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Presence of immunoreactive atrial natriuretic peptides in pericardial fluid of human subjects with congenital heart diseases
Life Sciences, Vol. 46, pp. 1977-1983 Printed in the U.S.A.
Pergamon Pres:
PRESENCE OF IMMUNOREACTIVE ATRIAL NATRIURETIC PEPTIDES IN PERICARDIAL FLUID OF HUMAN SUBJECTS WITH CONGENITAL HEART DISEASES Ja Hong Kuh*, Kong Soo Kim*, Suhn Hee Kim, Kyung Woo Cho, Kyung Hwan S e u l , and Gou Young Koh D e p a r t m e n t of P h y s i o l o g y and Chest S u r g e r y * Jeonbug N a t i o n a l U n i v e r s i t y M e d i c a l School Jeonju 5 6 0 - 1 8 0 , Korea (Received in final form April 24, 1990)
Summary The e p i c a r d i a l r e l e a s e of i m m u n o r e a c t i v e a t r i a l natriuretic p e p t i d e s ( i r - A N P s ) in i n s i d e - o u t p e r f u s e d rabb i t a t r i a has been r e p o r t e d . In o r d e r to d e t e r m i n e the p r e s e n c e of i r - A N P s in p e r i c a r d i a l f l u i d and to e v a l u a t e their biochemical characteristics, we measured the concentration of i r - A N P s in p e r i c a r d i a l fluid obtained from the p a t i e n t s w i t h c o n g e n i t a l h e a r t d i s e a s e s d u r i n g open h e a r t s u r g e r y . S e r i a l d i l u t i o n c u r v e s made w i t h t h e e x t r a t s of p e r i c a r d i a l f l u i d u s i n g Sep-Pak C18 c a r t r i d ges were p a r a l l e l w i t h s t a n d a r d c u r v e . The c o n c e n t r a t i o n of ir-ANPs in pericardial f l u i d was s i g n i f i c a n t l y lower than t h e c o r r e s p o n d i n g plasma concentration. On gel permeation and r e v e r s e - p h a s e high p e r f o r m a n c e liquid c h r o m a t o g r a p h y , the i r - A N P s in p e r i c a r d i a l fluid, plasma and atrial appendage showed both high and low m o l e c u l a r weights. The m a j o r peak of i r - A N P s in plasma was o b s e r ved a t the c o r r e s p o n d i n g f r a c t i o n to the alpha-human ANP and c o n s i d e r a b l e amount of high m o l e c u l a r w e i g h t form o f i r - A N P s was o b s e r v e d in p e r i c a r d i a l fluid. However, the major peak o f i r - A N P s in a t r i a l appendage was o b s e r v e d at the c o r r e s p o n d i n g f r a c t i o n to t h e r a t pro-ANP. The data s u g g e s t t h a t i r - A N P s e x i s t both high and low m o l e c u l a r w e i g h t forms in p e r i c a r d i a l fluid. Atrial m y o c y t e s have been known to c o n t a i n a t r i a l natriuretic peptides (ANPs), which caused p o t e n t n a t r i u r e s i s and diuresis, vasorelaxation and an i n h i b i t i o n of r e n i n and a l d o s t e r o n e secretions (I-5). However, l i t t l e i n f o r m a t i o n about the p h y s i o l o g i c a l c o n t r o l mechanisms f o r the ANP r e l e a s e from the a t r i a e x i s t s . Atrial d i s t e n s i o n or stretch (6-14), pressure (6, 8, 11, 1 4 ) , pacing frequency (10, 1 5 - 1 8 ) and humoral f a c t o r s (19-22) have been s u g g e s t e d f o r the r e g u l a t o r y f a c t o r s of the r e l e a s e o f ANP from the atria. The ANP i m m u n o r e a c t i v e s p e c i f i c granules in atrial m y o c y t e s were r e p o r t e d to be l o c a t e d in the s u b p e r i c a r d i a l Address a l l c o r r e s p o n d e n c e to : Dr. Kyung Woo Cho, Physiology, Jeonbug National University Medical Keum-Am-Dong-San, J e o n j u 5 6 0 - 1 8 0 , Korea 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
Department of School, 2-20
1978
ANP in Pericardial
Fluid
Vol. 46, No. 26, 1990
wall (23, 24) as w e l l as in t h e m y o c y t e s near the a t r i a l lumen (25, 26). Recently, Our group (27) r e p o r t e d the e p i c a r d i a l release of i r - A N P s in i n s i d e - o u t perfused rabbit atria. In this s t u d y we d e m o n s t r a t e d the p r e s e n c e o f i r - A N P in p e r i c a r d i a l fluid and c h a r a c t e r i z e d it. METHODS Subjects : Thirteen patients with congenital heart diseases (2 mitral stenosis, I Tetralogy of Fallot, 3 ventricular septal defect, 5 atrial septal defect, 2 aortic insufficiency) who u n d e r w e n t open h e a r t o p e r a t i o n s were s t u d i e d . E i g h t o f them was female and i n f o r m e d c o n s e n t was o b t a i n e d from the f a m i l y members or subjects. Blood samples were o b t a i n e d from healthy medical s t u d e n t s (n=20) f o r a c o m p a r i s o n . Preparation of samples: Pericardial fluid, plasma and atrial appendages were o b t a i n e d from s i x p a t i e n t s s u f f e r i n g congenital heart disease. Balanced f e n t a n y l a n e s t h e s i a was a d m i n i s t e r e d and t h o r a c o t o m y was p e r f o r m e d . A f t e r m e d i c a l s t e r n o t o m y the p e r i c a r dium was e x p o s e d . A s m a l l i n c i s i o n was a p p l i e d w i t h care and was electrocauterized. A c a n n u l a was i n t r o d u c e d i n t o the p e r i c a r d i a l space through i n c i s i o n and 15-20 ml o f pericardial fluid was immediately obtained into prechilled tubes c o n t a i n i n g 800 ul o f a mixture of ethylenediamine tetraacetic a c i d (EDTA) ( 2 . 7 mM i n pericardial fluid), phenylmethylsulfonyl fluoride (PMSF) (11.5 uM), a p r o t i n i n (200 K I U / m l ) , and soybean t r y p s i n i n h i b i t o r (SBTI) (50 BAEE/ml). In any case c o n t a m i n a t i o n from the b l o o d was n e g l i gible. Simultaneously, 10-20 ml o f blood was c o l l e c t e d from a radial artery into prechilled tubes c o n t a i n i n g a mixture of proteolytic enzyme i n h i b i t o r s as d e s c r i b e d above. Plasma samples from h e a l t h y p e r s o n s were t r e a t e d as the s a m e way. After centrifugation a t 4 C, 3,000 rpm f o r 15 m i n , the s u p e r n a t a n t and plasma were s e p a r a t e d . Atrial appendages were a l s o o b t a i n e d from patients during cardiac operation. After weighing, atrial appendages were b o i l e d i n i ml o f 0 . i M a c e t i c a c i d f o r I0 m i n , homogenized w i t h P o l y t ron h o m o g e n i z e r and c e n t r i f u g e d a t 4 C, I 0 , 0 0 0 g f o r 15 m i n . The ir-ANP in pericardial fluid, plasma and a t r i a l appendages was e x t r a c t e d u s i n g Sep-Pak C18 c a r t r i d g e s (Waters Associates, Milford, MA, U . S . A . ) as d e s c r i b e d p r e v i o u s l y (28-30). The e l u a t e s were lyophilized under g e n t l e s t r e a m of N2 g a s , reconstituted w i t h 0.1% t r i f l u o r o a c e t i c a c i d (TFA) and c e n t r i f u g e d a t lO,O00xg f o r 15 m i n . High P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y (HPLC): One hundred m i c r o liters o f the e l u a t e s were s u b j e c t e d to the reverse-phase high performance l i q u i d c h r o m a t o g r a p h y (HPLC) on a uBondapak (Waters Associates, Milford, MA, U . S . A . ) column as d e s c r i b e d p r e v i o u s l y (31). Elution was p e r f o r m e d w i t h the l i n e a r g r a d i e n t of 20 % to 60 % a c e t o n i t r i l e i n 0 . 1 % TFA f o r 40 min a t a f l o w r a t e of I m l / m i n and each f r a c t i o n volume was I ml . Gel p e r m e a t i o n HPLC was a l s o p e r f o r m e d on a TSK-GEL G2000 SW (Toyo Soda, Tokyo, Japan) column ( 7 . 5 x 300 mm) and e l u t e d w i t h 30 % a c e t o n i t r i l e in 0 . I % TFA. The f l o w r a t e was 0.3 m l / m i n and the fraction volume was 0.3 m l . The f r a c t i o n a t e d samples were lyophilized and a s s a y e d .
Vol. 46, No. 26, 1990
ANP in Pericardial Fluid
1979
Ra d i o i m m u n o a s s a y o f i r - A N P : The i r - A N P was measured by r a d i o i m m u noassay as d e s c r i b e d p r e v i o u s l y (27-30). Anti-ANP antibody was purchased from D i a g n o s t i c & Research A n t i b o d i e s Inc. (Berkely, CA, U.S.A.) and synthetic atriopeptin I I I (AP I I I ) (Peninsula Laboratories, B e l m o n t , CA, U . S . A . ) was i o d i n a t e d u s i n g c h l o r a m i ne-T m e t h o d , as d e s c r i b e d p r e v i o u s l y (29). The l y o p h i l i z e d samples were r e c o n s t i t u t e d w i t h I00 ul of T r i s - a c e t a t e buffer and incubated w i t h a n t i - A N P a n t i b o d y f o r 24 hrs a t 4 C. Following the additional incubation w i t h 125 I-ANP f o r 24 hrs at 4 C, separation o f f r e e from bound form was a c h i e v e d by a d d i n g 1.0 ml of Dextran-coated charcoal suspension. S t a t i s t i c s : S t a t i s t i c a l s i g n i f i c a n c e of d i f f e r e n c e was tested using Student's t - t e s t and was defined as a P-value of less than 0,05. The r e s u l t s were given as means + s.e.m.
RESULTS Serial d i l u t i o n of the e x t r a c t s of p e r i c a r d i a l f l u i d i n h i b i t e d the binding of 125 I-ANP to the antibody in p a r a l l e l with the standard curve, as shown in Figure 1. The concentration of ir-ANP in p e r i c a r d i a l f l u i d was s i g n i f i c a n t l y lower t h a n the corresponding plasma concentration (Table i ) . No s i g n i f i c a n t c o r r e l a t i o n between the concentrations of plasma and p e r i c a r d i a l f l u i d was observed (y=42.31x-O.071, r=-0.12, p=0.687). The ir-ANP p r o f i l e s of e x t r a c t s on reverse-phase and gel permeation HPLC are shown in Figure 2 and 3. I r - A N P in p e r i c a r d i a l f l u i d showed both high and low molecular weights. The one of major peaks of ir-ANP was observed at the corresponding f r a c t i o n to the s y n t h e t i c alpha human ANP (99-126) and the other of major peaks was correspondent to r a t pro-ANP. Two of s i x samples showed mainly high molecular weight of irANP in p e r i c a r d i a l f l u i d . In a t r i a l appendage and plasma, b o t h forms of ir-ANP were also observed. The major form of ir-ANPs in a t r i a l appendage was high molecular weight. Although the low molecular weight form of irANPs in plasma was major form, a considerable amount of high molecular weight form of ir-ANP was also detected.
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FIG. 1
A representative standard curve ( e - e ) and s e r i a l dilutions of pericardial fluid extracted (o-o, ~-~ )
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I 5 irANP
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1980
ANP in Pericardial
Fluid
Vol. 46, No. 26, 1990
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R e v e r s e - p h a s e HPLC p r o f i l e s of atrial natriuretic peptide in pericardial fluid (A), plasma ( B ) , and a t r i u m (C) on a uBondapak c o l u m n . Arrows indicate the peak p o s i t i o n s of elution of rat pro-atrial natriuretic p e p t i d e ( PRO) and a t r i o p e p t i n III (AP III), respectively. Rat pro-ANP was p u r i f i e d by the method o f T r i p p o d o e t al (38).
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FIG.
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Gel p e r m e a t i o n HPLC p r o f i l e s of atrial natriuretic peptide in pericardial fluid (A), plasma (B), and a t r i u m (C) on a TSKGel G2000 SW c o l u m n . Arrows indicate t h e peak p o s i t i o n s of elution of blue Dextran (Vo), b o v i n e serum a l b u m i n ( B S A ) , c y t o c h r o m e C (CC) and a t r i o p e p t i n III (APIII), respectively.
Vol. 46, No. 26, 1990
Table fluid, heart
ANP in Pericardial
Fluid
1981
1.
Immunoreactive atrial natriuretic p e p t i d e in p e r i c a r d i a l plasma and a t r i a l appendage from p a t i e n t s w i t h c o n g e n i t a l diseases Concentration Pericardial
fluid
Plasma Atrial
appendage
of
(pg/ml)
34.91
+ 10.45
(pg/ml)
103.00
+ 17.24
(ng/mg)
20.45
V a l u e s are t h e means + SEM o f
+
ir-ANP
8.25
9 patients.
DISCUSSION The p r e s e n t s t u d y shows the p r e s e n c e o f i r - A N P in p e r i c a r d i a l fluid of patients with congenital heart diseases. The concentration of i r - A N P in p e r i c a r d i a l f l u i d was l o w e r than i n plasma and no s i g n i f i c a n t correlation between them was f o u n d . In an a t t e m p t to f u t h e r c h a r a c t e r i z e the i r - A N P i n p e r i c a d i a l fluid, the ir-ANP profiles were i n v e s t i g a t e d by gel permeation and r e v e r s e - p h a s e HPLC. In p e r i c a r d i a l fluid and plasma, two major peaks o f i r - A N P c o r r e s p o n d i n g t o a l p h a - human ANP and r a t pro-ANP were found. The i r - A N P i n cerebrospinal fluid (33), ascitic fluid (32) and plasma ( 3 1 , 33) has been r e p o r t e d to be a low molecular weight form. Recently, Kim e t a l . (31) r e p o r t e d that a c o n s i d e r a b l e amount o f high m o l e c u l a r w e i g h t form o f irANP e x i s t s i n o v a r i a n f o l l i c u l a r fluid. A l s o , s e v e r a l papers have been shown the p r e s e n c e o f h i g h m o l e c u l a r w e i g h t form of ANP i n plasma (31, 34-37). The d i f f e r e n c e s i n irANP p r o f i l e s reported may be due to d i f f e r e n t specimen, unstability of pro-ANP, or activation of p r o t e o l y t i c enzymes d u r i n g e x t r a c t i o n . A l t h o u g h we do not know e x a c t l y the s i t e of o r i g i n of p e r i c a r d i a l ANP, i t may not be t a k e n up from the c i r c u l a t i o n . A special care was taken to o b t a i n pericardial fluid samples without contamination. We found no s i g n i f i c a n t correlation between t h e c o n c e n t r a t i o n of plasma and p e r i c a r d i a l ANP. Additionally, the ir-ANP profile in pericardial fluid is different from that in p l a s m a . The irANP in the p e r i c a r d i a l f l u i d may o r i g i n a t e from t h e ventricles (36) as w e l l as t h e a t r i a ( 2 7 ) . It is possible that the pro-ANP in p e r i c a r d i a l f l u i d may o r i g i n a t e from the v e n t r i c les. It i s w e l l known t h a t the a t r i a r e l e a s e mainly processed ANP. Recently, T h i b a u l t et al (36) r e p o r t e d t h a t the m a j o r p o r tion of the ANP in e f f l u e n t from the perfused ventricles is prohormone, and the v e n t r i c l e s are the m a j o r source of t h i s p r o ANP i n p l a s m a . The p r e s e n c e of high m o l e c u l a r w e i g h t of irANP in plasma from patients w i t h heart problems is consistent with previous reports (36,37). Plasma l e v e l s of ANP i n p a t i e n t s were significantly h i g h e r in h e a l t h y p e r s o n s ( 1 0 3 . 0 0 + 17.24 vs 44.80 + 2.97, p
1982
ANP in Perlcardial
Fluid
Vol. 46, No. 26, 1990
r e l e a s e of i r - A N P in p e r i c a r d i a l space. It is unclear at present t h a t the p r e s e n c e o f irANPs in p e r i c a r d i a l space i s p h y s i o l o g i c a l or p a t h o l o g i c a l in n a t u r e . ACKNOWLEDGMENTS We t h a n k Miss Kyung Mee S e u l , Eun J i n Nah and Kyung Hwa Nam f o r t h e i r e x p e r t t e c h n i c a l a s s i s t a n c e . T h i s work was s u p p o r t e d i n part by a g r a n t from the Korea S c i e n c e and Engineering Foundat i o n , Republic of Korea. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14 15 16 17 18 19 20. 21. 22. 23.
A . J . DEBOLD, H.B. BORENSTEIN, A . T . VERESS and H. SONNENBERG, L i f e S c i . 28 89-94 ( 1 9 8 1 ) . K.S. MISON~ R.T. GRAMMER, H. FUKUMI and T. INAGAMI, Biochem. B i o p h y s . Res. Commun. 123 444-451 (1984) M.C. CURRIE, D.M. GELLER, B.R. COLE, J . C . BOYLAR, W. YASHENG, S.W. HOLMBERG and P. NEEDLEMAN, S c i e n c e 221 71-73 ( 1 9 8 3 ) . K.ATARASHI, P . J . MURLOW, R. FRANCO-SAENZ, R. SHAPJAR and J. RAPP, S c i e n c e 224 992-994 ( 1 9 8 4 ) . M CANTIN and J. GENEST, Endocr. Rev. 6 107-127 ( 1 9 8 5 ) . R.E. LANG, H. THOELKEN, D. GANTEN, ~ . C . LUFT, H. RUSKOAHO, and T.H. UNGER, N a t u r e 314 264-266 ( 1 9 8 5 ) . J . R . LEDSOME, N. WILSON, C.A. COURNEYA and A . J . RANKIN, Can. J. P h y s i o l . P h a r m a c o l . 63 739-742 ( 1 9 8 5 ) . J . R . DIETZ, Am. J. P h y s i o l . 247 RIO93-RI096 ( 1 9 8 4 ) . A.J. DEBOLD, M.L. DEBOLD and I . R . SARDA, J. H y p e r t e n s i o n 4 ( s u p p l 2) s3-s7 ( 1 9 8 6 ) . G.E. BILDER, T . L . SCHOFIELD and E.H. BLAINE, Am. J. P h y s i o l . 251 F817-F821 ( 1 9 8 6 ) . H. RUSKOAHO, H. THOELKEN and R.E. LANG, P f l u g e r s A r c h . 407 170-174 ( 1 9 8 6 ) . R . J . SCHIEBINGER and J. LINDEN, C i r c . Res. 59 105-109 ( 1 9 8 6 ) . G, AGNOLETTI, A. RODELLA, R. FERRARI and P. HARRIS, J. Mol. C e l l . C a r d i o l . 19 249-253 ( 1 9 8 7 ) . D,E. UEHLINGER, T. ZAMAN, P. WEIDMAN, S. SHAW and M.P. GNADINGER, H y p e r t e n s i o n I0 249-253 ( 1 9 8 7 ) . R.J. SCHIEBINGER and J. LINDEN, Am. J. P h y s i o l . 251 HIO95N1099 ( 1 9 8 6 ) . M.G. ZIEGLER, S.R. SHACKFORD, K.D. WILNER and C.H. NORTON, E x p e r i e n t i a 43 1021-1022 ( 1 9 8 7 ) . E.L. SCHIFFRIN, J. GUTKOWSKA, O. KUCHEL, M. CANTIN and J. GENEST, N. E n g l . J. Med. 312 1196-1197 ( 1 9 8 5 ) . T. YAMAJI. M. ISHIBASHI, H. NAKAOKA, K. IMATAKA, M. AMANO and J. F U J I I , L a n c e t 1 1211 ( 1 9 8 5 ) . H. SONNENBERG, R.F. KREBS and A . T . VERESS, IRCS Med. S c i . 12 783-784 ( 1 9 8 4 ) . H. SONNENBERG and A.T. VERESS, Biochem. B i o p h y s . Res. Commun. 124 443-449 ( 1 9 8 4 ) . M.G. CURRIE and W.H. NEWMAN, Biochem. B i o p h y s . Res. Commun. 131 806-814 ( 1 9 8 6 ) . R. GARClA, W. DEVINSKI, J. GUTKOWSKA, O. KUCHEL, G. THIBAULT, J. GENEST and M. CANTIN, Biochem. B i o p h y s . Res. Commun. 131 806-814 ( 1 9 8 5 ) . M. CANTIN, J. GUTKOWSKA, G. THIBAULT, R.W. MILNE, S. LEDOUX, S. MINLI, C. CHAPEAU, R. GARClA, P. HAMET and J. GENEST, L
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Pergamon Pres:
PRESENCE OF IMMUNOREACTIVE ATRIAL NATRIURETIC PEPTIDES IN PERICARDIAL FLUID OF HUMAN SUBJECTS WITH CONGENITAL HEART DISEASES Ja Hong Kuh*, Kong Soo Kim*, Suhn Hee Kim, Kyung Woo Cho, Kyung Hwan S e u l , and Gou Young Koh D e p a r t m e n t of P h y s i o l o g y and Chest S u r g e r y * Jeonbug N a t i o n a l U n i v e r s i t y M e d i c a l School Jeonju 5 6 0 - 1 8 0 , Korea (Received in final form April 24, 1990)
Summary The e p i c a r d i a l r e l e a s e of i m m u n o r e a c t i v e a t r i a l natriuretic p e p t i d e s ( i r - A N P s ) in i n s i d e - o u t p e r f u s e d rabb i t a t r i a has been r e p o r t e d . In o r d e r to d e t e r m i n e the p r e s e n c e of i r - A N P s in p e r i c a r d i a l f l u i d and to e v a l u a t e their biochemical characteristics, we measured the concentration of i r - A N P s in p e r i c a r d i a l fluid obtained from the p a t i e n t s w i t h c o n g e n i t a l h e a r t d i s e a s e s d u r i n g open h e a r t s u r g e r y . S e r i a l d i l u t i o n c u r v e s made w i t h t h e e x t r a t s of p e r i c a r d i a l f l u i d u s i n g Sep-Pak C18 c a r t r i d ges were p a r a l l e l w i t h s t a n d a r d c u r v e . The c o n c e n t r a t i o n of ir-ANPs in pericardial f l u i d was s i g n i f i c a n t l y lower than t h e c o r r e s p o n d i n g plasma concentration. On gel permeation and r e v e r s e - p h a s e high p e r f o r m a n c e liquid c h r o m a t o g r a p h y , the i r - A N P s in p e r i c a r d i a l fluid, plasma and atrial appendage showed both high and low m o l e c u l a r weights. The m a j o r peak of i r - A N P s in plasma was o b s e r ved a t the c o r r e s p o n d i n g f r a c t i o n to the alpha-human ANP and c o n s i d e r a b l e amount of high m o l e c u l a r w e i g h t form o f i r - A N P s was o b s e r v e d in p e r i c a r d i a l fluid. However, the major peak o f i r - A N P s in a t r i a l appendage was o b s e r v e d at the c o r r e s p o n d i n g f r a c t i o n to t h e r a t pro-ANP. The data s u g g e s t t h a t i r - A N P s e x i s t both high and low m o l e c u l a r w e i g h t forms in p e r i c a r d i a l fluid. Atrial m y o c y t e s have been known to c o n t a i n a t r i a l natriuretic peptides (ANPs), which caused p o t e n t n a t r i u r e s i s and diuresis, vasorelaxation and an i n h i b i t i o n of r e n i n and a l d o s t e r o n e secretions (I-5). However, l i t t l e i n f o r m a t i o n about the p h y s i o l o g i c a l c o n t r o l mechanisms f o r the ANP r e l e a s e from the a t r i a e x i s t s . Atrial d i s t e n s i o n or stretch (6-14), pressure (6, 8, 11, 1 4 ) , pacing frequency (10, 1 5 - 1 8 ) and humoral f a c t o r s (19-22) have been s u g g e s t e d f o r the r e g u l a t o r y f a c t o r s of the r e l e a s e o f ANP from the atria. The ANP i m m u n o r e a c t i v e s p e c i f i c granules in atrial m y o c y t e s were r e p o r t e d to be l o c a t e d in the s u b p e r i c a r d i a l Address a l l c o r r e s p o n d e n c e to : Dr. Kyung Woo Cho, Physiology, Jeonbug National University Medical Keum-Am-Dong-San, J e o n j u 5 6 0 - 1 8 0 , Korea 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
Department of School, 2-20
1978
ANP in Pericardial
Fluid
Vol. 46, No. 26, 1990
wall (23, 24) as w e l l as in t h e m y o c y t e s near the a t r i a l lumen (25, 26). Recently, Our group (27) r e p o r t e d the e p i c a r d i a l release of i r - A N P s in i n s i d e - o u t perfused rabbit atria. In this s t u d y we d e m o n s t r a t e d the p r e s e n c e o f i r - A N P in p e r i c a r d i a l fluid and c h a r a c t e r i z e d it. METHODS Subjects : Thirteen patients with congenital heart diseases (2 mitral stenosis, I Tetralogy of Fallot, 3 ventricular septal defect, 5 atrial septal defect, 2 aortic insufficiency) who u n d e r w e n t open h e a r t o p e r a t i o n s were s t u d i e d . E i g h t o f them was female and i n f o r m e d c o n s e n t was o b t a i n e d from the f a m i l y members or subjects. Blood samples were o b t a i n e d from healthy medical s t u d e n t s (n=20) f o r a c o m p a r i s o n . Preparation of samples: Pericardial fluid, plasma and atrial appendages were o b t a i n e d from s i x p a t i e n t s s u f f e r i n g congenital heart disease. Balanced f e n t a n y l a n e s t h e s i a was a d m i n i s t e r e d and t h o r a c o t o m y was p e r f o r m e d . A f t e r m e d i c a l s t e r n o t o m y the p e r i c a r dium was e x p o s e d . A s m a l l i n c i s i o n was a p p l i e d w i t h care and was electrocauterized. A c a n n u l a was i n t r o d u c e d i n t o the p e r i c a r d i a l space through i n c i s i o n and 15-20 ml o f pericardial fluid was immediately obtained into prechilled tubes c o n t a i n i n g 800 ul o f a mixture of ethylenediamine tetraacetic a c i d (EDTA) ( 2 . 7 mM i n pericardial fluid), phenylmethylsulfonyl fluoride (PMSF) (11.5 uM), a p r o t i n i n (200 K I U / m l ) , and soybean t r y p s i n i n h i b i t o r (SBTI) (50 BAEE/ml). In any case c o n t a m i n a t i o n from the b l o o d was n e g l i gible. Simultaneously, 10-20 ml o f blood was c o l l e c t e d from a radial artery into prechilled tubes c o n t a i n i n g a mixture of proteolytic enzyme i n h i b i t o r s as d e s c r i b e d above. Plasma samples from h e a l t h y p e r s o n s were t r e a t e d as the s a m e way. After centrifugation a t 4 C, 3,000 rpm f o r 15 m i n , the s u p e r n a t a n t and plasma were s e p a r a t e d . Atrial appendages were a l s o o b t a i n e d from patients during cardiac operation. After weighing, atrial appendages were b o i l e d i n i ml o f 0 . i M a c e t i c a c i d f o r I0 m i n , homogenized w i t h P o l y t ron h o m o g e n i z e r and c e n t r i f u g e d a t 4 C, I 0 , 0 0 0 g f o r 15 m i n . The ir-ANP in pericardial fluid, plasma and a t r i a l appendages was e x t r a c t e d u s i n g Sep-Pak C18 c a r t r i d g e s (Waters Associates, Milford, MA, U . S . A . ) as d e s c r i b e d p r e v i o u s l y (28-30). The e l u a t e s were lyophilized under g e n t l e s t r e a m of N2 g a s , reconstituted w i t h 0.1% t r i f l u o r o a c e t i c a c i d (TFA) and c e n t r i f u g e d a t lO,O00xg f o r 15 m i n . High P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h y (HPLC): One hundred m i c r o liters o f the e l u a t e s were s u b j e c t e d to the reverse-phase high performance l i q u i d c h r o m a t o g r a p h y (HPLC) on a uBondapak (Waters Associates, Milford, MA, U . S . A . ) column as d e s c r i b e d p r e v i o u s l y (31). Elution was p e r f o r m e d w i t h the l i n e a r g r a d i e n t of 20 % to 60 % a c e t o n i t r i l e i n 0 . 1 % TFA f o r 40 min a t a f l o w r a t e of I m l / m i n and each f r a c t i o n volume was I ml . Gel p e r m e a t i o n HPLC was a l s o p e r f o r m e d on a TSK-GEL G2000 SW (Toyo Soda, Tokyo, Japan) column ( 7 . 5 x 300 mm) and e l u t e d w i t h 30 % a c e t o n i t r i l e in 0 . I % TFA. The f l o w r a t e was 0.3 m l / m i n and the fraction volume was 0.3 m l . The f r a c t i o n a t e d samples were lyophilized and a s s a y e d .
Vol. 46, No. 26, 1990
ANP in Pericardial Fluid
1979
Ra d i o i m m u n o a s s a y o f i r - A N P : The i r - A N P was measured by r a d i o i m m u noassay as d e s c r i b e d p r e v i o u s l y (27-30). Anti-ANP antibody was purchased from D i a g n o s t i c & Research A n t i b o d i e s Inc. (Berkely, CA, U.S.A.) and synthetic atriopeptin I I I (AP I I I ) (Peninsula Laboratories, B e l m o n t , CA, U . S . A . ) was i o d i n a t e d u s i n g c h l o r a m i ne-T m e t h o d , as d e s c r i b e d p r e v i o u s l y (29). The l y o p h i l i z e d samples were r e c o n s t i t u t e d w i t h I00 ul of T r i s - a c e t a t e buffer and incubated w i t h a n t i - A N P a n t i b o d y f o r 24 hrs a t 4 C. Following the additional incubation w i t h 125 I-ANP f o r 24 hrs at 4 C, separation o f f r e e from bound form was a c h i e v e d by a d d i n g 1.0 ml of Dextran-coated charcoal suspension. S t a t i s t i c s : S t a t i s t i c a l s i g n i f i c a n c e of d i f f e r e n c e was tested using Student's t - t e s t and was defined as a P-value of less than 0,05. The r e s u l t s were given as means + s.e.m.
RESULTS Serial d i l u t i o n of the e x t r a c t s of p e r i c a r d i a l f l u i d i n h i b i t e d the binding of 125 I-ANP to the antibody in p a r a l l e l with the standard curve, as shown in Figure 1. The concentration of ir-ANP in p e r i c a r d i a l f l u i d was s i g n i f i c a n t l y lower t h a n the corresponding plasma concentration (Table i ) . No s i g n i f i c a n t c o r r e l a t i o n between the concentrations of plasma and p e r i c a r d i a l f l u i d was observed (y=42.31x-O.071, r=-0.12, p=0.687). The ir-ANP p r o f i l e s of e x t r a c t s on reverse-phase and gel permeation HPLC are shown in Figure 2 and 3. I r - A N P in p e r i c a r d i a l f l u i d showed both high and low molecular weights. The one of major peaks of ir-ANP was observed at the corresponding f r a c t i o n to the s y n t h e t i c alpha human ANP (99-126) and the other of major peaks was correspondent to r a t pro-ANP. Two of s i x samples showed mainly high molecular weight of irANP in p e r i c a r d i a l f l u i d . In a t r i a l appendage and plasma, b o t h forms of ir-ANP were also observed. The major form of ir-ANPs in a t r i a l appendage was high molecular weight. Although the low molecular weight form of irANPs in plasma was major form, a considerable amount of high molecular weight form of ir-ANP was also detected.
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1980
ANP in Pericardial
Fluid
Vol. 46, No. 26, 1990
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R e v e r s e - p h a s e HPLC p r o f i l e s of atrial natriuretic peptide in pericardial fluid (A), plasma ( B ) , and a t r i u m (C) on a uBondapak c o l u m n . Arrows indicate the peak p o s i t i o n s of elution of rat pro-atrial natriuretic p e p t i d e ( PRO) and a t r i o p e p t i n III (AP III), respectively. Rat pro-ANP was p u r i f i e d by the method o f T r i p p o d o e t al (38).
0
I0 20 30 Fraction No.
FIG.
40
3
Gel p e r m e a t i o n HPLC p r o f i l e s of atrial natriuretic peptide in pericardial fluid (A), plasma (B), and a t r i u m (C) on a TSKGel G2000 SW c o l u m n . Arrows indicate t h e peak p o s i t i o n s of elution of blue Dextran (Vo), b o v i n e serum a l b u m i n ( B S A ) , c y t o c h r o m e C (CC) and a t r i o p e p t i n III (APIII), respectively.
Vol. 46, No. 26, 1990
Table fluid, heart
ANP in Pericardial
Fluid
1981
1.
Immunoreactive atrial natriuretic p e p t i d e in p e r i c a r d i a l plasma and a t r i a l appendage from p a t i e n t s w i t h c o n g e n i t a l diseases Concentration Pericardial
fluid
Plasma Atrial
appendage
of
(pg/ml)
34.91
+ 10.45
(pg/ml)
103.00
+ 17.24
(ng/mg)
20.45
V a l u e s are t h e means + SEM o f
+
ir-ANP
8.25
9 patients.
DISCUSSION The p r e s e n t s t u d y shows the p r e s e n c e o f i r - A N P in p e r i c a r d i a l fluid of patients with congenital heart diseases. The concentration of i r - A N P in p e r i c a r d i a l f l u i d was l o w e r than i n plasma and no s i g n i f i c a n t correlation between them was f o u n d . In an a t t e m p t to f u t h e r c h a r a c t e r i z e the i r - A N P i n p e r i c a d i a l fluid, the ir-ANP profiles were i n v e s t i g a t e d by gel permeation and r e v e r s e - p h a s e HPLC. In p e r i c a r d i a l fluid and plasma, two major peaks o f i r - A N P c o r r e s p o n d i n g t o a l p h a - human ANP and r a t pro-ANP were found. The i r - A N P i n cerebrospinal fluid (33), ascitic fluid (32) and plasma ( 3 1 , 33) has been r e p o r t e d to be a low molecular weight form. Recently, Kim e t a l . (31) r e p o r t e d that a c o n s i d e r a b l e amount o f high m o l e c u l a r w e i g h t form o f irANP e x i s t s i n o v a r i a n f o l l i c u l a r fluid. A l s o , s e v e r a l papers have been shown the p r e s e n c e o f h i g h m o l e c u l a r w e i g h t form of ANP i n plasma (31, 34-37). The d i f f e r e n c e s i n irANP p r o f i l e s reported may be due to d i f f e r e n t specimen, unstability of pro-ANP, or activation of p r o t e o l y t i c enzymes d u r i n g e x t r a c t i o n . A l t h o u g h we do not know e x a c t l y the s i t e of o r i g i n of p e r i c a r d i a l ANP, i t may not be t a k e n up from the c i r c u l a t i o n . A special care was taken to o b t a i n pericardial fluid samples without contamination. We found no s i g n i f i c a n t correlation between t h e c o n c e n t r a t i o n of plasma and p e r i c a r d i a l ANP. Additionally, the ir-ANP profile in pericardial fluid is different from that in p l a s m a . The irANP in the p e r i c a r d i a l f l u i d may o r i g i n a t e from t h e ventricles (36) as w e l l as t h e a t r i a ( 2 7 ) . It is possible that the pro-ANP in p e r i c a r d i a l f l u i d may o r i g i n a t e from the v e n t r i c les. It i s w e l l known t h a t the a t r i a r e l e a s e mainly processed ANP. Recently, T h i b a u l t et al (36) r e p o r t e d t h a t the m a j o r p o r tion of the ANP in e f f l u e n t from the perfused ventricles is prohormone, and the v e n t r i c l e s are the m a j o r source of t h i s p r o ANP i n p l a s m a . The p r e s e n c e of high m o l e c u l a r w e i g h t of irANP in plasma from patients w i t h heart problems is consistent with previous reports (36,37). Plasma l e v e l s of ANP i n p a t i e n t s were significantly h i g h e r in h e a l t h y p e r s o n s ( 1 0 3 . 0 0 + 17.24 vs 44.80 + 2.97, p
1982
ANP in Perlcardial
Fluid
Vol. 46, No. 26, 1990
r e l e a s e of i r - A N P in p e r i c a r d i a l space. It is unclear at present t h a t the p r e s e n c e o f irANPs in p e r i c a r d i a l space i s p h y s i o l o g i c a l or p a t h o l o g i c a l in n a t u r e . ACKNOWLEDGMENTS We t h a n k Miss Kyung Mee S e u l , Eun J i n Nah and Kyung Hwa Nam f o r t h e i r e x p e r t t e c h n i c a l a s s i s t a n c e . T h i s work was s u p p o r t e d i n part by a g r a n t from the Korea S c i e n c e and Engineering Foundat i o n , Republic of Korea. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14 15 16 17 18 19 20. 21. 22. 23.
A . J . DEBOLD, H.B. BORENSTEIN, A . T . VERESS and H. SONNENBERG, L i f e S c i . 28 89-94 ( 1 9 8 1 ) . K.S. MISON~ R.T. GRAMMER, H. FUKUMI and T. INAGAMI, Biochem. B i o p h y s . Res. Commun. 123 444-451 (1984) M.C. CURRIE, D.M. GELLER, B.R. COLE, J . C . BOYLAR, W. YASHENG, S.W. HOLMBERG and P. NEEDLEMAN, S c i e n c e 221 71-73 ( 1 9 8 3 ) . K.ATARASHI, P . J . MURLOW, R. FRANCO-SAENZ, R. SHAPJAR and J. RAPP, S c i e n c e 224 992-994 ( 1 9 8 4 ) . M CANTIN and J. GENEST, Endocr. Rev. 6 107-127 ( 1 9 8 5 ) . R.E. LANG, H. THOELKEN, D. GANTEN, ~ . C . LUFT, H. RUSKOAHO, and T.H. UNGER, N a t u r e 314 264-266 ( 1 9 8 5 ) . J . R . LEDSOME, N. WILSON, C.A. COURNEYA and A . J . RANKIN, Can. J. P h y s i o l . P h a r m a c o l . 63 739-742 ( 1 9 8 5 ) . J . R . DIETZ, Am. J. P h y s i o l . 247 RIO93-RI096 ( 1 9 8 4 ) . A.J. DEBOLD, M.L. DEBOLD and I . R . SARDA, J. H y p e r t e n s i o n 4 ( s u p p l 2) s3-s7 ( 1 9 8 6 ) . G.E. BILDER, T . L . SCHOFIELD and E.H. BLAINE, Am. J. P h y s i o l . 251 F817-F821 ( 1 9 8 6 ) . H. RUSKOAHO, H. THOELKEN and R.E. LANG, P f l u g e r s A r c h . 407 170-174 ( 1 9 8 6 ) . R . J . SCHIEBINGER and J. LINDEN, C i r c . Res. 59 105-109 ( 1 9 8 6 ) . G, AGNOLETTI, A. RODELLA, R. FERRARI and P. HARRIS, J. Mol. C e l l . C a r d i o l . 19 249-253 ( 1 9 8 7 ) . D,E. UEHLINGER, T. ZAMAN, P. WEIDMAN, S. SHAW and M.P. GNADINGER, H y p e r t e n s i o n I0 249-253 ( 1 9 8 7 ) . R.J. SCHIEBINGER and J. LINDEN, Am. J. P h y s i o l . 251 HIO95N1099 ( 1 9 8 6 ) . M.G. ZIEGLER, S.R. SHACKFORD, K.D. WILNER and C.H. NORTON, E x p e r i e n t i a 43 1021-1022 ( 1 9 8 7 ) . E.L. SCHIFFRIN, J. GUTKOWSKA, O. KUCHEL, M. CANTIN and J. GENEST, N. E n g l . J. Med. 312 1196-1197 ( 1 9 8 5 ) . T. YAMAJI. M. ISHIBASHI, H. NAKAOKA, K. IMATAKA, M. AMANO and J. F U J I I , L a n c e t 1 1211 ( 1 9 8 5 ) . H. SONNENBERG, R.F. KREBS and A . T . VERESS, IRCS Med. S c i . 12 783-784 ( 1 9 8 4 ) . H. SONNENBERG and A.T. VERESS, Biochem. B i o p h y s . Res. Commun. 124 443-449 ( 1 9 8 4 ) . M.G. CURRIE and W.H. NEWMAN, Biochem. B i o p h y s . Res. Commun. 131 806-814 ( 1 9 8 6 ) . R. GARClA, W. DEVINSKI, J. GUTKOWSKA, O. KUCHEL, G. THIBAULT, J. GENEST and M. CANTIN, Biochem. B i o p h y s . Res. Commun. 131 806-814 ( 1 9 8 5 ) . M. CANTIN, J. GUTKOWSKA, G. THIBAULT, R.W. MILNE, S. LEDOUX, S. MINLI, C. CHAPEAU, R. GARClA, P. HAMET and J. GENEST, L
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