The in vitro intestinal absorption of enterostatin is limited by brush-border membrane peptidases

ELSEVIER

Regulatory Peptides 54 (1994)495-503

The in vitro intestinal absorption of enterostatin is limited by brush-border membrane peptidases J . F . H u n e a u a'*, C. E r l a n s o n - A l b e r t s s o n

b, C . B e a u v a l l e t a, D . T o m 6 a

INRA, UnitO de Nutrition Humaine et Physiologie Intestinale, Facult~ de Pharmacie, 75006 Paris, France bDepartment of Medical and Physiological Chemistry, University of Lund, S-221 O0 Lund, Sweden Received 27 October 1993; revised version received and accepted 5 September 1994

Abstract

The intestinal metabolism and absorption of enterostatin was studied using brush-border membrane vesicles and an in vitro model of intestinal segments from rabbit ileum mounted in Sweetana-Grass diffusion chamber. Hydrolysis of enterostatin was observed with both epithelial sheets and brush-border membranes. The main metabolite was found to be des-arginine-enterostatin. Dipeptidylpeptidase IV was found to play a minor role in enterostatin degradation, whereas carboxypeptidase P activity accounted for the initial step of peptide hydrolysis. More than 50?o of the amount of enterostatin added to the mucosal compartment of the Sweetana-Grass diffusion chamber was degraded after 30 min. Enterostatin was mainly absorbed as degradation products but a small transepithelial passage of des-arginine-enterostatin and immunoreactive enterostatin was also detected. Although immunoreactive enterostatin exhibits a low apparent permeability coefficient in rabbit ileum, the luminal production of this peptide may be of physiological importance in the control of appetite.

Keywords: Peptide absorption; Colipase; Satiety signal; Proline residue; Des-arginine-enterostatin

1. Introduction

Enterostatin is the amino-terminal pentapeptide of pancreatic procolipase released in the intestine by trypsin activity [ 1 ]. This peptide has the sequence A P G P R in man, chicken and rabbit and V P D P R in pig and ox [ 1,2 ]. This tryptic cleavage simultaneously generates both colipase, which serves as a protein * Corresponding author. Fax: + 33 1 43255653. 0167-0115/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 0 1 1 5 ( 9 4 ) 0 0 0 8 8 - 3

cofactor for pancreatic lipase necessary for intestinal fat digestion [ 1], and enterostatin, which has been shown to be a powerful anorectic peptide [3,4] with a specific ability to suppress the intake of dietary fat [5,6]. Furthermore, this regulation of fat intake by enterostatin appears to be of physiological significance based on observations of a high peptide production in rats with a low voluntary fat intake, S5B/PI rats, and a low production of the peptide in rats with a high voluntary fat intake, Osborne-

496

J.F. Huneau et al.

Regulato D, Peptides 54 (1994) 495-503

Mendel rats [7]. Since enterostatin exerts its anorectic effect when given either centrally [8] or peripherally [9], and since enterostatin is produced in the intestine [ 10], the intestinal uptake of enterostatin is probably of importance in the expression and control of its physiological action. The intestinal absorption of bioactive peptides and their subsequent delivery to endogenous targets is usually hindered by their hydrolysis by brush-border membrane peptidases [11-16]. The aim of the present study was to evaluate the intestinal metabolism and transepithelial transport of enterostatin. As a model for the study of hydrolysis and transepithelial passage of the peptide, brush-border membrane vesicles and in vitro preparation of rabbit ileum mounted in Sweetana-Grass diffusion chambers were used.

2. Materials and methods

2. I. Substances Enterostatin (alanylprolylglycylprolylarginine, A P G P R ) was a product of Ferring AB (Malm0, Sweden). [2-3H-Pro]enterostatin, obtained by tritiation of Ala-Dehydropro-Gly-Pro-Arg, was synthesized by Axis Research (Oslo, Norway). Diprotin A and benzoylcarbonylprolyltryptophan (CBZ-ProTrp) were obtained from Bachem (Basel, Switzerland). Phosphoramidon, captopril, leupeptine, diisopropylfluorophosphate (DFP), 1,10-phenanthroline and EDTA were obtained from Sigma chemicals (La Verpilli6re, France). All other products were of research grade.

2.2. Transport experiments Male New Zealand White rabbits weighing 2.53.5 kg were killed by i.v. injection of sodium pentobarbital. Segments of the distal ileum were removed, rinsed free of intestinal contents, and the serosal and external muscular layers stripped off with fine forceps. The tissue was opened along the mesenteric

border and 4-cm-long segments mounted between two halves of Sweetana-Grass diffusion chambers [17] (Precision Instruments design, Los Altos, CA, USA). Care was taken to avoid Peyer's patches. Each side of the tissue (exposed area 2.51 cm 2) was bathed with 7.5 ml of isotonic Ringer solution consisting of(in mM) 140 Na +, 5.2 K ~ , 1.2 Ca 2~ , 1.2 Mg 2+, 120 C1 , 25 H C O 3 , 2.4 HPO42 and 0.4 H2PO 4 (pH 7.4). Oxygenation of the tissue was ensured by a gas lift of 02/C02 (95:5) and the temperature was maintained at 37°C throughout the study with an aluminium block heater. The spontaneous potential difference was short-circuited by an automatic voltage clamp (World Precision Instruments, Sarasota, FL, USA) and the short-circuit current corrected for fluid resistance. Tissue conductance was calculated according to Ohm's law. After stabilizing the electrical parameters for 15 min, enterostatin was added to the mucosal compartment with simultaneous addition of 5 #Ci of [2-3H Pro]enterostatin. Samples of 500 ~tl were withdrawn every 10 min from both mucosal and serosal compartments. The amount of radioactivity was determined in a 50 #1 aliquot by liquid scintillation counting. ZnC12 (final concentration 2.10 -2 M) was added to the remaining sample. After a 10,000 g centrifugation, 100 gl of the supernantant were analysed using reverse phase HPLC. The remaining fraction was lyophylized and used for immunoassay detection of enterostatin. The transport rate of immunoreactive enterostatin was used to calculate the apparent permeability coefficient of the peptide (Papp, in cm s l) according to the formula Papp = (dQ/dt)x 1/(A'Co) where dQ/dt is the transport rate of enterostatin, C o the initial concentration in the mucosal compartment and A the exposed area of mucosa.

2.3. Brush-border membrane vesicle experiments Brush-border membrane vesicles (BBMV) were prepared from everted rabbit intestine by calcium precipitation and differential centrifugation, as

J.F. Huneau et al. / Regulatory Peptides 54 (1994) 495-503

previously described [ 11]. Enterostatin degradation was measured in the presence of purified BBMV. The buffer consisted of 10 mM Hepes-Tris pH 7.5 and 300 mM mannitol. The reaction was initiated by mixing 50/~1 of diluted BBMV (37/~g proteins) with 400/~1 of enterostatin solution (final concentration 1 mM). The reaction proceeded at 37°C and was terminated by the addition of 1 volume of 50~o trifluoroacetic acid (TFA). After centrifugation at 15,000 g for 20 min, the supernatant was analysed by HPLC and the rate of hydrolysis calculated from the disappearance of the peak corresponding to enterostatin. The effect of various peptidase inhibitors on enterostatin hydrolysis was examined by preincubation of BBMV with inhibitors at 37°C for 20 min.

497

for 30 min. A colour reaction was obtained by adding p-nitrophenyl phosphate. The optical density was measured by reading the absorbance at 405 nm on an Emax precision microplate reader (Molecular Devices Corporation, Menlo Park, CA, USA). The concentration of enterostatin of the unknown samples were calculated by the use of a Softmax computer program (Softmax version 2.01, Molecular Devices Corporation, Menlo Park, CA, USA).

2.6. Calculations Results were expressed as means + S.D. Statistical comparisons were made using the non-parametric Wilcoxon test (NPAR1WAY, SAS 6.03, SAS Institute, Cary, NC, USA).

2.4. High pressure liquid chromatography 3. Results

HPLC analysis was run on a Waters gradient HPLC system equipped with an ultrabase C18 reverse phase column (5 x 250 mm) (Shandon, Les Ulis, France) and a UV detector at 214 nm. Peptides were eluted at 1 ml min-1 with a 20 min linear gradient of 4 to 45 ~o acetonitrile in 0.1 ~o TFA. When necessary, 1 ml fractions were collected and the amount of radioactivity determined by liquid scintillation counting.

2.5. ELISA analysis of enterostatin (APGPR) To quantify immunoreactive enterostatin, a competitive-enzyme-linked immunosorbent assay was used [18]. Briefly, microtiter 96-well plates were coated with 100 #1 of albumin-CGG-enterostatin, 0.2/~g/ml. 50/~1 of enterostatin (standard solution, 10-6-10 -8 M) or 50 #1 of samples at the correct dilution, were added to the plates followed by 50/al of antiserum (R5181 1:2000). After incubating for one hour at room temperature, anti-rabbit IgG-biotin conjugate was added and the plates incubated for 30 min at room temperature, followed by incubation with an extravidine alkaline phosphatase solution

3.1. Brush-border membrane hydrolysis of enterostatin In an initial experiment the hydrolysis of enterostatin by intestinal peptidases was investigated using brush-border membrane vesicles and HPLC analysis. The main hydrolytic product detected by HPLC was the tetrapeptide des-arginine-enterostatin (Fig. 1). The identification of the des-arginineenterostatin peak was confirmed by amino acid analysis (data not shown). A small peak which coeluted with alanyl-proline and glycyl-proline was also observed. The rate of hydrolysis measured after 10 min was 6.44+ 1.13 nmol min-1 #g-x of membrane protein (Table 1). The most efficient inhibitors of enterostatin degradation were the chelating agents phenantroline and EDTA. DFP and diprotin A (dipeptidyl peptidase IV inhibitors) slightly inhibited the degradation of enterostatin, whereas leupeptin (proteinase inhibitor) and captopril (angiotensinconverting enzyme inhibitor) had no effect on enterostatin degradation. The addition of 1 mM CBZ-Pro-Trp to the incubation medium partially prevented the hydrolysis of the peptide.

498

J.F. Huneau et al. / Regulator 3' Peptides 54 (1994) 495-503 1

Table 2

2

T Y

Effect of mucosal enterostatin on the electrical parameters of the rabbit ileum in Sweetana-Grass diffusion chambers

==

Mucosal enterostatin (M)

r-

lsc b~c

e-

fiPd G 6G

c-

F Fig. 1. HPLC analysis ofenterostatin hydrolysis by brush-border membrane vesicles (BBMV). Enterostatin (1 mM) was incubated for 10 min with 37/~g BBMV proteins at 37°C. Peaks 1, 2 and 3 correspond to enterostatin, des-arginine-enterostatin and a mixture of alanyl-proline and glycyl-proline, respectively.

Table 1 Hydrolysis of enterostatin by brush-border membrane peptidases Addition

None Phenanthroline 1 mM EDTA 1 mM Diprotin A 1 mM D F P 1 mM Leupeptine 10/~M Captopril 10 #M CBZ-Pro-Trp 1 mM

Enterostatin hydrolysis nmol/#g protein per min

~o of control

6.44 ± 1.13 0.97 ±0.01" 2.40 ± 0.33* 4.61 ± 0.61" 5.49±0.61 5.95 + 0.31 9.58 ± 1.58 2.94 ± 0.36*

100 15 37 71 85 92 148 56

Means + S.D. of 5 determinations. * Significantly different from control, P < 0.05.

10 -3

10 -4

10 -5

10-~,

18.5±9.3 4.3±1.8" -1.9±1.1 -0.4±0.1" 13.0±8.2 -1.7±3.6

18.3±9.1 2.0±1.6 -2.6±1.7 -0.2±0.2 8.2±2.8 0.1±0.2

24.5±7.5 0.3±1.0 -3.5±0.3 -0.2±0.2 7.1±1.9 -0.3±0.3

21.7±4.4 0.1±1.8 -1.9±0.4 0.0±0.1 11.6±0.9 0.2±1.3

Values are means + S.D. of 5 to 6 animals. Short-circuit current (Isc) is expressed in p A cm 2, potential difference (Pd) is expressed in mV, and conductance (G) is expressed in mS cm z. Isc, b Pd and b G correspond to changes in Isc, Pd and G induced by 10- 3 to 10- 6 M enterostatin, measured 10 min after its mucosal addition. * Significantly different from 0, P < 0.05.

Grass diffusion chamber induced a slight but significant increase in the short-circuit current and the potential difference, without affecting the tissue conductance (Table 2). This effect was not observed with more diluted solutions of the peptide. H P L C analysis of aliquots collected in the mucosal compartment at various times following the mucosal addition of 10-3 M enterostatin showed that peptide hydrolysis had occurred. The more hydrophobic tetrapeptide des-arginine-enterostatin was recovered in the incubation medium (Fig. 2). Smaller hydrolytic products, including dipeptides and free proline, were also observed. The rate of hydrolysis of enterostatin was constant, 58 + 9~o of the initial amount of radiolabelled material being hydrolysed after 30 min for 10-3 M A P G P R (Fig. 3). 3.3. Mucosal-to-serosal transport of enterostatin

3.2. Mucosal hydrolysis of enterostatin The addition of 10-3 M enterostatin to the mucosal side of the rabbit ileum mounted in Sweetana-

The absorption of A P G P R was studied for 30 min after mucosal addition of 10 -6 to 10 -3 M enterostatin to the rabbit ileum in the Sweetana-Grass diffusion chamber by following the appearance of

499

J.F. Huneau et al. / Regulatory Peptides 54 (1994) 495-503

0 min

12.000

lOOM

lk X2

4.000

"~

80

0

o ~

60

10min

12.000

2

40

A

0 ~ 1

T~

8.000

---'0 E " 1

4.000

f

0

e~

2o 0-

~1

~

20min

~

10

1 182'~000

20

30

40

50

60

70

flme (mTn)

~

~

~

j

4.0~

.~0

0 4 ~ 12.000

<

1

30 min

0

.L

,

I0

Fig. 2. HPLCanalysisofmucosalcompartmentatdifferenttimes following the addidon of 1 mM enterostatin. Absorbance at 214 nm was recorded directly with a UV detector and 1 ml fractions were collected for radioactivity determination (histogram). Peaks 1 and 2 corresponded to enterostadn and desarginine-enterostatin, respectively. Peak 3 coeluted with free proline, alanyl-proline and glycyl-proline.

radiolabelled material in the serosal compartment. The amount of radioactivity in the serosal compartment increased as a function of time for the different concentrations (Fig. 4). Moreover, the amount of transported material recovered after 30 min was proportional to the amount of enterostatin present in the mucosal compartment. The transported material was then analysed by reverse phase H P L C (Fig. 5). Most of the radioactivity recovered in the serosal compartment corresponded to free [3H ]proline and alanyl-[3H]proline (peak 1). Radiolabelled material was also recovered in fractions 15-16, cor-

Fig. 3. Rate of hydrolysis of 1 mM enterostatin in the mucosal compartment of the Sweetana-Grass diffusion chambers. The percentages of initial mucosal radioactivity recovered as intact enterostatin (m), des-arginine-enterostatin (A) and alanylproline + proline (O) were determined by HPLC analysis. Results are means + S.D. of 5 experiments.

responding to a mixture of enterostatin and desarginine-enterostatin (peak 2). This fraction represented 1 to 3 ~ of the transported material (Table 3). Intact enterostatin recovered in the serosal compartment after 30 min was also quantified by specific ELISA and found to be 20 + 10 pmol c m - 2 for 10- 3 M mucosal enterostatin. The apparent permeability coefficient of immunoreactive enterostatin was 1.1.10-8 cm s-1. For mucosal concentrations ranging from 10 - 6 to 10 - 4 M , the amount of immunoreactive enterostatin recovered in the serosal Table 3 Amount of enterostatin and des-arginine-enterostatin across the intestinal epithelium (in pmol cm- 2) 10 -3 M Total 9993+5889 A P G P R + A P G P 296+ 108

10 -4 M

10 -5 M

absorbed

10 -6 M

1330_+803 186+96 16+10 20+ 13 6+3 0.2+0.1

Results are expressed as means +_S.D. of 4 to 6 determinations.

J,F. Huneau et al. / Regulatory Peptides 54 (1994) 495-503

500

]

4000 i

3.0

E o

"~

3500

2.5

o 2.0

1

2

f

f

4, >

3000

1.5

e- 2500 O

tO

1.0

7~ ~ 2ooo

'~

0.5

& o

0.0I0

20

i 30

1500

q

time (mln)

Fig. 4. Mucosal to serosal transport of radiolabelled material across isolated rabbit ileum. Initial concentrations of the peptide in the mucosal compartment were 10 - 6 (O), 10- 5 (O), 10 - 4 ( i ) and 10 - 3 M (A). The amount ofradiolabelledmaterial recovered in the serosal compartment was compared to the amount of peptide added in the mucosal compartment at the begining of the experiment and results are expressed as % of mucosal peptide absorbed per cm2 of mucosa. Means + S.D. of 6 experiments.

1000

500

| 2

4

6

nail III n n n ~ III ~ |

8

10

12

14

16

n nim

18

20

4. D i s c u s s i o n

fraction number Fig. 5. HPLC analysis of the serosal compartment of the Sweetana-grass diffusion chamber 30 min after the addition of 1 mM enterostatin to the mucosal compartment. Absorbance at 214 nm was recorded directly with a UV detector and 1 ml fractions were collected and the radioactivity determined by liqtfid scintillation counting. Peak 1 is a mixture of dipeptides and free proline, whereas peak 2 is composed of enterostatin and des-arginine-entero statin.

The a b s o r p t i o n o f intact enterostatin across the intestinal m u c o s a is an i m p o r t a n t clue in u n d e r s t a n d ing the m e c h a n i s m o f action for enterostatin in the regulation o f energy intake [ 1 , 3 - 1 0 ] . This peptide, being p r o d u c e d in the intestinal lumen, has been shown to display anorectic activity when injected into the circulation [6]. The present results d e m o n strate that although enterostatin is extensively hyd r o l y s e d by b r u s h - b o r d e r m e m b r a n e p e p t i d a s e s and a b s o r b e d largely as d i p e p t i d e s a n d free amino acids, an a b s o r p t i o n o f b o t h 'des-arginine-enterostatin a n d intact enterostatin m a y occur across the rabbit ileum in vitro.

T h e first step in enterostatin hydrolysis by intestinal m e m b r a n e p e p t i d a s e s , according to our studies, is a loss o f the C-terminal arginine residue, as indicated by the recovery o f high a m o u n t s o f desarginine-enterostatin. Since pancreatic enzymes are unable to hydrolyse proline-containing peptides, c a r b o x y p e p t i d a s e P, a m e m b r a n e - b o u n d p e p t i d a s e specifically cleaving C-terminal amino acids adjacent to a proline residue [19,20], is suggested to play a key role in the d e g r a d a t i o n o f enterostatin. F u r t h e r evidence for a role o f c a r b o x y p e p t i d a s e P in the d e g r a d a t i o n o f enterostatin was the significant

c o m p a r t m e n t s was in no case different from the b a s a l value.

J.F. Huneau et al. / Regulatory Peptides 54 (1994) 495-503

prevention of hydrolysis obtained with the chelating agents phenantroline or EDTA [ 19] known to inhibit carboxypeptidase P. Moreover, a carboxypeptidase substrate peptide containing proline residue in the penultimate position, as with the N-blocked CBZ-Pro-Trp, was found to significantly retard the rate of enterostatin degradation. On the other hand, captopril, an inhibitor of angiotensin-converting enzyme, was unable to prevent enterostatin hydrolysis, thus indicating that the angiotensin-converting enzyme was not implicated in this process. A surprising feature was the poor effect of DFP and diprotin A in stabilizing enterostatin, indicating a minor role of dipeptidylpeptidase IV (DPP IV) in the degradation process. DPP IV is known to play a major role in the hydrolysis of peptides with prolyl residues in position 2 and 4 such as fl-casomorphin and substance P [11,21]. Using intact porcine procolipase as a substrate, Heymann et al. [22] reported purified DPP IV to be able to release the N-terminal dipeptide Val-Pro from pancreatic procolipase, corresponding to the N terminal sequence of porcine enterostatin. However, no attempt was made to study the hydrolysis of the activation pentapeptide enterostatin. More recently, Bowyer and his coworkers have reported that phenylmethylsulphonylfluoride, a DPP IV inhibitor, failed to stabilize enterostatin immunoreactivity in human serum [ 18]. Our results are thus in agreement with those of Bowyer et al. [ 18] and suggest carboxypeptidase P to be the main enzyme responsible for the initial degradation of enterostatin by the intestinal mucosa, DPP IV playing only a minor role in the degradation of the pentapeptide. The gradual disappearance of des-arginine-entrostatin observed in the mucosal compartment of the Sweetana-Grass diffusion chamber is associated with an increase in the amount of radiolabelled material recovered in the fraction corresponding to dipeptides, thus suggesting that des-arginine-enterostatin is further hydrolysed by DPP IV. The absorption of enterostatin across rabbit ileum was studied for 30 min in Sweetana-Grass diffusion

501

chamber. Rabbit ileum mounted in Ussing chambers or related devices such as Sweetana-Grass diffusion chambers, has been shown to be a valuable model for studying the intestinal absorption of peptides and proteins. This intestinal segment has a satisfactory viability in vitro and provides accurate and reproducible results for up to 120 min of incubation [ 11,23,24]. However, due to extensive hydrolysis of enterostatin by brush-border membrane peptidases, transport experiments were limited to 30 min. At that time, more than 55~o of the initial amount of peptide was recovered as des-arginine-enterostatin, dipeptides and amino acids. Since the absorption of dipeptides and amino acids is far more efficient than that of tetra- and pentapeptides [ 14], the recovery of radiolabelled material in the serosal compartment of the Sweetana-Grass diffusion chambers may represent the absorption of the degradation products rather than that of the intact peptide. The gradual accumulation of hydrolysis products in the mucosal compartment could therefore be responsible for the acceleration in the transport rate observed between 10 and 30 min. An active transport of the degradation products could also account for the slight increase in transepithelial potential difference and short-circuit current observed following the mucosal addition of 1 mM enterostatin. Using H P L C analysis, we were able to observe a transport of tetra-and pentapeptide representing up to 3~o of the total absorption. The amount of immunoreactive enterostatin recovered in the serosal compartments after 30 min represented 0.2~o of the total absorption, thus suggesting that des-arginineenterostatin accounted for the major part of the tetraand pentapeptide fraction collected by HPLC. Immunoreactive enterostatin was found to have an apparent permeability coefficient of 1.1.10 - 8 cm s - 1 in rabbit ileum. This coefficient is 10-times smaller than that of immunoreactive oxytocin measured in rat ileum [ 12], probably because of a higher rate of hydrolysis of enterostatin compared with that of oxytocin. It has been shown that in vivo poorly absorbed peptides have low apparent permeability

502

J.F. Huneau et al. / Regulatory Peptides 54 (1994) 495-503

coefficients in vitro [25], this coefficient being even lower when the peptides are hydrolysed by epithelial peptidases [ 12]. Although only very small amounts of intact enterostatin were absorbed under basal conditions across the rabbit ileal sheets in vitro, intestinal absorption of luminal enterostatin may be of physiological importance in vivo. Enterostatin is mainly produced in the proximal part of the intestine [10], although it has also been recovered in h u m a n distal ileum (Maht, S. and Erlanson-Albertsson, C., unpublished results). Since the paracellular permeability of the jejunum is usually slightly higher than that of the ileum [26,27], the overall absorption of intact enterostatin m a y be higher than that predicted by the in vitro experiment. Moreover, it has been shown that luminal factors, such as bile salts or high glucose concentration (25 mM), can increase intact peptide transport by promoting paracellular absorption [13,16,28,29]. Such factors are present in the intestinal lumen after meal ingestion and m a y thus p r o m o t e the absorption of intact enterostatin. Lastly, our results suggest that the concomitant release of other proline-containing peptides during the luminal digestion of endogenous or exogenous proteins can retard the hydrolysis of enterostatin by m e m b r a n e peptidases and m a y also contribute to an increased enterostatin absorption. Indeed, an anorectic effect of enterostatin has been observed after oral administration in the form of procolipase pellets [8 ], suggesting that intestinal absorption m a y also occur in vivo. In conclusion, the above experiments demonstrate poor absorption of intact enterostatin by the rabbit ileum in vitro, a large fraction of the peptide being degraded by the removal of the C-terminal arginine residue by carboxypeptidase P. This hydrolysis may be of physiological significance since the removal of the C-terminal arginine is associated with a loss of the anorectic effect [9]. Further studies are needed to characterize the absorption of enterostatin in vivo as well as the regulation of its absorption.

Acknowledgements This work was made possible thanks to a scholarship for C E A from I N S E R M , Paris, France and the housing offered at Centre Culturel Sutdois, Paris. by the Swedish Institute, Stockholm, Sweden.

References [ 1] Erlanson-Albertsson, C., Pancreatic colipase. Structural and physiological aspects, Biochim. Biophys. Acta, 1125 (1192) 1-7. [ 2] Colwell, N.S., Aleman-Gomez, J.A., Sasser, T. and Kumar, V.B., Cloning and characterization of rabbit pancreatic colipase, Int. J. Biochem., 25 (1993) 885-890. [ 3] Erlanson-Albertsson, C. and Larsson, A., A possible function of pancreatic procolipase activation peptide in appetite regulation, Biochimie, 70 (1988) 1245-1250. [ 4] Erlanson-Albertsson, C. and Larsson, A., The activation peptide of pancreatic procolipase decreases food intake in rats, Regul. Pept., 22 (1988) 325-331. [ 5] Erlanson-Albertsson, C., Mei, J., Okada, S., York, D., and Bray, G.A., Pancreatic procolipase propeptide, enterostatin, specifically inhibits fat intake, Phys. Behav., 49 (1991) 1191-1194. [ 6] Okada, S., York, D.A., Bray, G.A., and ErlansonAlbertsson, C., Enterostatin (Val-Pro-Asp-Pro-Arg), the activation peptide of procolipase, selectively reduces fat intake, Phys. Behav., 49 (1991) 1185-1189. [ 7] Okada, S., York, D.A., Bray, G.A., Mei, J., and ErlansonAlbertsson, C., Differential inhibition of fat intake in two strains of rat by the peptide enterostatin, Am. J. Physiol., 262 (1992) R l l l I - R l l l 6 . [8] Shargill, N.S., Tsujii, S., Bray, G.A. and ErlansonAlbertsson, C., Enterostatin suppresses food intake following injection into the third ventricle of rats, Brain Res., 544 (1991) 137-140. [ 9] Mei, J. and Erlanson-Albertsson, C., Effect of enterostatin given intravenously and intracerebroventricularly on highfat feeding in rats, Regul. Pept., 23 (1992) 209-218. [10] Mei, J., Bowyer, R.C., Jehanli, A.M.T., Patel, G., and Erlanson-Albertsson C., Identification of enterostatin, the pancreatic procolipase activation peptide, in the intestine of rat; effect of CCK-8 and high-fat feeding, Pancreas, 8 (1993) 488-493. [11] Maht, S., Tom6 D., Dumontier, A.M. and Desjeux, J.F., Absorption of intact morphiceptin by diisopropylfluorophosphate-treated rabbit ileum, Peptides, 10 (1989) 45-52.

J.F. Huneau et al. / Regulatory Peptides 54 (1994) 495-503

[12] Lundin, S., Pantzar, N., Broeders, A., Ohlin, M. and WestrOm, B., Differences in transport rate of oxytocin and vasopressin analogues across proximal and distal isolated segments of the small intestine of the rat, Pharm. Res., 8 (1991) 1274-1280. [13] Ungell, A.L., Andreasson, A., Lundin, K. and Utter, L., Effect of enzymatic inhibition and increased paracellular shunting on transport of vasopressin analogues in the rat, J. Pharm. Sci., 81 (1992) 640-645. [14] Matthews, D.M., Absorptions of peptides. In D.M. Matthews (Ed.), Protein absorption. Development and present state of the subject, Wiley-Liss, New York, 1991, pp. 235-319. [15] Takaori, K., Burton, J., and Donowitz, M., The transport of an intact oligopeptide across adult mammalian jejunum, Biochem. Biophys. Res. Commun., 137 (1986) 682-687. [16] Aungst, B., Blake, J.A. and Hussain, M.A., An in vitro evaluation of metabolism and poor membrane permeation impending intestinal absorption of leucine enkephalin, and methods to increase absorption, J. Pharmacol. Exp. Ther., 259 (1991) 139-145. [17] Grass, G.M. and Sweetana, S.A., In vitro measurement of gastrointestinal tissue permeability using a new diffusion cell, Pharm. Res., 5 (1988) 372-376. [18] Bowyer, R.C., Jehanli, A.M.T., Patel, G. and HermonTaylor, J., Development of enzyme-linked immunosorbent assay for free human procolipase activation peptide (APGPR), Clin. Chim. Acta, 200 (1991) 137-152. [ 19] Erickson, R.H., Song, I.S., Yoshioka, M., Gulli, R., Miura, S. and Kim Y.S., Identification of proline-specific carboxypeptidase localized to brush-border membrane of rat small intestine and its possible role in protein digestion, Dig. Dis. Sci., 34 (1989) 400-406. [20] Yoshioka, M., Erickson, R.H. and Kim, Y.S., Digestion and assimilation of proline-containing peptides by rat intestinal brush-border membrane carboxypeptidases. Role of the combined action of angiotensin-converting enzyme and carboxypeptidase P, J. Clin. Invest., 81 (1988) 1090-1095.

503

[21] Conlon, J.M. and Sheehan, L., Conversion of substance P to C-terminal fragments in human plasma, Regul. Pept., 7 (1983) 335-345. [22] Heymann, E., Mentlein, R., Nausch, 1., Erlanson-Albertsson, C., Yoshimoto T. and Feller, A.C., Processing of procolipase and trypsinogen by pancreatic dipeptidyl peptidase IV, Biomed. Biochim. Acta, 45 (1986) 575-584. [23] Caillard, I. and Tomr, D., Different routes for the transport of ~-lactalbumin in the rabbit ileum, J. Nutr. Biochem., 3 (1992) 653-658. [24] Brachet, P., Gaertner, H., Tom6 D., Dumontier, A.M., Guidoni, A. and Puigserver, A., Transport of N-~t(or N-e)L-methionyl-L-lysine and acetylated derivatives across the rabbit intestinal epithelium, J. Nutr. Biochem., 2 (1991) 387-394. [25] Artursson, P. and Karlsson, J., Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells, Biochem. Biophys. Res. Commun., 175 (1991) 880-885. [26] Artursson, P., Ungell, A.-L. and Lrfroth, J.E., Selective paracellular permeability in two models of intestinal absorption: cultured monolayers of human intestinal epithalial cells and rat small intestinal segments, Pharm. Res., 10 (1993) 1123-1129. [27] Davis, G., Santa Ana, S., Morawski, S. and Fordtran. J., Permeation characteristics of human jejunum, ileum proximal colon and distal colon: results of potential difference measurements and unidirectional fluxes, Gastroenterology, 83 (1982) 844-850. [28] Atisook, K. and Madara, J.L., An oligopeptide permeates intestinal tight junctions at glucose-elicited dilations. Implications for oligopeptide absorption, Gastroenterology, 100 (1991) 719-724. [29] Fasano, A., Budillon, G., Guandalini, S., Cuomo, R., Parrilli, G., Cangiotti, A.M., Morroni, M. and Rubino, A., Bile acids reversible effects on small intestinal permeability. An in vitro study in the rabbit, Dig. Dis. Sci., 35 (1990) 801-808.