete immunoreactive hANF peptides into the medium at about 500 μg/liter. The hANF is the first human gene product to be secreted from B. subtilis using this signal peptide. We have used promoters active during vegetative growth or sporulation and hosts deficient in several extracellular proteases but some proteolysis of the secretion products still occurs. In addition, both cell growth and sporulation are adversely affected by hANF production. Possible explanations for this observation are inefficient secretion of the atrial hormone or toxicity of the precursor or mature peptide.