Analysis of atrial natriuretic factor biosynthesis and secretion in adult and neonatal rat atrial cardiocytes

Life Sciences, Vol. 41, pp. 1953-1959 Printed in the U.S.A.

Pergamon Journals

ANALYSIS OF ATRIAL NATRIURETIC FACTOR BIOSYNTHESIS AND SECRETION IN ADULT AND NEONATAL RAT ATRIAL CARDIOCI~fES

Jerome B. Zisfein, Diana Sylvestre, Charles J. Homcy, and Robert M. Graham Cellular and Molecular Research Laboratory, Cardiac Unit, Massachusetts General Hospital, and Harvard Medical School Boston, Massachusetts 02114 (Received in final form August 20, 1987) Summary Atrial natrluretic factor (ANF) is stored in atrial cardiocytes as the 126 amino acid polypeptide, proANF, which is later cleaved to the 24-28 amino acid carboxyterminal peptides, the major circulating forms. Earlier studies have demonstrated that isolated, cultured neonatal rat cardiocytes both store and secrete proANF, which can be cleaved to the smaller circulating form(s) by a serum protease. Since differences may exist between neonatal and adult cardiocytes with respect to ANF synthesis and processing, we compared the forms of ANF stored and secreted by neonatal rat cardiocytes with those of adult cells. Using four to five day cultures of isolated atrial cardiocytes prepared from the hearts of neonatal and adult rats, pulse-chase studies were performed with 3SS-cysteine and 35S-methionine. Analysis of ANF stored and secreted by these cells was performed by immunoprecipitation of cell extracts and culture media using antibodies directed to either the carboxyterminus or aminoterminus of proANF followed by SDS-PAGE and autoradiogarphy. Cell extracts from both adult and neonatal cultures were found to contain only a 17-kDa polypeptide, previously identified as proANF. The predominant form found in the culture media was also the 17-kDa peptide, with smaller quantities of its 3-kDa carboxyterminal and 14-kDa aminoterminal cleavage products. We conclude from these studies that proANF is the major form stored and secreted by both adult and neonatal cardiocytes in culture; the activity of the protease that cleaves proANF to the smaller forms found in the circulation is either attenuated or is overwhelmed by high ANF-secretory rates in these cultures. Alternatively, the ANF processing and secretory pathways may be somehow altered in culture such that proANF escapes protease cleavage. Further studies will elucidate the nature and location of this protease. Atrial natriuretic factor (ANF) is synthesized in rat atrial cardiocytes as a 152-amino acid "preprohormone" (1-3). Following cleavage of the amino-terminal signal peptide and two carboxy-terminal arginine residues, it is stored in atrial granules as a 126-amino acid polypeptide, proANF, also known as y-rANP, ANF(I-126), and cardionatrin IV (4-8). Analysis of immunoreactive and bioactive ANF in blood by gel filtration chromatography, HPLC and by amino-terminal sequencing indicates that a 24- and a 28-amino acid peptide consisting of the C-terminal sequences of proANF constitute the predominant 0024-3205/87 $3.Q0 + .00 Copyright (c) 1987 Pergamon Journals Ltd.

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circulating forms (9-12). These peptides have similarly been demonstrated in the perfusates of isolated perfused heart preparations (13,14), and there is some evidence that cleavage of proANF to the smaller C-terminal peptide is necessary for full biological activity (15,16). Recently, it has been demonstrated by intrinsic labeling techniques that ANF is both stored and secreted by cultured neonatal atriocytes as the intact precursor, proANF, a 17-kDa peptide by SDS-PAGE analysis. ProANF can be cleaved in serum to a 3-kDa cysteine-containing fragment, believed to be the C-terminal 24- and 28-amino acid peptide(s), and a 14-kDa N-terminal fragment (17,18). The obvious discrepancy between these findings and those obtained using models of intact adult hearts, suggested that there might be differences between adult and neonatal cardiocytes with respect to ANF biosynthesis and processing. To test this hypothesis, we compared the biosynthesis and secretion of ANF in simultaneously prepared and labeled adult and neonatal rat cardiocytes. Methods C a r d i o c y t e C u l t u r e P r e p a r a t i o n . Neonatal r a t a t r i a l c e l l s were prepared as previously desc~(17). The h e a r t s of ten 250-300 gm female SD r a t s were excised immediately following d e c a p i t a t i o n . The a t r i a l appendages were removed, minced and placed immediately i n t o t r y p s i n - v e r s e n e - c o n t a i n i n g media ( B i o f l u i d s ) supplemented with an a d d i t i o n a l 35 mgZ t r y p s i n ( W o r t h i n g t o n ) ( " d i s p e r s i o n media") a t room t e m p e r a t u r e . The minced a t r i a l t i s s u e was then t r a n s f e r r e d to a 40 ml Falcon f l a s k , to which was added 5 ml of d i s p e r s i o n media, and s t i r r e d with a " f l e a " magnetic s t i r bar f o r 30 m i n u t e s . The cloudy media was then a s p i r a t e d and d i s c a r d e d , and the a t r i a l t i s s u e was a g a i n s t i r r e d in 5 ml of fresh dispersion media. When the media again became cloudy, it was aspirated and diluted into 30 ml of DMEM containing 20% Nu serum, thymidlne (0.6gmlL), penicillin (20 U/ml) and streptomycin (20 ~g/ml) ("growth media"). This cell-dlspersion step was repeated six to ten times until the media no longer became cloudy on addition of fresh dispersion media. The diluted cell suspensions were centrifuged at 650 x g for 15 minutes and following aspiration of the supernatant, the cells were resuspended in growth media and incubated in a 60 mm culture dish at 37°C for 20 hours. Following gentle agitation, the cells were aspirated from the dish and subjected to separation by centrlfugation with Ficoll-Paque at 1600 x g for 20 minutes. The cells at the medla-Ficoll interface were then aspirated, resus#ended in growth media and placed into a 6-well Falcon culture plate (1-3 x 10 ° cells/well). Intrinsic ~ . L a b e l i n g e x p e r i m e n t s were performed on a d u l t and neonatal cells a~ter four to five days of culture as previously described (17). Briefly, the growth media was aspirated and replaced first with fresh DNEM without serum and then with RPMI media deficient in either cysteine or methionine. After 30 minutes incubation the media was aspirated and replaced with the identical media containing either 35S-cystelne or ~SS-methionine (200 ~Ci/ml in each culture well) for 30 minutes. The labeling media was then aspirated and replaced vlth complete RPMI media (1 ml/well) for variable lengths of time after which the media was aspirated, centrifuged and stored at 4°C with aprotinin (I00 KIU/ml) and phenylmethylsulfonylfluoride (PMSF) (0.5 mM) for the duration of the experiment. At specified time points the cells in each well were harvested and lysed in 1 ml of I0 mM Tris buffer containing 0.5 M NaCl, 0.5% NP-40, aprotinin (100 KIU/ml) and PMSF (0.5 mM), centrifuged and stored at 4°C. Immunoprecipitation o__ff 35S-ANF. Intrinsically labeled ANF was immunoprecipitated from culture media and cell extracts with two n o n - c r o s s r e a c t i v e r a b b i t a n t i s e r a r a i s e d to two s y n t h e t i c ANF p e p t i d e s ( 7 ) . Anti-ANF(103-126) was r a i s e d by immunizing r a b b i t s with r a t a t r i o p e p t i n I I I ( P e n i n s u l a ) , the 2 4 - r e s i d u e C - t e r m i n a l fragment of r a t proANF, c o n j u g a t e d to b o v i n e t h y r o g l o b u l i n and suspended i n complete or i n c o m p l e t e F r e u n d ' s a d j u v a n t .

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Anti-ANF(57-68) was similarly raised against a synthetic peptide corresponding to residues 57-68 of rat proANF. Immunoprecipitation of 3SS-cysteine-ANF was carried out with anti-ANF(103-126) and 3SS-methionine-ANF with anti-ANF(57-68). In a 1.5 ml plastic microfuge tube 400 ~l of culture media or cell extract was incubated with 5 ~l antiserum, phenylmethylsulfonylfluoride (PMSF) (0.5 mM) and 500 ~l 0 . 1 M sodium phosphate buffer containing 0.05 M NaC1, 0.1Z BSA, 0.1% Triton-X and O.02Z NaN 3 ("IP buffer") for 2 hours at room temperature. PMSF at this concentration has previously been shown to completely block the cleavage of proANF by serum (20). To each tube was then added 50 ~l goat anti-rabbit IgG serum, and incubation was continued for 2 hours at 4°C. The tubes were then centrifuged in an Eppendorf 5414 centrifuge for 5 minutes and decanted. The pellets were washed with 1 ml cold IP buffer, centrifuged for 5 minutes, decanted, resuspended in 50 ~i SDS-PAGE sample buffer (see below) and boiled for 5 minutes prior to electrophoresis. SDS-PAGE. Polyacrylamide gel electrophoresis was carried out in 17Z polyacry-'l-~de containing 0.1% sodium dodecyl sulfate as previously described (17). Gel slabs were incubated in Autofluor (National Diagnostics) for 1 hour prior to drying and autoradiographs prepared by 24-48 hour exposure at -70°C. Densitometry was carried out on autoradlographs with an LKB laser densitometer. Quantltatlon of specific 3sS-labeled ANF peptides was also performed by excising specific bands from the gels and counting them in a Beckman LS 1800 beta scintillation counter in Liquisclnt (National Diagnostics). Corresponding areas from lanes in which 3ss-labeled ANF had been immunoprecipitated in the presence of excess competing ANF ("nonspecific Immununoprecipitatatlon") were similarly counted and used as background values. The lanes generated from two separate experiments involving both 3SS-cysteine (total of 7 lanes) and 3SS-methionine (6 lanes) labeling were so analysed. Results A f t e r f o u r days i n c u l t u r e , both n e o n a t a l and a d u l t r a t c a r d i o c y t e s were f i r m l y a t t a c h e d to the growth p l a t e s i n f l a t t e n e d , s t e l l a t e c o n f i g u r a t i o n s and beat s p o n t a n e o u s l y . Less than 5Z of the a t t a c h e d c e l l s took up t r y p a n b l u e . The results of pulse-chase labeling experiments with 3SS-cystelne are shown in Figure i. Following a 20 minute labeling period, both adult and neonatal c e l l e x t r a c t s were found to c o n t a i n a 17-kDa c y s t e i n e - c o n t a i n i n g p r o t e i n specifically i m m u n o p r e c i p i t a t e d by the a n t i s e r u m d i r e c t e d to the c a r b o x y t e r m i n a l p e p t i d e ( a n t i - A N F [ 1 0 3 - 1 2 6 ] ) . The p r e s e n c e of t h i s p e p t i d e p e r s i s t e d i n the c e l l e x t r a c t s f o r the d u r a t i o n of the 2 hour chase p e r i o d . The media from these cells, similarly analyzed, revealed the appearance of a 17-kDa protein after a 20 minute chase period, which accumulated in the media over time. Smaller quantities of a 3-kDa peptlde were detected at the end of the 2 hour chase period. Analysis of gels by scintillation counting and by scanning densitometry revealed that the 3 kDa band constituted 25.9 +8.2~ (mean ~SD) of the total specifically identified ANF, and that this percentage remained constant over time (up to 4 hours). Figure 2 shows the results of a similar experiment in which labeling was c a r r i e d out with 3SS-methionine and i m m u n o p r e c i p i t a t i o n of c e l l e x t r a c t s and media performed with a n t i s e r u m d i r e c t e d to a sequence c o n t a i n e d i n the middle of proANF ( a n t i - A N F [ 5 7 - 6 8 ] ) . F o l l o w i n g the 20 minute l a b e l i n g p e r i o d e x t r a c t s of both n e o n a t a l and a d u l t a t r i o c y t e s were found to c o n t a i n the 17-kDa p e p t i d e , which p e r s i s t e d f o r the 2 hour chase p e r i o d . A n a l y s i s of the c u l t u r e media r e v e a l e d the a c c u m u l a t i o n of the 17-kDa p e p t i d e as w e l l as r e l a t i v e l y s m a l l e r q u a n t i t i e s of a 14-kDa p e p t i d e , which c o n s t i t u t e d 31 +5.7% (mean +SD) of the t o t a l of the two bands, as d e t e r m i n e d by d e n s i t o m e t r y and s c ~ n t i l l a t i o n c o u n t i n g of g e l s l i c e s . This p e r c e n t a g e did not vary with l o n g e r chase p e r i o d s (up to 4 h o u r s ) .

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FIG. I 35S-cysteine labeling of cultured atrial cardiocytes. Autoradiograms of SDS-polyacrylamide gels of cell extracts and culture media following pulse-chase labeling with 35S-cysteine and immunoprecipitation with the carboxy-terminal-directed anti-ANF antiserum. Immunoprecipitation of the 17-kDa band identified in cell extracts and media is blocked in the presence of excess added ANF(I03-126) ("120 + AP IIl"), whereas other, nonspecific bands, including a light 14-kDa band, are not.

FIG. 2 35S-methionine labeling of cultured atrial cardiocytes. Autoradiograms of SDS-polyacrylamide gels of cell extracts and culture media following pulse-chase labeling with 35S-methionine and immunoprecipitation with the N-terminal-directed anti-ANF antiserum. Immunoprecipitation of the 17-kDa band in cell extracts and media and the 14-kDa band in media is blocked in the presence of excess added proANF peptide 57-68 ("120 + PP").

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Discussion Based on immunostaining, gel filtration, HPLC and sequence analysis of ANF stored in atrial cardiocytes, it is generally agreed that ANF is stored predominantly as the 126-amino acid proANF (4-8), which is later cleaved to the smaller 24-28-amino acid carboxy-terminal peptides, the major circulating forms of ANF (9-12). The site of this cleavage, however, is a matter of dispute. Analysis of ANF released from the atria of isolated perfused hearts has suggested that cleavage takes place in the absence of blood proteases (13,14). These findings have led to the notion that proANF is cleaved during or just a f t e r i t s r e l e a s e from the a t r i o c y t e by a p r o t e a s e r e s i d i n g in the a t r i a l or e n d o t h e l i a l c e l l membrane. However, s t u d i e s in our l a b o r a t o r y ( 1 7 ) , as w e l l as o t h e r s (1 9 ) , have shown t h a t c u l t u r e d n e o n a t a l a t r i a l c e l l s r e l e a s e proANF intact. F u r t h e r m o r e , a serum p r o t e a s e has been i d e n t i f i e d that effects a specific e n d o l y t i c c l e a v a g e of proANF to form a c y s t e i n e - c o n t a i n i n g 3-kDa fragment c o n s i s t i n g o f the 28- and 24-amino a c i d C - t e r m i n a l p e p t i d e s , and a 14-kDa m e t h i o n i n e - c o n t a i n i n g fragment r e p r e s e n t i n g the N - t e r m i n a l r e m a i n d e r o f proANF (17,19-21). The physiologic role of this serum protease in ANF processing is not yet clear. This obvious discrepancy between the forms of ANF seemingly secreted from intact adult atria and cultured neonatal atrial cells raised the possibility that differences may exist between adult and neonatal cardiocytes with respect to ANF biosynthesis and secretion. Initially, we used a neonatal cardiocyte culture system to investigate the biosynthesis of ANF because of the facility of preparing viable cells with high yields. In contrast, preparation of cardiocyte cultures from adult rat hearts is somewhat more difficult and affords lower yields of viable cells. In the present study, we have demonstrated that proANF is the major form stored in and released from cultured atriocytes prepared, not only from neonatal rat hearts, but also from the atria of adult rats. Analysis of cell extracts with antisera to the tarboxy-terminal peptide after intrinsic labeling of these cells with 35S-cysteine demonstrated a predominant 17-kDa peptide, previously identified by N-terminal radiosequence analysis as proANF (17). This peptide was similarly detected in the media by antisera directed to the amino-terminus after 35S-methionine labeling. Although the 17-kDa proANF was the major form secreted by the cardiocytes, the 3-kDa and 14-kDa cleavage products were also found in the culture media, accounting for less than one third of the total immunoprecipitated ANF. Since the C-termlnal 3-kDa and N-terminal 14-kDa cleavage products of proANF appear in comparably small quantities in the culture media, it is unlikely that preferential degradation of the 3-kDa peptide is responsible for the predominant appearance of the 17-kDa proANF in the culture media. Most investigators have found the cleaved form of ANF in plasma with relatively small quantities of uncleaved proANF (9-12). Although it is possible that ANF biosynthesis differs in cultured versus in situ cardiocytes, considered together, these data suggest that proANF is se'cret--~ intact from atrial cardiocytes and that cleavage to the biologically active 24-28-amino acid ~ ~erminal fragments occurs after secretion. Another possibility is that there are inherent differences between intact atria and cultured cardiocytes with respect to ANF processing and secretion. This possibility will require further investigation. The finding of relatively small quantities of cleaved ANF in the media of cultured cardiocytes suggests that either the low molecular weight fragments are generated non-specifically or that there is a protease in these cultures that processes only a fraction of the newly synthesized proANF. This in vitro protease activity may represent an attenuated version of an enzyme t-h--at, in vivo, is capable of processing all of the synthesized ANF or may be one of--a number of proANF-cleaving enzymes. The specificity of this enzyme and whether or not its activity is attenuated in culture will require further study. It is

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u n l i k e l y t h a t the c l e a v a g e fragments a r e g e n e r a t e d d u r i n g i m m u n o p r e c i p i t a t i o n with antiserum since this step is carried out with small quantities of antiserum and in the presence of PMSF, which has previously been shown to block the cleavage of proANF by serum (20,21). Although the c l e a v e d form of ANF has been the major form i d e n t i f i e d i n the plasma of normal or a c u t e l y s t i m u l a t e d a n i m a l s , r e c e n t l y , t h e r e have been several r e p o r t s of i n c r e a s e d q u a n t i t i e s of proANF c i r c u l a t i n g i n i n d i v i d u a l s with c h r o n i c c o n g e s t i v e h e a r t f a i l u r e (22) and i n a n i m a l s s u b j e c t e d to p r o l o n g e d volume e x p a n s i o n (23). These o b s e r v a t i o n s imply t h a t t h e r e may be a proANF c l e a v i n g enzyme, e i t h e r i n t r a c e l l u l a r or e x t r a c e l l u l a r , whose p r o c e s s i n g c a p a c i t y i s overwhelmed i n the face of prolonged high ANF s e c r e t i o n r a t e s . It i s q u i t e p o s s i b l e t h a t the c u l t u r e d a t r i a l c a r d i o c y t e too, i s i n a c o n s t a n t high ANF-secretory s t a t e r e s u l t i n g i n the a c c u m u l a t i o n of mostly proANF i n the culture media, accompanied by minor q u a n t i t i e s of the c l e a v e d forms. A l t e r n a t i v e l y , the pathways for p r o c e s s i n g and s e c r e t i o n of ANF may be somehow a l t e r e d i n c a r d i o c y t e c u l t u r e s ( e . g . , c o n s t i t u t i v e vs. r e g u l a t e d s e c r e t i o n ) , such t h a t newly s y n t h e s i z e d proANF escapes p r o t e a s e c l e a v a g e p r i o r to r e l e a s e from the c e l l . I n c o n c l u s i o n , the 126-amino a c i d p o l y p e p t i d e , proANF i s the major form of ANF s t o r e d and s e c r e t e d by a d u l t as w e l l as n e o n a t a l r a t a t r i a l c a r d i o c y t e s i n culture. Thus, the d i s c r e p e n c y between s i m i l a r f i n d i n g s of e a r l i e r s t u d i e s u s i n g c u l t u r e d n e o n a t a l r a t c a r d i o c y t e s a l o n e and those d e m o n s t r a t i n g s m a l l e r ANF p e p t i d e s i n the c i r c u l a t i o n and i n p e r f u s a t e s of i s o l a t e d a d u l t r a t h e a r t s cannot be e x p l a i n e d on the b a s i s of d i f f e r e n c e s between a d u l t and n e o n a t a l ANF p r o c e s s i n g per se. Whether proANF i s indeed s e c r e t e d i n t a c t i n vivo with subsequent cleavage by a circulating or membrane-bound p r o t e a s e or a l t e r n a t i v e l y , i s c l e a v e d p r i o r to s e c r e t i o n has not y e t been d e t e r m i n e d . AcknowledGements We a r e g r a t e f u l to Dr. G. Matsueda for s y n t h e s i s of p e p t i d e s and D. R o l l i n s for secretarial assistance. This work was supported by a National Institutes of Health Grant HL-35642, and by a grant from the R.J.R. Nabisco Company. RMG is an Established Investigator, AlIA (Grant 82-240). References i. 2. 3. 4. 5. 6. 7. 8. 9.

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( 19%6).