474

Biochimica et Biophysica Acta, 544 (1978) 474--481

© Elsevier/North-Holland Biomedical Press

BBA 28730 C H A R A C T E R I Z A T I O N OF A VASOACTIVE INTESTINAL PEPTIDE-SENSITIVE A D E N Y L A T E CYCLASE IN RAT INTESTINAL EPITHELIAL CELL MEMBRANES

B. AMIRANOFF, M. LABURTHE, C. DUPONT and G. ROSSELIN Unit~ de Recherche de Diabdtologie et d'Etudes Radio-lmmunologiques des Hormones Protdiques (INSERM, U.55-CNRS, ERA 494), H6pital Saint-Antoine, 184, rue du Faubourg Saint-Antoine, 75571 Paris Cedex 12 (France)

(Received May 29th, 1978) Summary A vasoactive intestinal peptide-sensitive adenylate cyclase in intestinal epithelial cell membranes was characterized. Stimulation of adenylate cyclase activity was a function of vasoactive intestinal peptide concentration over a range of 1 . 1 0 - 1 ° - - 1 • 10 -7 M and was increased six-times b y a maximally stimulating concentration of vasoactive intestinal peptide. Half-maximal stimulation was observed with 4.1 _+0.7 nM vasoactive intestinal peptide. Fluoride ion stimulated adenylate cyclase activity to a higher extent than did vasoactive intestinal peptide. Under standard assay conditions, basal, vasoactive intestinal peptide- and fluoride-stimulated adenylate cyclase activities were proportional to time of incubation up to 15 min and to membrane concentration up to 60 gg protein per assay. The vasoactive intestinal peptide-sensitive enzyme required 5--10 mM Mg 2+ and was inhibited b y 1 • 10 -s M Ca 2+. At sufficiently high concentrations, both ATP (3 mM) and Mg 2+ (40 mM) inhibited the enzyme. Secretin also stimulated the adenylate cyclase activity from intestinal epithelial cell membranes b u t its effectiveness was 1/1000 that of vasoactive intestinal peptide. Prostaglandins Et and E2 at 1 - 1 0 -s M induced a two-fold increase of cyclic AMP production. Vasoactive intestinal peptide was the most p o t e n t stimulator of adenylate cyclase activity, suggesting an important physiological role of this peptide in the cyclic AMP-dependent regulation of the intestinal epithelial cell function.

Introduction Cyclic AMP is implicated in the regulation of intestinal epithelial cell function, since in different in vitro preparations of gut mucosa, it elicits a stimula-

475 tion of chloride and bicarbonate secretion [1] and an inhibition of sodium absorption [2], contributing to a net fluid secretion [3]. Furthermore, cyclic AMP was shown to stimulate the uptake by jejunal mucosa of neutral and dibasic amino acids [4]. The effect of VIP in stimulating small intestine electrolytic secretion [5,6] appears to be due to the presence of specific receptors for vasoactive intestinal peptide (VIP), acting through adenylate cyclase (EC 4.6.1.1): specific binding sites for VIP were previously characterized in rat intestinal epithelial cells [7,8] and in human malignant colonic cells [8,9]; moreover, VIP induced cyclic AMP accumulation in these cells [ 7--9]. Attempts to isolate adenylate cyclase in systems such as homogenates of stripped colonic mucosa [10,11] and membranes prepared from ileal mucosa scraping [12], resulted in obtaining an enzyme sensitive to high VIP concentration (1 • 10 -7 M--1 • 10 -s M). In this paper, we report a m e t h o d for obtaining an adenylate cyclase extremely sensitive to VIP (1" 10-1°--1 • 10 -7 M) using membranes prepared from isolated epithelial cells from rat jejuno-ileum. We describe the properties of this VIP-sensitive adenylate cyclase regarding its substrate and ionic requirement. Furthermore, we demonstrate that among many substances tested, VIP is the most potent and efficient stimulator of the adenylate cyclase activity, suggesting it is a key substance in the regulatory control of the intestinal enzyme. Materials and Methods

Chemicals and reagents. Cyclic AMP and creatine phosphate (A grade) were purchased from Calbiochem, phosphocreatine kinase from Boehringer, EDTA from Prolabo. Bacitracin, isoproterenol, epinephrine, norepinephrine, 3-isc~ butyl-l-methylxanthine (iBuMeXan), prostaglandins El and E2 were obtained from Sigma Chemical Company. Bovine serum albumin (BSA, fraction V) was purchased from Miles laboratories. Other chemicals were of reagent grade. Synthetic neurotensin and substance P (bovine) were purchased from Beckman. Porcine VIP was a gift of the National Institutes of Health (USA) and Professor V. Mutt (Karolinska Institute Stockholm). Synthetic secretin was generously supplied by E. Wiinsch (Max Planck Institute, Miinchen, G.F.R.), synthetic octapeptide of cholecystokinin by M. Ondetti (Squibb, U.S.A.). Preparation of membranes from intestinal epithelial cells. Membranes were obtained in two main steps: in a first step, cells were isolated from jejuno-ileum of male Sprague-Dawley rats (200--250 g body weight), according to Mitjavila et al. [13] with slight modifications [7]. Briefly, the jejuno-ileum was everted and shaken by hand during 10 rain in 100 ml of 14%oNaC1, EDTA 2.5 raM, pH 7.5 at 4°C. The everted bowel was removed from the cell suspension. A pellet of cells was obtained after centrifugation at 2000 × g for 5 rain [7]. In a second step, epithelial cells were disrupted in a glass-teflon homogenizer (clearance 0.095--0.115 mm, Braun G.m.b.H., Melsungen, G.F.R.) and membranes were obtained following exactly the m e t h o d described by Miirer et al. [14]. The membranes used represented the "crude plasma membrane" of the Miirer et al. m e t h o d [14]. The washed pellet was resuspended in Krebs-Ringer phosphate buffer (pH 7.5) at amembrane protein concentration of 3 mg/ml and distributed

476

into aliquots which were stored at --80°C. Aliquots were used once and discarded. Adenylate cyclase assay. Unless otherwise mentioned, incubation conditions were adapted from those described previously [15]. In the standard conditions, the incubation solution contained in a 250-pI final volume, 0.8 m M ATP, the ATP-regenerating system (20 m M creatine phosphate, 1 mg/ml phosphocreatine kinase), 0.2 m M iBuMeXan, 25 m M Tris-HCl (pH 7.5), 4 mg/ml bovine serum albumin, 1 m M E D T A and 200 pg/ml bacitracin. Reactions were started with addition of membrane preparation at approx. 40/~g (in 50 pl) of protein, i.e. 160 pg/ml final concentration. Incubations were performed at 30°C, for 15 min. The reaction was stopped and cyclic A M P determined by radioimmunoassay as described previously [15]. Protein determination. Proteins were estimated by the method of Lowry et al. [16] using bovine serum albumin as the standard.

Results

Assay conditions for adenylate cyclase activity Fig. 1 shows the time-course of adenylate cyclase activity. Under standard conditions (see Methods), the VIP-stimulated adenylate cyclase activity was proportional to time of incubation within a period of 15 min. This was also true for fluoride-stimulated adenylate cyclase activity. So subsequent experiments were performed within a 15-min period of incubation and enzyme activity was expressed as pmol cyclic AMP produced per min. Fig. 2 presents the adenylate cyclase activity with varying concentrations of membrane proteins assayed in the medium. Under standard conditions, the basal and stimulated production of cyclic AMP were proportional to protein concentration up to 240 gg/ml. Thus, adenylate cyclase was further assayed in the range of membrane concentrations between 60 and 240 pg protein/ml.

40

.,= E

1000 EE o

a." ,,$. 20

500 ...a

0 0

5

10

15

TIME,minutes

0 60

240

420

[PROTEIN],p9/ml

Fig. 1. T i m e - c o u r s e o f t h e a d e n y l a t e c y c l a s e a c t i v i t y . N o a d d i t i o n ( s ) , 1 • 1 0 - 8 M V I P (@), 5 m M N a F (o). The memb1~ne concentration was 120 #g protein per m]. Assay conditions were specified under Methods. E a c h v a l u e is t h e m e a n ± S . E . o f t r i p l i c a t e d e t e r m i n a t i o n s . Fig. 2. E f f e c t o f v a r y i n g m e m b r a n e c a n c e n t r a t i o n s o n a d e n y l a t e e y c l a s e a c t i v i t y . N o a d d i t i o n ( s ) , 1 • 1 0 -8 M V I P (@), 5 m M N a F (o). A s s a y c o n d i t i o n s w e r e s p e c i f i e d u n d e r M e t h o d s . E a c h v a l u e is t h e m e a n + S . E . of triplicate determinations.

477

o

E

~- 150

200

.=_ E

150

m o

~E m

- 0

20

40

o0

1

2

3

[A T P] ,ram Fig. 3. E f f e c t o f v a r y i n g Mg 2+ c o n c e n t r a t i o n s o n a d e n y l a t e cyclase activity. M e m b r a n e s w e r e i n c u b a t e d in m e d i a c o n t a i n ' r a g n o s t i m u l a n t (1), 1 • I 0 - $ M V I P ( e ) , 5 m M N a F ( o ) a n d v a z y i n g c o n c e n t r a t i o n s o f MgCI 2 . T h e m e m b r a n e c o n c e n t r a t i o n w a s 1 2 0 Dg p r o t e i n per ml. O t h e r c o n d i t i o n s w e r e as d e s c r i b e d u n d e r M e t h o d s . Each v a l u e is t h e m e a n +S.E. o f triplicate d e t e r m i n a t i o n s . Fig. 4. Effect of v a r y i n g A T P c o n c e n t r a t i o n s o n a d e n y l a t e cyclase activity. M e m b r a n e s w e r e i n c u b a t e d in m e d i a c o n t a i n i n g n o s t i m u l a n t (B), 1 • 1 0 - 8 M V I P ( e ) , 5 m M N a F ( o ) a n d v a r y i n g c o n c e n t r a t i o n s o f A T P . T h e m e m b r a n e c o n c e n t x a t i o n w a s 1 8 0 / ~ g p r o t e i n per ml. MgCI 2 c o n c e n t r a t i o n w a s 1 0 m M . O t h e r c o n d i t i o n s w e r e as d e s c r i b e d u n d e r M e t h o d s . Each v a l u e is t h e m e a n +S.E. o f triplicate d e t e r m i n a t i o n s .

The optimal pH for the adenylate cyclase stimulation by VIP and fluoride is between 7.2 and 7.5 (not shown). The presence of a phosphodiesterase inhibitor, iBuMeXan, slightly enhanced the VIP- and fluoride-stimulated adenylate cyclase activity: 0.2 mM iBuMeXan, a maximally effective concentration (data not shown), produced a 22% and 25% increase of adenylate cyclase activity stimulated by 1 • 10 -8 M VIP and 5 mM fluoride, respectively. This slight effect suggested that under experimental conditions used, the phosphodiesterase activity of the membrane preparation was low. The effect of varying magnesium and ATP concentrations on cyclase activity are shown in Figs. 3 and 4, respectively. Under standard conditions, absence of MgC12 in the assay resulted in a complete inhibition of basal, VIP- and fluoridestimulated activity (Fig. 3). 10 mM MgC12 gave optimal basal and VIP-stimulated production of cyclic AMP. Fluoride-stimulated adenylate cyclase was maximal at 20 mM MgC12. High concentrations of MgC12 (40 mM) inhibited both VIP- and fluoride-stimulated enzyme activity. The effect of ATP concentration was tested in the presence of 10 mM MgC12 (Fig. 4). Without any substrate, no enzyme activity could be detected. Both basal and stimulated adenylate cyclase activity increased with ATP concentration, with an optimum at 0.8--1.6 mM ATP. Above 1.6 mM ATP, VIP and fluoride-stimulated cyclic AMP production decreased. The reason of this inhibition by high concentrations of ATP, could be explained by the fact that ATP in excess is not complexed with MgC12 but exists in a free form as previously suggested [17]. This free form of substrate could be a strong inhibitor of adenylate cyclase activity [18]. Calcium is a potent inhibitor of basal and stimulated adenylate cyclase activity (Fig. 5). Calcium at a concentration as low as 1 • 10 -s M inhibited the production of cyclic AMP. This inhibition increased with increasing CaC12 concentration in the assay. 2.5 mM calcium salt in the medium almost completely

478

*u

4O

o eL.

~- 150

2O s=

~ 100

c=-

0

o."

50

0

OJ

0.5

0

1 2.5

[C'CI21,mM

-10 10-9 10-8 10-7 IV' P]. M

Fig. 5. E f f e c t o f v a r y i n g c o n c e n t r a t i o n s o f CaC12 o n a d e n y l a t e c y c l a s e a c t i v i t y . M e m b r a n e s w e r e incub a t e d i n m e d i a c o n t a i n i n g n o s t i m u l a n t (m), 1 • 1 0 -8 M V I P ( * ) 5 rnM N a F ( o ) a n d v a r y i n g c o n c e n t r a t i o n s o f CaCI 2. T h e m e m b r a n e c o n c e n t r a t i o n w a s 2 4 0 / ~ g p r o t e i n p e r m l . E D T A w a s o m i t t e d f r o m t h e m e d i u m . O t h e r c o n d i t i o n s w e r e as d e s c r i b e d u n d e r M e t h o d s . E a c h v a l u e is t h e m e a n +S.E. o f t r i p l i c a t e d e t e r m i n a tions. Fig. 6. E f f e c t o f v a r y i n g V I P c o n c e n t r a t i o n s on a d e n y l a t e c y c l a s e a c t i v i t y . A s s a y c o n d i t i o n s w e r e described u n d e r Methods. The values represent the m e a n s of seven experiments.

abolished basal and stimulated adenylate cyclase activity. To prevent the inhibitory effect of endogenous calcium in membrane preparation, studies were performed in the presence of 1 mM EDTA.

TABLE I E F F E C T OF V A R I O U S SUBSTANCES ON A D E N Y L A T E CYCLASE OF I N T E S T I N A L E P I T H E L I A L CELL MEMBRANES M e m b r a n e s w e r e i n c u b a t e d in c o n d i t i o n s d e s c t i b e d u n d e r M e t h o d s . E a c h v a l u e is t h e m e a n + S.E. o f triplicate determinations. Substances added

Concentration (M)

None

--

VIP

1 • 10 -8

Secretin

1 •

1 1 Isoproterenol Epinephrine Norepinephrine Prostagland/n E 1 Prostaglandin E 2 Glucagon Neurotensin Substance P Octapeptlde of eholecystoklnln NaF

1

1 1 I

1 I 1 1

1 5

Adenylate cycl~e activity (pmol cyclic AMP/rain per rag protein) 6.54

10 - 7 • 10 -6 • 10 -5 • 10 -5 • 1 0 -5 • 10 -5 • I 0 -5 • 10 -5 • 10-6 • 10-6 • 10-6 • 10-6 • 10 - 3

* N o t s / g n i f l c a n t l y i n c r e a s e d o v e r basal a c t i v i t y .

± 1.02

24.79

± 6.66

13.54 16.70 24.70 9.68 9.16 7.74 13.96 15.46 6.42 8.32

± + ~ + ± ± ± ± ± ±

0.01 1.56 1.59 0.51 0.37 0.86 1.87 1.14 0.07 1.08 6.69 ± 0.33 10.24 + 1.98 7 7 . 1 2 + 7.13

479

Effect of vasoactive intestinal peptide and other substances on adenylate cyclase activity Membranes prepared from intestinal epithelial cells from rat jejuno-ileum exhibited an adenylate cyclase sensitive to very low concentration of VIP (Fig. 6). Adenylate cyclase activity was a function of VIP concentration over a range of 1 • 10-1°--1 • 10 -7 M. VIP, at 1 • 10 -7 M, maximally stimulated adenylate cyclase activity which was increased 6-fold above basal activity. Half-maximal stimulation of enzyme activity was observed with 4.1 + 0.7 nM VIP. VIP produced the largest stimulation of the adenylate cyclase activity of any agents which we have tested (Table I). None of adrenergic substances tested displays any significant stimulation of enzyme activity, whereas prostaglandins E1 and E2, at 1 • 10 -s M, exhibited a similar 2-fold stimulation over the basal cyclic AMP production. A variety of other polypeptlde hormones tested: glucagon, neurotensin, substance P, octapeptide of cholecystokinin, failed to stimulate the adenylate cyclase activity. Secretin elicited a stimulation of adenylate cyclase activity similar to that observed with VIP. However, secretin was effective over a 1000-fold higher range of concentration than was VIP (1 • 10-7--1 • 10 -s M), making unlikely the interference of secretin in the physiological regulation of intestinal epithelial cells adenylate cyclase. Discussion

We succeeded in characterizing an adenylate cyclase highly sensitive to VIP, further demonstrating that the early step of VIP action on intestinal epithelial cells is the activation of a membrane-bound adenylate cyclase. This effect of VIP is in agreement with its effectiveness in stimulating cyclic AMP accumulation in different cells [7--9] or mucosa preparations [10--12] from the gut. Several arguments led us to conclude that gut adenylate cyclase is remarkable by the preponderance of VIP in its regulatory control. Indeed, the characteristics of this enzyme related to its substrate, pH and ionic dependence, were similar to the properties initially described for the glucagon-sensitive adenylate cyclase in liver [19] and for other adenylate cyclase systems [20]. Gut adenylate cyclase appeared to be unique for its sensitivity and its specificity to VIP. In our preparation, adenylate cyclase is sensitive to concentrations of VIP as low as 1 • 10 -l° M. This finding correlates well with the dose of VIP which induced stimulation of cyclic AMP accumulation in intestinal epithelial cells [7,8]. The sensitivity to VIP of these two systems was much higher than that obtained with other intestinal preparations such as rabbit ileal mucosa [12] and rat colonic mucosa [10,11]. As an explanation of these discrepancies, we suggest that the VIP content of these preparations was different. VIP has been demonstrated to be present in the deep strata of the gut wall [21], i.e., the mucosal-muscular fraction (excluding epithelium) which contains the totality of VIP a m o u n t measured in whole jejuno-ileum [21,22]. We verified that intestinal epithelial cells we used for the membrane preparation do not contain VIP [22]. Consequently, membrane adenylate cyclase obtained with such preparations can be fully stimulated by exogenous VIP, whereas sensitivity of the mucosa system to VIP could be masked by the presence of endogenous VIP. Other explanations are also possible: a 5-fold stimulation of adenylate

480

cyclase has been observed in homogenates of intestinal epithelial cells from hamster [23]. This effect is obtained with VIP concentrations which are in a ten-fold higher range than in our system, suggesting that these discrepancies may be related to species differences in VIP-intestinal epithalial cell interaction. Other VIP-sensitive adenylate cyclases have been characterized in plasma membranes of liver [24,25], exocrine pancreas [26], adipose tissue [24,25] and recently in brain synaptosomes [27], arguing for the VIP action via cyclic AMP production. However, in these tissues, other hormones are more efficient and/or more potent than VIP in stimulating enzyme activity. In liver membranes, maximal cyclic AMP production induced by VIP is approx. 5-fold lower than that induced by glucagon [24]. In pancreas acinar cell membranes, secretin and VIP display the same efficiency in maximally stimulating adenylate cyclase activity, but with a concentration 15-fold lower for secretin than VIP [26]. In that respect, receptors in membranes of intestinal epithelial cells exhibit a characteristic pattern in which the VIP-sensitive adenylate cyclase predominates, since among all other compounds tested, only secretin stimulates the intestinal epithelial cell adenylate cyclase activity to the same extent as did VIP, but with concentrations 1000-fold higher than VIP. Existing evidence indicating that the adenylate cyclase is present only at the contraluminal membrane of the intestinal epithelial cell [14], together with the observation of VIP immunoreactive nerves in deep strata of the gut wall [28] and in mucosa-muscular tissue [21] strongly suggest action of VIP near the site where it is released justifying the term of local hormone given to this peptide. Therefore, the discovery of specific binding sites for VIP on rat intestinal epithelial cells [7,8] coupled to the presence of a VIP-sensitive adenylate cyclase in plasma membranes argue for a physiological role of VIP in intestinal cell function. Acknowledgments In addition to those who gave us hormone (see Methods) we would like to thank C. Brunet for her careful preparation of the manuscript. This work was supported by the Institut National de la Sant~ et de la Recherche M6dicaie (Contrat libre 78.5.030.4), by the D~l~gation G6n6rale ~ la Recherche Scientifique et Technique (Grant 77.7.0465) and by the Centre National de la Recherche Scientifique. References 1 2 3 4 5 6 7

Field, M., Plotkin, G.R. and Silen, W. (1968) Nature 217, 469--471 Field, M., F r o m m , D., A1-Awqatt, Q. and Greenough, W.B. (1972) J. Clin. Invest. 5 1 , 7 9 6 - - 8 0 4 Field, M. (1974) Gastroenterology 66, 1 0 6 3 - - 1 0 8 4 Kinzie, J.L., Ferrendelli, J.A. and Alpess, D.H. (1973) J. Biol. Chem. 248, 7 0 1 8 - - 7 0 2 4 Barbezat, G. and Grossman, M. (1971) Science 174, 422--424 Krejs. G.J., Barkley, R.M., Read. N.W. and Fordtran, J.S. (1978) J. Clin. Invest. 61, 1337--1345 Laburthe, M., Besson, J., Hui Bon Hoa, D. and Rosselin, G. (1977) C.R. Acad. Sci. Paris 234, 2139-21 42 8 Laburthe. M., Bataille, D., Rousset. M., Besson, J., Broer, Y.. Zweibaum, A. and Rosselin, O. (1978) in Proceedings o f the Membrane Proteins section of the 11 th FEBS Meeting Copenhagen 1977 (Nicholls, P., Moiler, J.V., Leth, P. and MoodY, A.J., eds.), Vol. 45, Pp. 271--290, Pergamon Press, Oxford

481 9 Laburthe, M., Rousset, M., Bolssard, C., Chevalier, G., Zweibaum, A. and Rosselin, G. (1978) Proc. Natl. Acad. Sci. U.S. 75, 2772--2775 10 Waidman, D.B., Gardner, J.D., Zfass, A.M. and Makhlouf, G.M. (1977) Gastroenterology 73, 518-523 11 Racusen, L.C. and Binder, H.J. (1977) Gastroenterology 73, 790--795 12 Schwartz, C., Kimberg, D., Sheerin, H., Field, M. and Said, S.L. (1974) J. Clin. Invest. 54, 536--544 13 Mitjavila, M.T., Mitjavila, S. and Derache, R. (1973) Toxicology 1,237--248 14 M/irer, H., A m m a n n , E., Biber, J. and Hopfer, U. (1976) Biochim. Biophys. Acta 433, 509--519 15 Rosselin, G. and Freychet, P. (1973) Biochim. Biophys. Acta 304, 541--551 16 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265---275 17 De Hahn, C. (1974) J. Biol. Chem. 249, 2756--2762 18 Lin, M.C., Salomon, Y., Rendell, M. and Rodbell, M. (1975) J. Biol. Chem. 250, 4246---4252 19 Pohl, S.L., Birnbaumer, L. and Rodbell, M. (1971) J. Biol. Chem. 246, 1849--1856 20 Robison, G.A., Butcher, R.W. and Sutherland, E.W. (1971) Cyclic AMP, pp. 79--82, Academic Press, New York 21 L a r ~ o n , L.I., Fahrenkrug, J., Schaffaiitsky de MuckadeB, O., Sundler, F., Hakanson, R. and Rehfled, J.F. (1976) Proc. Natl. Acad. ScL U.S. 73, 3197--3200 22 Besson, J., Laburthe, M., Bataille, D , Dupont, C. and Rosselin, G. (1978) Acta Endocrinol. 87, 799-810 23 Gaginella, T.S., Phillips, S.F., Dozois, R.R. and Go, V.L. (1978) Gastroenterology 74, 11--15 24 Desbuquots, B., Laudat, M.H. and Laudat, P. (1973) Biochem. Biophys. Res. Commun. 53, 1187-1194 25 Bataille, D., Freychet, P. and Rosselin, G. (1974) Endocrinology 95, 713--721 26 Milutinovi~, S., Schulz, I., Rosselin, G. and Fasold, H. (1977) First International Symposium on Hormonal Receptors in Digestive Tract Physiology, INSERM symposium No. 3 (Bonfils, S., Fromageot, P. and Rosselin, G., eds.), pp. 213--226, Elsevier, Amsterdam 27 Deschodt-Lanckman, M., Robberecht, P. and Christophe, J. (1977) FEBS Lett. 83, 76---80 28 Larsson, L.I. (1977) Histochemistry 54, 173--176

Characterization of a vasoactive intestinal peptide-sensitive adenylate cyclase in rat intestinal epithelial cell membranes.

474 Biochimica et Biophysica Acta, 544 (1978) 474--481 © Elsevier/North-Holland Biomedical Press BBA 28730 C H A R A C T E R I Z A T I O N OF A VAS...
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