Processing and transfer of epidermal growth factor in developing rat jejunum and ileum

Peptides, Vol. 11, pp. 1093-1102. ©Pergamon Press plc, 1990. Printed in the U.S.A.

0196-9781/90 $3.00 + .00

Processing and Transfer of Epidermal Growth Factor in Developing Rat Jejunum and Ileum R. K. R A O , * O. K O L D O V S K ~ ' , * t M. KORC,:~ P. F. P O L L A C K , * S. W R I G H T t A N D T. P. D A V I S §

Departments of *Pediatrics, i'Physiology, .~Medicine, and §Pharmacology University of Arizona College of Medicine, Tucson, AZ 85724 R e c e i v e d 5 M a r c h 1990

RAO, R. K., O. KOLDOVSK'Y, M. KORC, P. F. POLLACK, S. WRIGHT AND T. P. DAVIS. Processing and transfer of epidermal growthfactor in developing rat jejunum and ileum. PEPTIDES 11(6) 1093-1102, 1990.--Using everted sac technique we demonstrated the transfer of ~25I-mEGF across the jejunal and ileal walls of suckling, weanling and adult rats. The transfer by the suckling rat jejunum and ileum was significantly inhibited by the presence of dinitrophenol and sodium azide or by the replacement of sodium with potassium or choline. RP-HPLC analysis detected carboxy-terminal processing of 125I-mEGF in suckling and adult rat jejunum and ileum. Suckling rat jejunum produced ~2SI-des(53)mEGF and 125I-des(49-53)mEGF, whereas ~25I-des(48-53)mEGF was detected in suckling rat ileum or adult rat jejunum and ileum. All three forms of ~25I-mEGF bound to anti-EGF antibody and EGF receptors. The receptor binding of 125I-des(53)mEGF was higher than that of ~251-mEGF, but those of ~25I-des(49-53)mEGF and 125I-des(48-53)mEGF were greatly diminished. Results indicate a carboxy-terminal processing of mouse EGF during uptake and transfer in the small intestine of developing and adult rats, and the resulting products showed altered receptor binding. An identical amino acid sequence of the C-terminal pentapeptide of EGF from mouse, human and possibly rat may suggest a biological significance of C-terminal processing of EGF in the small intestine. Peptide

Metabolism

Neonatal

Small intestine

METHOD

EPIDERMAL growth factor (EGF), a polypeptide originally detected in mouse submaxillary glands (6), is found in salivary (17) and duodenal (18) secretions as well as in milk and colostrum of several species (12). Orogastrically administered EGF exhibits trophic effects on the gastrointestinal tract (including liver and pancreas) in newborn rabbits and suckling rats (12,13). In adult rats, it was shown that intraluminally administered EGF stimulated ornithine decarboxylase activity and DNA synthesis in the small intestine (34). We demonstrated previously that orogastrically administered 125I-labeled mouse EGF (mEGF) is absorbed, appearing in various organs (liver, lung and kidney) of suckling (31) and weanling (33) rats. This was later confirmed by both orogastric administration in suckling mice (20) and administration to isolated intestinal segments in suckling rats (7,21). Degradation products of L25I-mEGF were detected in the gastrointestinal tract and other tissues. However, the role of the small intestine in the metabolism of 125I-mEGF is unclear. In our present studies, of which a preliminary report has been published (22), we have characterized the in vitro uptake and transfer of ~25I-mEGF by jejunum and ileum of suckling, weanling and adult rats. Using RP-HPLC analysis we also characterized the degradation products of 12SlmEGF.

Chemicals All chemicals used were of analytical grade. Chloramine-T, bovine serum albumin (BSA), carboxypeptidase B (C-7011), trypsin (T-8642), N'ethylmaleimide (N'EM), dinitrophenol (DNP), sodium azide (NAN3) and cyanogen bromide activated sepharose were purchased from Sigma Chemical Company (St. Louis, MO) and receptor grade mEGF from Collaborative Research (Lexington, MA). 125I-Sodium iodide was purchased from Amersham (Arlington Heights, IL); 3H-polyethylene glycol (PEG; mol.wt. 4000) and 14C-mannitol from New England Nuclear (Boston, MA). All other chemicals were from Fisher Scientific (Tustin, CA). Triple distilled water, with a final distillation in the presence of 0.1% potassium permanganate and 0.2% potassium hydroxide to remove hetero-organic water contaminants, was used throughout the experiments.

Animals Sprague-Dawley rats were reared in our animal colony and culled to 10 pups at the age of 2 days. Experiments were performed at the age of 12 (suckling) and 31 (weanling) days regard-

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less of sex. For adult animals, male rats were weaned at the age of 30 days and fed with a standard laboratory chow (Lab Blox, Allied Mills, Chicago, IL) until 82 days of age. Rats of all ages were fasted for 2 hours before killing. Suckling rats were fasted in a cage with half of the cage resting on a heating pad to help maintain normal body temperature. Experiments were performed between 9 and l l a.m.

Preparation of 12Sl-Labeled mEGF Mouse EGF was labeled with ~25I using the chloramine-T method (4). Stock 125I-mEGF (0.72 mCi/nmole) containing 95% immunoreactive radioactivity was used. 125I-mEGF was purified by RP-HPLC for characterization of degradation products. ~25Ides(53)m-EGF, 15I-des(49-53)mEGF and 125I-des(48-53)mEGF were prepared from ~25I-mEGF as described by Schaudies and Savage (26).

Tissue Uptake and Serosal Transfer of 1251-EGF Animals were killed by decapitation, and the small intestine from the ligament of Treitz to the ileocecal junction was quickly removed and divided into 3 equal segments. The proximal (jejunum) and distal (ileum) segments were flushed with 3 ml (suckling) or 5 ml (weanling or adult) of 0.9% saline. The central 6-7 cm section of each segment was excised and everted using a glass rod (for weanling and adult) or polyethylene (Intramedic PE-50) tubing (for suckling). Everted sacs were prepared and incubated with 2 ng/ml of 125I-mEGF in mucosal fluid for 30, 60 and 120 min. This concentration of EGF was at least 10-fold lower than that of rat milk, and well below that of salivary and duodenal secretions (17,18). Everted sacs were prepared and incubated as described previously (14) with the following modifications. Krebs Ringer bicarbonate, pH 7.4, containing 10 mM fructose and 0.2% BSA, gassed with 95% O 2 was used as mucosal and serosal fluids. The composition of the buffer was modified in some experiments as described in the Results section. The gas mixture (95% 0 2 -}- 5 % C02) was constantly delivered to the surface of the mucosal fluid (pH of mucosal fluid remained stable at 7.4 until the end of incubation). After incubation, sacs were rinsed three times with 0.9% saline (5 ml) at room temperature and blotted with wet tissue paper. Weight of empty and filled sacs were recorded before and after incubation. The serosal transfer of fluid was calculated from the difference between the weight of the fluid content before and after incubations. Radioactivity in the serosal fluid (serosal transfer of mEGF) as well as in intestinal wall (tissue uptake of mEGF) was measured in a gamma counter. Active transport of D-glucose was measured by the incubation of everted sacs in the presence of 10 mM D-glucose in both serosal and mucosal fluids, and by estimating the glucose accumulation on serosal fluids by the glucose oxidase method. Uptake and transfer of 3H-PEG and 14C-mannitol were studied under similar conditions to evaluate the simple passive diffusion. Integrity of intestinal tissues during incubations was assessed by exclusion of poly-R 478, a nonabsorbable marker (28). Poly-R 478 was estimated spectrophotometrically at 510 nm.

pernatant was collected and neutralized with 1 N sodium hydroxide, then centrifuged at 3000 rpm for 10 rain at 4°C to remove the formed precipitate. This procedure extracted more than 90% of the radioactivity. 125I-mEGF, in extracts of initial and final mucosal fluid, intestinal wall and serosal fluid were analyzed by anti-EGF antibody affinity chromatography as described previously (31) or by RP-HPLC. RP-HPLC analysis of ~25I-mEGF added during homogenization and extraction demonstrated an absence of any degradation of EGF under these conditions.

RP-HPLC ~25I-mEGF in mucosal fluids, tissue and serosal fluids was extracted in 100 mM phosphate buffer, pH 2.4. A curvilinear gradient of 3--40% acetonitrile against 100 mM phosphate buffer, pH 2.4, monitored at a flow rate of 2 ml/min for 60 min (40°C) was used to elute the samples from Beckman ultrasphere C18ODS column (5 micrometer). Fractions of 0.6 rain were collected and radioactivity was measured in a gamma counter.

Receptor Binding of IeSI-mEGF Human placental membranes were prepared as described by Hock et al. (8). Labeled EGF or its degradation products (approximately 10,000 cpm), with or without 1 microgram unlabeled mEGF (4000-fold excess), were incubated with placental membranes (100 microgram protein) in 0.3 ml of 0.05 M phosphate buffer, pH 7.4, containing 0.1% BSA. After one hour at room temperature, the membranes were filtered and washed on GF/B glass microfiber filter (Whatman, Ltd; England) using a filtration manifold (Hoefer Scientific Instruments, San Francisco, CA). Under these conditions only 5-10% nonspecific binding was detected. Specific binding of stock ~2SI-mEGF was approximately 35%. Binding of degradation products of ~25I-mEGF was compared with that of intact '25I-mEGF.

Statistical Analysis All values are expressed as radioactivity (total or immunoreactive) per gram intestinal segment. All statistical analyses were performed by ANOVA followed by range test (Fisher PLSD) using program, Statview, in Macintosh microsoft computer. RESULTS

In our present studies we detected a time-dependent increase of sodium- and energy-dependent transport of fluid and glucose in everted sacs of all segments studied up to 60 min. In addition, a lack of transfer of poly-R 478, a nonabsorbable marker (28), suggested the viability of everted sacs for up to 60 min. At 120 min, diffusion of poly R478 was elevated particularly in adult rat intestinal segments, indicating the loss of viability. Transfer of ~25I-mEGF across the intestinal wall (serosal transfer) was determined by measuring the radioactivity in serosal fluids. Radioactivity in the wall of everted sacs was analyzed to characterize the binding and uptake of ~25I-mEGF (tissue uptake).

Tissue Uptake and Serosal Transfer of 1251-mEGF Tissue Extraction and Biochemical Analysis Samples were homogenized in 2 ml (suckling) or 4 ml (weanling and adult) water at 4°C using a Potter Elvehjem homogenizer (T-line laboratory stirrer, Emerson, NJ) at 4000 rpm for 1 rain. Homogenates of tissue, mucosal fluids and serosal fluids were mixed with a one-ninth vol. of 0.5 M HC1 and kept on ice for one hour; it was then centrifuged at i05,000 × g for 30 rain. The su-

Tissue uptake (Fig. 1) of radioactivity showed no age-dependent differences except for a slight, but significant difference at 120 min between suckling and weanling rats (p<0.05). Serosal transfer was highest in suckling rat jejunum and lowest in adult rat jejunum or ileum. Activities capable of removing iodine from proteins were described previously in developing rat intestine. It is possible that some of the label appearing in these samples may

INTESTINAL PROCESSING OF EPIDERMAL GROWTH FACTOR

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FIG. 1. Tissue uptake (A, jejunum; B, ileum) and serosal transfer (C, jejunum; D, ileum) of 125ImEGF (total radioactivity) by everted sacs of jejunum or ileum from suckling (O), weanling ([3) and adult (A) rats. Everted sacs were incubated at 37°C for varying times in the presence of 125I-mEGF (2 ng/ml). Radioactivity in the intestinal wall (tissue uptake) and serosal fluids (serosal transfer) is expressed as cpm per 0.1 mg wet weight of intestine. Values (mean +-SEM; n = 3-7) are significantly different from those for weanling (*) or adult rats (@) (p<0.05).

represent free iodine. In our present studies, only minor fractions (less than 8%) of radioactive material present in tissues and serosal fluids of suckling rat intestinal segments were free iodine. It was slightly higher in serosal fluid of adult rat intestinal segments (15-17%). Therefore, it should be noted that differences in transfer by different segments could be partially due to differences in formation and transfer of free iodine. Characterization of radioactivity by anti-EGF antibody affinity chromatography indicated considerable degradation of t25ImEGF. Immunoreactive radioactivity in tissue extracts at different time periods varied from 22-52%, 31-57% and 69-75% of total radioactivity, and in serosal fluids 9-28%, 9-55% and 45-68% in suckling, weanling and adult rats, respectively. Figure 2 shows time-dependent increases of immunoreactive radioactivity in tissue extracts with F(2,5)= 27.8, p<0.002 for jejunum, F(2,5)= 9.6, p<0.019 for ileum of suckling rats and F(2,6)=29.8, p<0.0008 for jejunum of weanling rats, and in serosal fluids with F(2,6) = 25.5, p<0.0012 for jejunum, F(2,6) = 92.5, p<0.0001 for ileum of suckling rats and F(2,6)= 7.3, p<0.025 for weanling rat jejunum.No significant time-dependent increase was detected in weanling rat ileum and adult rat jejunum and ileum. In tissue extracts of suckling rat jejunum, immunoreactive radioactivities were significantly lower than those observed in weanling or adult rat jejunum with F(2,7)=9.2, p<0.011 at 30 min and F(2,9) = 19.5, p < 0 . 0 0 0 5 at 60 min; this difference was not seen in ileum. There were no significant segmental or age-dependent differences in immunoreactive radioactivity of serosal fluids ex-

cept for a higher value in suckling rat jejunum at 60 min with F(2,8)=9.7, p<0.007, and in suckling rat ileum at 120 min (p<0.006).

Characterization of Uptake and Transfer of 1251-mEGF in Suckling Rat Jejunum and Ileum Comparison with transfer of PEG and mannitol. Tissue uptake of ~25I-mEGF (total or immunoreactive radioactivity) by jejunum and ileum was several times greater than that of 3H-PEG and t4C-mannitol (Table 1). Serosal transfer of t25I-mEGF (total radioactivity) was also 4-6 times greater; however, the transfer of immunoreactive t25I-mEGF was not different from those of 3HPEG or '4C-mannitol. Effects of serosal osmolarity. Data presented in Table 2 show that presence of 600 mM mannitol in serosal fluid significantly increased the fluid transfer (approximately two-fold) by everted sacs of suckling rat jejunum and ileum, whereas serosal transfer of immunoreactive " I-mEGF was unaffected. Replacement of sodium with potassium inhibited the serosal transfer of both fluid and immunoreactive '25I-mEGF; however, 34--35% of t2sImEGF transfer (but only 3-10% of fluid transfer) was independent of sodium. Effects of metabolic inhibitors and sodium replacement. The serosal transfer of t2SI-mEGF (both total and immunoreactive) by jejunum sacs was significantly inhibited by the presence of 0.05 mM DNP or 0.5 mM NaN 3 (Table 3). In case of ileal sacs, serosal transfer of total radioactivity was inhibited; however, transfer

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FIG. 2. Tissue uptake (A, jejunum; B, ileum) and serosal transfer (C, jejunum; D, Ileum) of immunoreactive 12~I-mEGF (irEGF) by everted sacs of jejunum or ileum from suckling (O), weanling (D) and adult (&) rats. Radioactivities extracted from tissues and serosal fluids were analyzed by anti-mEGF antibody affinity chromatography and expressed as cpm per 0.1 mg wet weight of intestine. *Represents values that are significantly different from those for weanling and adult rats (p<0.05).

o f i m m u n o r e a c t i v e radioactivity w a s unaffected by both D N P and N a N 3. R e p l a c e m e n t o f s o d i u m with either p o t a s s i u m or choline significantly decreased the serosal transfer o f 125I-mEGF (total or

i m m u n o r e a c t i v e ) in both j e j u n u m and ileum. U n d e r these conditions, serosal transfer o f fluid and g l u c o s e (as well as tissue uptake o f glucose) w a s m a r k e d l y attenuated by the presence o f D N P

TABLE 1 UPTAKE AND TRANSFER OF 125 I-mEGF BY EVERTED SACS OF SUCKLING RAT JEJUNUM AND ILEUM Tissue Uptake Jejunum ~25I-mEGF Total CPM (n = 7) irCPM (n = 3) 3H-PEG (n = 4) J4C-Mannitol (n=4)

Serosal Transfer Ileum Jejunum (CPM × 10-4/g tissue)

Ileum

15.15 ± 1.50

14.60 ± 1.05

10.50 ± 0.95

7.30 ± 0.70

3.95 ± 0.40

6.55 ± 0.65

2.10 ± 0.05

2.00 -+ 0.10

1.30 ± 0.05"?

1.40 ± 0.10"~"

1.90 ± 0.15"

1.85 -+ 0.15"

1.15 _+ 0.10"?

1.25 ± 0.10"?

1.80 -+ 0.10"

1.80 ± 0.10"

Everted sacs were incubated with 5 × 10 -5 CPM/ml of ~25I-mEGF with concentration (100 nM) adjusted by adding unlabeled mEGF, 3H-PEG (mol.wt. 4000, 100 nM) or 14C-mannitol (5 mM) at 37°C for 60 min.Values (mean ~ SEM) are significantly different from those corresponding for ~25I-mEGF, total CPM (*) or immunoreactive (ir) CPM (t) (p<0.05).

INTESTINAL PROCESSING OF EPIDERMAL GROWTH FACTOR

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TABLE 2 EFFECT OF SEROSALOSMOLARITY ON J2~I-mEGFTRANSFER BY EVERTED SACS OF SUCKLING RAT JEJUNUMAND ILEUM Fluid Transfer (microliter/g tissue wt.) Serosal Osmolarity Isotonic Hypertonic Isotonicsodium free

mEGF Transfer (irCPM x 10-4/g tissue wt.)

Jejunum

Ileum

Jejunum

Ileum

62.5 +- 4.6 (5) 128.2 +_ 16.7" (4) 2.2 _+ 2.2* (3)

47.8 -*- 11,7 (5) 105.0 ± 10.0 (4) 4.8 ± 1.3" (3)

2.43 ___ 0.02 (3) 2.87 ± 0.79* (4) 0.82 ± 0.16" (3)

2.74 ±_ 0.06 (3) 2.95 ___ 0.30 (4) 0.95 ___ 0.17" (3)

Serosal compartment of everted sacs was filled with the same buffer used for mucosal fluid with (hypertonic) or without (isotonic) 600 mM mannitol. For isotonic-sodium free conditions, sodium chlroide from mucosal and serosal fluids was replaced with equimolar potassium chloride. Everted sacs were incubated with 125I-mEGF (2 ng/ml) and 10 mM D-glucose at 37°C for 60 min. Values are mean ± SEM; n shown in parentheses. *Values that are significantly different from corresponding values for "isotonic" conditions (p<0.05).

or NaN 3 as well as by sodium depletion (data not shown). Competition by unlabeled EGF. The effects of increasing doses of unlabeled EGF (4.2-166 nM) on tissue uptake and serosal transfer of 12SI-mEGF by suckling rat jejunum and ileum were studied in the absence (Fig. 3A, B) or the presence (Fig. 3C, D) of 1.0 mM N'ethylamaleimide (N'EM). Presence of N ' E M completely inhibited the degradation of 125I-mEGF. Serosal transfer of total radioactivity was unaffected by N ' E M . In the absence of N ' E M , tissue uptake and serosal transfer of immunoreactive 125ImEGF were not affected by unlabeled mEGF except for a 28% decrease in tissue uptake by 166 nM unlabeled mEGF in jejunum (Fig. 3A). Serosal transfer was increased by 166 nM unlabeled mEGF; however, this increase was not statistically significant. Data presented in Fig. 3C show that in the presence of N ' E M , tissue uptake of ~25I-mEGF by suckling rat jejunum was dosedependently inhibited by unlabeled mEGF, F(6,26)=11.1,

p<0.0001. A maximum of about 50% inhibition was demonstrated by 83 nM mEGF. Only 12% of tissue uptake was inhibited in ileum which was found to be statistically insignificant by variance analysis in multiple comparisons and the F(5,12)= 2.1, p > 0 . 1 3 . Serosal transfer in both jejunum and ileum showed a 5 20% decrease; however, no statistical significance was established.

Characterization of Degradation Products ~25I-mEGF extracted from mucosal fluids, tissue and serosal fluids of everted sacs (after 30-min incubation) prepared from jejunum and ileum of suckling (Fig. 4) or adult (Fig. 5) rats was analyzed by RP-HPLC. Intact 125I-mEGF (standard) was eluted in fraction # 5 2 (0.6-rnin fractions); this elution position remained unaltered when the standard I2~I-mEGF was mixed with extracts

TABLE 3 EFFECTS OF METABOLICINHIBITORS ON TRANSFER OF 125I-mEGFBY EVERTEDSACS OF SUCKLING RAT INTESTINE Total Radioactivity Jejunum

Control (n=7) Dinitrophenol (0.05 raM; n= 3) Sodium azide (0.5 mM; n=3)

Immunoreactive Radioactivity

Ileum Jejunum (CPM x 10 - 4/g tissue)

Ileum

8.95 ___ 0.50 6.52 + 0.24*

9.79 - 0.40 6.80 ~ 0.65*

2.35 ±_ 0.31 0.82 ± 0.06*

1.98 _ 0.20 1.87 _+ 0.61

7.01 ± 0.09*

7.56 --- 0.56*

1.03 _+ 0.30*

1.21 ___ 0.29

Replacement of sodium with: Potassium (n=9) 1.50 - 0.05* Choline (n=5) 2.31 - 0.21"

2.10 --- 0.12" 2.38 +-- 0.17"

0.62 "4- 0.13" 0.68 --- 0.07*

0.70 +- 0.14" 0.86 ± 0.08*

Everted sacs were incubated with 125I-mEGF (2 ng/ml) and 10 mM glucose for 60 min at 37°C, with or without the presence of 0.05 mM dinitrophenol or 0.5 mM sodium azide, or with the sodium replaced by potassium or choline. Total and immunoreactive a25I-mEGF in serosal fluids were measured. *Values (mean -+ SEM) significantly different from corresponding control values (p<0.05).

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FIG. 3. Effects of unlabeled EGF on tissue uptake (A and C) and serosal transfer (B and D) of immunoreactive 125I-mEGF (irCPM) by suckling rat jejunum ([q) and ileum (A). Everted sacs were incubated with 0.33 nM ~25I-mEGFin the presence of 0-166 nM unlabeled EGF (control is the first point in each figure which represents zero unlabeled mEGF). Experiments were conducted in the absence (A and B) or presence (C and D) of 1.0 mM N'EM. Values are mean_+ SEM (n=3 for A and B; 3-9 for C and D). *Values that are significantly different from corresponding control (no added unlabeled EGF) values (p<0.05).

of mucosal fluid or tissue prior to analysis. 12Sl-mEGF was mixed with extracts of mucosal fluid or tissue prior to analysis. 125Ides(49-53)mEGF, 125I-des(48-53)mEGF and 1251-des(53)mEGF (standard, see the Method section) were eluted in fractions #39, 44 and 59, respectively. Data presented in Figs. 4 (suckling) and 5 (adult) show that 125I-mEGF undergoes proteolytic processing in everted sacs of suckling and adult rat intestinal segments. Intact 125I-mEGF was detected in mucosal fluid and serosal fluid but not in the tissue extracts of suckling rat jejunum (Fig. 4). In the case of suckling rat ileum, no intact 125I-mEGF was detected in mucosal fluid or tissue extracts; however, the major peak of radioactivity in serosal fluid was identified as intact 125I-mEGF. There was no intact 125I-mEGF detected in adult rat jejunum or ileum. Three different forms of carboxy-terminally processed ~25I-mEGF (identified by their coelution with pure standards) and several smaller fragments (unidentified) were detected in the everted sacs of suckling rat jejunum and ileum. In mucosal fluid of suckling rat jejunum, a second major peak of radioactivity (which also forms the major peak of radioactivity in serosal fluid) coeluted with 125Ides(53)mEGF. This peak was absent in everted sacs of suckling rat ileum or adult rat jejunum and ileum. The major portion of radioactivity in tissue extracts of suckling rat jejunum (eluting in fraction #39) was identified as 125I-des(49-53)mEGF which also formed a small peak of radioactivity in serosal fluid. 125I-des(4953)mEGF also was absent in suckling rat ileum and adult rat je-

junum or ileum. In contrast to suckling rat jejunum, radioactivity in mucosal fluids and tissue extracts of everted sacs from suckling rat ileum and adult rat jejunum or ileum was identified as 125I-des(48-53)mEGF; this formed only a minor peak in suckling rat jejunum. In addition, several minor unidentified peaks of radioactivity (eluting in fractions #4, 10, 11, 12, 22 and 38) were detected in the everted sacs of suckling and adult rat intestinal segments (Figs. 4 and 5).

Binding to Antibody and Receptor More than 95% of intact 1251-mEGF and carboxy-terminally processed 125I-mEGF (both standards and those isolated from everted sacs) bound to anti-EGF antibody column (Table 4). Specific binding of 125I-des(53)mEGF and peak D (isolated from mucosal fluid of suckling rat jejunum) to placental membrane receptors was 18-20% higher than that of intact 125I-mEGF. Conversely, bindings of standard 125I-des(49-53)mEGF and ~25I-des(48-53) mEGF to the receptors were lower (58% and 89%, respectively) than that of intact ~25I-mEGF. Similarly, bindings of peak A (isolated from suckling rat jejunal tissue) and peak B (isolated from mucosal fluids of suckling rat ileum and adult rat jejunum or ileum) were lower by 59% and 80-83%, respectively. RP-HPLC analysis of the radioactivity in the receptor binding medium after incubations demonstrated the absence of degradation of ~25ImEGF and its derivatives. Other minor fragments (eluting in frac-

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TABLE 4 BINDINGOF ~2SI-mEGFAND ITS DEGRADATIONPRODUCTSTO ANTI-mEGFANTIBODYAND EGF RECEPTOR Antibody-Bound CPM

Receptor-Bound CPM

Authentic Products ~25I-mEGF ~25I-des(53)mEGF ~25I-des(49-53)mEGF 125I-de(48-53)mEGF

100.0 99.2 98.0 97.3

--- 1.5 ± 0.8 ± 0.4 ± 1.6

100.0 117.5 42.1 11.0

Products: Everted Sacs Peak C (Su-jej-MF) Peak D (Su-jej-MF) Peak A (Su-jej-TIS) Peak B (Su-il-MF) Peak B (Ad-jej-MF) Peak B (Ad-il-MF)

98.6 97.3 96.9 96.5 96.4 96.5

- 1.2 --- 1.5

106.0 ± 3.4 128.0 ± 4.4* 45;42 17.7 ~ 1.1" 19.1 +-- 1.2" 21.0 ± 0.7*

- 1.0 --- 1.3 ± 1.6

± ± ± ±

1.5 1.6" 1.8" 3.1"

RP-HPLC peaks C, D, A and B, isolated from either suckling (Su) or adult (Ad) rat jejunum (jej) or ileum (il), mucosal fluids (MF) or tissue (TIS) were coeluted with authentic 12SI-mEGF, 125I-des(53)mEGF, 125Ides(49-53)mEGF and 125I-des(48-53)mEGF, respectively. Different samples (of 10,000 CPM) were bound to anti-EGF antibody or EGF-specific receptors; binding values are presented as percentage of values for ~:sImEGF (9842 --- 50 antibody-bound CPM and 3320 --- 51 receptor-bound CPM). All values are mean ± SEM of triplicate determinations except for peak A of Su-jej-TIS. *Values significantly different from those for authentic 125I-mEGF(p<0.05).

tions # 4 , 10, 11, 12 and 22) did not bind to anti-mEGF antibody or to placental membrane receptors. DISCUSSION Using the everted sac technique, we have shown the uptake and transfer of ~2~I-mEGF in immunoreactive and receptor binding forms by jejunum and ileum of suckling, weanling and adult rats. Serosal transfer of immunoreactive ~25I-mEGF exhibited a time-dependent increase in suckling rat jejunum and ileum or in weanling rat jejunum. This supports previous reports regarding gastrointestinal absorption of orogastrically or intraintestinally administered ~25I-mEGF in suckling rats and mice (20, 21, 33). Neonatal mammalian small intestine absorbs gamma globulins and other milk proteins (1,3); this absorption is considerably diminished at weaning. However, there is evidence to indicate that adult animals continue to absorb macromolecules in antigenic and biologically active quantities (29,35). We characterized further the intestinal uptake and transfer of ~25I-mEGF in suckling rats and examined the degradation products formed in suckling and adult rats. Uptake and transfer of ~25I-mEGF, measured as radiolabeled material in the wall and serosal fluids of everted sacs, were several-fold greater than those of 3H-PEG or ~4C-mannitol, indicating a facilitated EGF uptake in suckling rat small intestine. The transfer of ~25I-mEGF in jejunum and ileum was significantly inhibited by metabolic inhibitors (DNP or NaN 3) and replacement of sodium with potassium or choline. These results indicate that intestinal transfer of ~25ImEGF is energy and sodium dependent. Interdependence of sodium transfer and oxygen consumption with intestinal transfer of serum proteins in young rats was demonstrated in vitro (1,3). The transfer of horse radish peroxidase in adult rats was also shown to be energy dependent (35). In contrast, recent studies have shown that intestinal transfer of smaller oligopeptides in adult ro-

dents was independent of sodium and energy (10,30). The energy- and sodium-dependent transfer of mEGF in suckling rat small intestine may be relevant to previous studies on in vivo absorption of orally administered mEGF (31) and on the effects of orally administered mEGF on intestinal growth (12,13). Tissue uptake of 125I-mEGF was dose-dependently inhibited by unlabeled EGF, indicating the presence of specific binding sites for mEGF in suckling rat jejunum and ileum. Specific binding sites for ~25I-mEGF were previously localized by electron microscopic autoradiography on apical membranes of suckling rat ileal sheets (7). Our present study shows that approximately 50% of tissue uptake by suckling rat jejunum is receptor mediated, whereas only 12% is receptor mediated in the ileum. Specific receptors for IgG which mediate the transepithelial transport of intact antibodies have been also detected in absorptive cells of proximal intestine (15). Degradation of 125I-mEGF occurred in jejunum and ileum of suckling, weanling and adult rats. We have previously demonstrated in in vitro studies using isolated enterocytes that both villus and crypt cells of suckling rat jejunum degraded ~25I-mEGF whereas in adult rats degradative activity was restricted to crypt cells (23). In contrast, our previous in vivo studies have indicated that there is a greater degradation of ~25I-mEGF in the gastrointestinal tract of weanling rats than in suckling rats (33). The apparent discrepancy between the results obtained in in vivo and in vitro studies suggests that proteolytic activities of pancreatic origin in gastrointestinal lumen may play a major role in the mEGF degradation in the in vivo studies. This is supported by a low degradation of EGF by suckling rat pancreatic homogenates (32) and by duodenojejunal juice collected from infants (2) from suckling rats as compared to that of adult rats. Previous studies (31,33) have shown that absorbed ~2SI-mEGF is a molecule large enough to exclude in the void volume of Sephadex G-25 columns, and is capable of binding to anti-mEGF

1100

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FIG. 4. RP-HPLC of J25I-mEGFin everted sacs of suckling rat jejunum and ileum incubated for 30 min at 37°C. 125I-mEGFextracted from mucosal fluids (MF), tissue (TIS) and serosal fluids (SF) was analyzed by RP-HPLC as described in the Method section. Radioactivity was measured in 0.6-min fractions. Exclamation marks designate the elution positions of 125I-EGF (C), 12~I-des(53)mEGF(D), 12~I-des(49-53)mEGF(A), and 125I-des(48-53)mEGF(B). antibodies and EGF-specific receptors. Studies by other investigators (7) indicated a decrease in the size of 125I-mEGF in isolated ileal loops of suckling rats. However, the structural and functional characteristics of absorbed ~25I-mEGF is unclear. In our present studies, RP-HPLC analysis indicated that ~25I-EGF undergoes proteolytic processing at the carboxy-terminus prior to transfer into serosal fluid. Intact ~251-mEGF was detected only in the serosal fluid of suckling rat ileum although previous in vivo studies (7) did not indicate a transfer of intact EGF. ~25I-mEGF transferred by suckling rat jejunum was identified as laSI-des (53)mEGF in which carboxy-terminal arginine was removed by carboxypeptidase B-like activity. ~25I-des(53)mEGF, isolated from everted sacs of suckling rat jejunum, binds to anti-mEGF antibodies and EGF-specific receptors; receptor binding is significantly greater than that of intact ~zSI-mEGF. On the other hand, radiolabeled material transferred by adult rat jejunum and ileum was identified as 125I-des(48-53)mEGF in which the carboxyterminal polypeptide of 6 amino acids was deleted. This modified

form of mEGF was fully immunoreactive, but receptor binding was greatly diminished. Diminished affinity of EGF for its receptors by removal of carboxy-terminal polypeptide was first reported by Carpenter et al. (5). Formation of carboxy-terminally processed mEGF forms with altered receptor binding affinity was later demonstrated in other laboratories using cultured fibroblasts (19,26). A lower affinity of des(49-53)mEGF to sheep skin membrane receptors was correlated with its diminished potency to induce precocious eyelid opening in mice (36). The physiological significance of carboxy-terminal processing in fibroblasts (19,26) and in rat small intestine is unclear. However, interesting studies by Hollenberg and Gregory (9) have shown that des(48-53)mEGF is as potent as intact mEGF in inhibiting gastric acid secretion, but it is several-fold less potent in stimulating thymidine incorporation to DNA in fibroblasts. On the other hand, C-terminally processed mEGF derivatives were as potent as intact mEGF when tested by mouse bioassay (25). Therefore, it is possible that the mEGF derivatives formed in

INTESTINAL PROCESSING OF EPIDERMAL GROWTH FACTOR

1101 ILEUM

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FIG. 5. RP-HPLC of ~25I-mEGF in evened sacs of adult rat jejunum and ileum incubated for 30 min at 37°C. ~251-mEGFextracted from mucosal fluid (MF), tissue (TIS) and serosal fluids (SF) was analyzed by RP-HPLC as described in the Method section. Radioactivity was measured in 0.6-min fractions. Exclamation marks designate the elution positions of t2sImEGF (C) and ]25I-des(48-53)mEGF (B).

everted sacs of suckling and adult rat intestine are biologically active. In tissue extracts of suckling rat jejunum, ]25I-mEGF was identified as ~25I-des(49-53)mEGF which possesses complete immunoreactivity (with diminished receptor binding). This form of mEGF was not detected in suckling rat ileum or adult rat jejunum and ileum. Thus formation of specific products may indicate a distinct role for suckling rat jejunum in mEGF processing. Interestingly, sequence at C-terminal pentapeptide is identical in mouse and human EGF. Studies on complete amino acid sequencing of rat EGF (27) have demonstrated that rEGF lacks the 5 C-terminal residues present in mEGF and human EGF. This may raise questions about the biological significance of C-terminal processing of EGF in rat small intestine. However, it is possible that the truncated C-terminus of rat EGF may have arisen due to the activity of contaminating proteases during the isolation procedure. For instance, when mEGF was extracted from submaxillary glands at pH 3.2 instead of pH 4.5, the predominant form of EGF obtained

lacked the C-terminal dipeptide (24). And hence there is considerable doubt about the primary sequence of rat EGF particularly at the C-terminus. In addition, recent studies (16) have demonstrated that rat urine collected without any additives contains only low molecular weight form of EGF (48 residues), whereas urine collected in the presence of serine proteases inhibitors contains a second EGF molecule (51 residues) in which residues 49 and 50 are identical to those of mouse and human EGF. Therefore, it can be safely stated that C-terminal processing of EGF may have biological significance in rat, mouse and human small intestine. ~25I-mEGF in the mucosal fluids and tissue extracts of suckling rat ileum and adult rat jejunum or ileum was identified as ~25I-des(48-53)mEGF. This indicates carboxy-terminal cleavage of 125I-mEGF by brush border enzymes or luminal enzymes adhering to the brush border surface. In contrast to other segments, only a minor amount of ]25I-des(53)mEGF was detected in the tissue extract of suckling rat jejunum, although it was present in

RAO ET AL.

1102

mucosal and serosal fluids; ~25I-des(49-53)mEGF was detected in tissue extracts. This may suggest that processing to J25Ides(53)mEGF occurs at the apical membrane surface of suckling rat jejunum, whereas processing to 1251-des(49-53)mEGF may occur intracellularly. However, understanding the localization of EGF processing and uptake and transfer pathways needs further study. In conclusion, the presented studies indicate that mEGF undergoes carboxy-terminal processing during uptake and transfer by the small intestine of developing and adult rats. Suckling rat jejunum transfers mEGF in a form which shows greater receptor

binding, whereas adult rat intestine metabolizes mEGF to a form with diminished receptor binding. An identical amino acid sequence of C-terminal pentapeptide of EGF from mouse, human and possibly rat may suggest a biological significance of Cterminal processing of EGF in the small intestine of different species. ACKNOWLEDGEMENTS This work was supported by National Institutes of Health grants DK27624 (O.K.); DK36289 (T.P.D.); MH42600 (T.P.D.); and CA 40162 (M.K.).

REFERENCES 1. Bamford, D. R. Studies in vitro of the passage of serum proteins across the intestinal wall of young rats. Proc. R. Soc. Biochem. 166: 30-45; 1966. 2. Britton, J. R.; Koldovsk3~, O. Epidermal growth factor degradation by gastrointestinal fluid from suckling and weanling rats. Pediatr. Res. 21:210A; 1987. 3. Brown, P.; Smith, M. W.; Witty, R. Interdependence of albumin and sodium transport in the foetal and new-born pig intestine. J. Physiol. 198:365-381; 1968. 4. Carpenter, G.; Cohen, S. 1251-labeledhuman epidermal growth factor, binding, internalization and degradation in human fibroblasts. J. Cell Biol. 71:159-171; 1976. 5. Carpenter, G.; Lembach, K. J.; Morrison, M. M.; Cohen, S. Characterization of the binding of lZSI-labeledepidermal growth factor to human fibroblasts. J. Biol. Chem. 250:4297-4304; 1975. 6. Cohen, S. Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal. J. Biol. Chem. 237:1555-1562; 1962. 7. Gonnella, P. A.; Siminoski, K.; Murphy, R. A.; Neutra, M. R. Transepithelial transport of epidermal growth factor by absorptive cells of suckling rat ileum. J. Clin. Invest. 80:22-32; 1987. 8. Hock, R. A.; Nex0, E.; Hollenberg, M. D. Solubilization and isolation of the human placenta receptor for epidermal growth factor-urogastrone. J. Biol. Chem. 255:10737-10743; 1980. 9. Hollenberg, M. D.; Gregory, H. Epidermal growth factor-urogastrone: Biological activity and receptor binding of derivatives. Mol. Pharmacol. 17:314-320; 1980. 10. Hunjan, R. J.; Evgred, D. F. Absorption of glutathione from the gastro-intestinal tract. Biochim. Biophys. Acta 815:185-188; 1985. 11. Kleinman, R. E.; Walker, W. A. Antigen processing and uptake from the intestinal tract. Clin. Rev. Allergy 2:25-37; 1984. 12. Koldovsk~, O.; Thornburg, W. Hormones in milk. J. Pediatr. Gastroenterol. Nutr. 6:172-196; 1987. 13. Koldovsk3), O. Search for role of milk-borne biologically active peptides for the suckling. J. Nutr. 119:1543-1551 ; 1989. 14. Leichter, J.; Goda, T.; Bhandari, S. D.; Bustamante, S.; Koldovsk3~, O. Relation between dietary-induced increase of intestinal lactase activity and lactose digestion and absorption in adult rats. Am. J. Physiol. 247:G729-G735; 1984. 15. Morris, B.; Morris, R. The digestion and transmission of labelled immunoglobulins G by enterocytes of the proximal and distal regions of the small intestine of young rats. J. Physiol. 203:427-442; 1977. 16. Nex¢, E.; J0rgensen, P. E.; Thim, L.; Roepstorff, P. Purification and characterization of a low and a high molecular weight form of epidermal growth factor from rat urine. Biochim. Biophys. Acta 1037:388-393; 1990. 17. Olsen, P. S.; Kirkegaard, P.; Poulsen, S. S.; NexO, E. Adrenergic effects on exocrine secretion of rat submandibular epidermal growth factor. Gut 25:1234-1240; 1984. 18. Olsen, P. S.; Next, E. Quantitation of epidermal growth factor in the rat. Identification and partial characterization of duodenal EGF. Scand. J. Gastroenterol. 18:771-776; 1983. 19. Planck, S. R.; Finch, J. S.; Magun, B. E. Intracellular processing of epidermal growth factor. II. Intracellular cleavage of the COOH-terminal region of lZSI-epidermal growth factor. J. Biol. Chem. 259:

3053-3057; 1984. 20. Popliker, M.; Shatz, A.; Avivi, A.; Ullrich, A.; Schlessinger, J.; Webb, C. G. Onset of endogenous synthesis of epidermal growth factor in neonatal mice. Dev. Biol. 119:38-44; 1987. 21. Rao, R. K.; Davis, T. P.; Grimes, J.; Koldovsk3), O. Gastrointestinal absorption of epidermal growth factor: Dose-dependency and regional differences. Pediatr. Res. 25:1234A; 1989. 22. Rao, R. K.; Thornburg, W.; Grimes, J.; Koldovsk~, O. Transfer and processing of epidermal growth factor in vitro in everted jejunal and ileal sacs of suckling, weanling and adult rats. Pediatr. Res. 20:248A; 1986. 23. Rao, R. K.; Thornburg, W., Matrisian, L., Magun, B. E.; Korc, M.; Koldovsk~, O. Processing of epidermal growth factor by suckling and adult rat intestinal cells. Am J. Physiol. 250:G850~3855; 1986. 24. Savage, C. R.; Cohen, S. Epidermal growth factor and a new derivative. J. Biol. Chem. 247:7609-7611; 1972. 25. Savage, C. R.; Inagami, T.; Cohen, S. The primary structure of epidermal growth factor. J. Biol. Chem. 247:7612-7621; 1972. 26. Schaudies, R. P.; Savage, C. R., Jr. Intracellular modification of ~25I-labeled epidermal growth factor by normal human foreskin fibroblasts. Endocrinology 118:875-882; 1986. 27. Simpson, R. J.; Smith, J. A.; Moritz, R. L.; O'Hare, M. J.; Rudland, P. S.; Morrison, J. R.; Lloyd, C. J.; Grego, B.; Burgess, A. W.; Nice, E. E. Rat epidermal growth factor: Complete amino acid sequence. Eur. J. Biochem. 153:629-637; 1985. 28. Stahl, G. E.; Fayer, J. C.; Ling, S.; Watkins, J. B. Comparison of non-absorbable markers (NAMs) for use in developmental absorption studies. Pediatr. Res. 25:124A; 1989. 29. Stern, M.; Pang, K. Y.; Walker, W. A. Food proteins and gut mucosal barrier. II. Differential interaction of cow's milk proteins with the mucous coat and the surface membrane of adult and immature rat jejunum. Pediatr. Res. 18:1252-1257; 1984. 30. Takori, K.; Burton, J.; Donowitz, M. The transport of an intact oligopeptide across adult mammalian jejunum. Biochem. Biophys. Res. Commun. 137:682-687; 1986. 31. Thornburg, W.; Matrisian, L.; Magun, B. E.; Koldovsk3), O. Gastrointestinal absorption of epidermal growth factor in suckling rats. Am. J. Physiol. 246:G80-G85; 1986. 32. Thornburg, W.; Planck, S.; Matrisian, L.; Korc, M.; Pollack, P.; Magun, B.; Koldovsk3~,O. Hydrolysis of epidermal growth factor by rat pancreas and liver in vitro. Fed. Proc. 44:685; 1985 (abstract). 33. Thornburg, W.; Rao, R. K.; Magun, B. E.; Matrisian, L.; Koldovsk~, O. Effect of maturation on gastrointestinal absorption of epidermal growth factor in rats. Am. J. Physiol. 253:G68-G71; 1987. 34. Ulshen, M. H.; Lyn-Cook, L. E.; Raasch, R. H. Effects of intraluminal ,epidermal growth factor on mucosal proliferation in the small intestine of adult rats. Gastroenterology 91:1134-1140; 1986. 35. Walker, W. A.; Cornell, R.; Davenport, L. M.; Isselbacher, K. J. Macromolecular absorption. Mechanism of horseradish peroxidase uptake and transport in adult and neonatal rat intestine. J. Cell Biol. 54:195-205; 1972. 36. Wynn, P. C.; Maddocks, I. G.; Moore, G. P. M.; Panaretto, B. A.; Djura, P.; Ward, W. G.; Fleck, E.; Chapman, R. E. Characterization and localization of receptors for epidermal growth factor in ovine skin. J. Endocrinol. 121:81-90; 1989.